Neuronal cells appeared rarely in GNE KO ES cultures and did not reach an advanced differentiated stage. Although primary cardiac cells appeared at the same time in both normal and GNE KO ES cultures, GNE KO cardiac cells degraded very soon and their beating capacity decayed rapidly. Furthermore very rare skeletal muscle committed cells were detected in the GNE KO ES cultures at any stage of differentiation, as assessed by analysis of the expression of either Pax7, MyoD and MyHC markers. Beyond the supporting evidence that GNE plays an important role in neuronal cell and brain development, these results show that GNE is strongly involved in cardiac tissue and skeletal muscle early survival and organization. These findings could open new avenues in the understanding of muscle function mechanisms in health and in disease.UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamime kinase (GNE) is a bifunctional enzyme which catalyzes the two key sequential steps in the biosynthetic pathway of sialic acid, the most abundant terminal monosaccharide on glycoconjugates of eukaryotic cells. GNE knock out (GNE KO) mice are embryonically lethal at day E8.5. Although the role of GNE in the sialic pathway has been well established as well as the importance of sialylation in many diverse biological pathways, less is known about the involvement of GNE in muscle development. To address this issue we have studied the role of GNE during  UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamime kinase (GNE) is a bifunctional enzyme with 2 distinct domains which catalyze the two key sequential steps in the biosynthetic pathway of sialic acid: the epimerase activity synthesizes ManNAc from UDP-GlcNAc, followed by ManNAc kinase phosphorylation of ManNAc to generate ManNAc 6-phosphate Sialic acid is the most abundant terminal monosaccharide on glycoconjugates of eukaryotic cells Interestingly, an overall reduction of 25% in membrane bound sialic acid was observed in various organs of heterozygous GNE knock out mice Therefore we and others have hypothesized that GNE plays additional roles that could eventually explain the relatively mild and skeletal muscle specific phenotype of HIBM. Knocking out the GNE gene in mice results in mouse embryos lethality at day E8.5 and therefore these mice cannot be used as a tool for our purpose, although the cause of death has not been determined.In an attempt to unravel novel role(s) of GNE in normal cells, we have used the embryonic stem cells from wild type and GNE−/− mice embryos, to study the differentiation patterns of these cells during very early development, to various tissues, in particular skeletal and cardiac muscle.Embryonic stem cells were the kind gift of Dr Ruediger Horstkorte . GNE−/− cells had been fully characterized as having no GNE enzymatic activity −5 M β-mercaptoethanol )Bio-Rad, USA) and with 5×105 units of recombinant human leukemia inhibitory factor (LIF)/500 ml.Cultures were established essentially as described by Wobus A. et al For differentiation, cells were grown in hanging drops to allow the formation of embryoid bodies (EB). On day 3, 1% of DMSO  was added to the medium in order to favor skeletal muscle specific differentiation and EBs were grown in suspension for 3 more days. At day 5, EBs were plated on gelatin coated tissue culture plates for further differentiation. On average, 2 EBs were plated per well of a 6 well plate. For immunofluorescence, EBs were plated on LAB-TEC chambers slides . Cells were kept in culture up to 45 days and examined at different time points for morphology and specific markers expression. Time point 0 refers to proliferating ES cells before EB formation as a hanging drop. Day 5 refers to the plating day of EBs onto gelatin coated tissue culture plates.RNA was extracted from 2 wells of the 6 wells plate, with Tri-Reagent  at each time point, both from GNE KO and WT ES differentiated cells, as described by the manufacturer. RNA was reverse transcribed using random hexamer primers  by the Masterscript reverse transcriptase enzyme  following the protocol supplied by the manufacturer. The cDNA products were denatured for 5 min at 95°C, followed by 40 cycles of amplification with the appropriate primers: 45 sec denaturation at 95°C, 30 sec annealing at 55 to 72°C (depending on the primer used), and 30–120 sec elongation at 72°C followed by a final elongation at 72°C for 5 min. For β-tubulin detection, 30 cycles of amplification were performed and 25 cycles for NFM. One-fifth of each RT–PCR reaction was electrophoretically separated on 2% agarose gels. For MyoR detection the 7500 real-time PCR system  was used, together with mouse GAPDH, using power SYBR green master mix . The expression of the following genes was studied: Oct4, NFM, βMHC, Pax7, MyoD, MyoR , permeabilized and fixed with ice-cold methanol (15 min at −20°C). Cells were blocked in 2% BSA/PBS for 60 min at RT and incubated for 30 min at RT with mouse monoclonal anti Myosin Heavy Chain antibody (MF-20 hybridoma)  and mouse monoclonal anti desmin antibody  diluted in 2% BSA/PBS. After washing in PBS, cells were incubated 30 min at RT with goat anti-mouse Cy3-conjugated antibody  diluted in 1% BSA/PBS. Nuclei were stained by Hoechst .Light microscope pictures were viewed in CKX41 system inverted microscope ,and photographed by a C5060 digital camera (Olympus). Immunofluorescence was viewed in a BX41 system microscope , photographed by a DP70 microscope digital camera and analyzed by the DP software (Olympus).For glycan analyses, cell pellets were dissolved in 0.2% SDS, 1% beta-mercaptoethanol, 0.1% NP40 and homogeneized.exc 373 nm and λem 448 nm.WT and GNE ko stem cell samples were subjected to sialic acid analysis Mouse ES cells can proliferate and differentiate in vitro. In certain culture conditions, these cells can differentiate spontaneously in to various lineages, in particular to neuronal cells, cardiac muscle cells and skeletal muscle cells During cell culture expansion prior to the differentiation process, we noticed that the proliferation rate was about twice higher in GNE−/− cells than in normal ES (results not shown), in line with the observations reported by Weidemann et al At day 5+20, the formation of long fibers could be observed  beating Molecular markers representing the different differentiation stages of the different tissues were analyzed at various time points. cDNA was synthesized from RNA extracted from cell cultures of both GNE+/+ and GNE−/− origin, at several differentiation time points, and analyzed by PCR for the expression of various relevant genes: Oct4 as a marker of proliferating ES cells; NF-M (medium neurofilament) as a very early marker of neuronal precursor; cardiac β-MHC (cardiac myosin heavy chain beta) which is expressed mostly during embryonic development; Pax7 as a very early marker of skeletal muscle satellite cells commitment; MyoD, which is one of the earliest markers of myogenic commitment and is expressed in activated satellite cells, representing the beginning of the skeletal muscle differentiation process, and MyoR, a regulator of MyoD The results are showed in Myosin heavy chain (MyHC), a marker of late differentiation of skeletal muscle, was examined by immunofluorescence. As seen in Sialic acid analysis of the membrane glycoprotein fractions of 300,000 cells showed the presence of Neu5Ac as well as trace amounts of Neu5Gc in both samples, the analysis was performed in duplicate and the average values are presented in In the context of the recessive disease Hereditary Inclusion Body Myopathy (HIBM), caused by missense mutations in GNE, we were interested in elucidating possible roles of GNE specifically in skeletal muscle. The role of GNE in the sialic acid pathway has been well established, as well as the importance of sialylation in many diverse biological pathways mostly involving cell-cell recognition mechanisms, in particular in brain development. In an attempt to clarify whether GNE is directly involved in muscle development, we have take advantage of the availability of GNE KO ES cells derived from homozygous GNE KO embryos at day E3.5 In this study, we could recapitulate the time course development of normal ES cells as reported before As expected, we found that GNE has a great impact in the development of neural cells; indeed the role of GNE and sialic acid in brain development and plasticity has been well established. It has been shown that the polysialic acid chain bound to the neural cell adhesion molecule (NCAM) is responsible for the neural plasticity during fetal development and in the brain of the adult Skeletal muscle development in GNE−/− ES cultures seems to be affected even earlier. Normal ES cells can differentiate to satellite skeletal muscle cells, as revealed by the expression of Pax7, MyoD MyHC and MyoR in GNE+/+ cell cultures. Although we could not detect clearly cells with skeletal muscle morphology in either the GNE+/+ or the GNE−/− cultures, probably because the density of the cultures at this stage  and GNE KO ES (KO ES) cells at day 5+12 post differentiation. Both cell types contract at similar pace.(MPG)Click here for additional data file.Video S2Functional cardiac cells in cultures of normal (N ES) and GNE KO ES (KO ES) cultures at day 5+30 post differentiation. Normal cells contract at the same pace than at day 5+12, in contrast GNE KO cells contractions are much slower.(MPG)Click here for additional data file.Table S1Primer sequences used for analysis of expression markers.(DOC)Click here for additional data file.


It has been suggested that age-related impairments in learning and memory may be due to age-related deficits in long-term potentiation of glutamatergic synaptic transmission. For example, olfactory discrimination learning is significantly affected by aging in mice and this may be due, in part, to diminished synaptic plasticity in piriform cortex. In the present study, we tested for alterations in electrophysiological properties and synaptic transmission in this simple cortical network. Whole-cell recordings were made from principal neurons in slices of anterior piriform cortex from young (3–6 months old) and old (24–28 months) C57Bl/6 mice. Miniature excitatory postsynaptic currents (mEPSCs) mediated by AMPA receptors were collected from cells in presence of tetrodotoxin (TTX) and held at -80 mV in voltage-clamp. Amplitudes of mEPSCs were significantly reduced in aged mice, suggesting that synaptic AMPA receptor expression is decreased during aging. In a second set of experiments, spontaneous excitatory postsynaptic currents (s/mEPSCs) were recorded in slices from different cohorts of young and old mice, in the absence of TTX. These currents resembled mEPSCs and were similarly reduced in amplitude in old mice. The results represent the first electrophysiological evidence for age-related declines in glutamatergic synaptic function in the mammalian olfactory system. Changes in the nervous system with aging are profound and mysterious. The brain exhibits subtle alterations in cellular morphology, synaptic structure, gene expression patterns, and electrophysiological characteristics as it ages; less subtle, perhaps, are the sensory, motor, and cognitive declines that accompany the aging process. Understanding the relationships between neurobiological changes and functional outcomes is one of the fundamental challenges of aging neuroscience. Considerable progress has been made in correlating age-dependent changes in hippocampal circuitry to spatial learning and memory deficits in aging animals. Two general principles have emerged from studies of hippocampal long-term potentiation (LTP) in aged animals : first, The olfactory system has a number of advantages for neurobiological studies of sensory and cognitive functions in aging. Olfactory discrimination ability declines markedly in human aging  and appeThe primary olfactory (piriform) cortex receives monosynaptic input from the mitral (and tufted) cells of the olfactory bulb, which themselves receive monosynaptic input from primary sensory neurons in the olfactory epithelium. Layer II pyramidal neurons of piriform cortex appear to be situated to form combinatorial representations of the different olfactory bulb glomeruli that respond to distinct molecular features of the chemicals comprising an odor . Piriforad libitum and routine veterinary visits ensured that animals were in good health prior to experimentation. Any animals that displayed physical ailments or lethargy were excluded from this study. Electrophysiological experiments were conducted on brain slices obtained from mice aged 3–6 months  or 24–28 months . All procedures were in accordance with NIH guidelines and protocols were approved by the Animal Care Committee of the University of Illinois at Chicago (UIC ACC #09-232).C57Bl/6J mice were bred in the Psychiatric Institute vivarium from breeding stock obtained from Jackson Laboratories . Mice were weaned at 21 days of age and males were separated and housed in groups of two to four per cage until sacrificed for experiments. Food and water were available 2HPO4 (1.25), NaHCO3 (26), dextrose (5.0), L-ascorbate (2), MgCl2 (1.0), CaCl2 (2.0) at ~4°C. The brain was then sectioned into blocks (2–3 mm thick), mounted on a cutting stage, and sliced on a vibrating cutter . The slices were incubated at 32°C for 1 h and then allowed to cool to room temperature (~25–27°C). Slices were then transferred to a submerged recording chamber and perfused with aCSF. Recordings were obtained from principal cells in layer II at room temperature, as described previously (2 (1), HEPES (10), BAPTA (1.1), MgATP (5), and phosphocreatine (20) adjusted to pH 7.2 with CsOH, 290 mOsm. Cells were visualized with differential interference contrast (DIC) optics on a Nikon Eclipse E600FN “PhysioStation” . Twisted bipolar electrodes (custom made) with a tip diameter ~50 μm were positioned in layer Ia and Ib 150–200 μm rostral from the recorded cell. Stimulation (0.1 ms pulses 1–200 μA) in these layers activate afferent lateral olfactory tract (LOT) and intrinsic association (ASSN) fibers synapsing onto principal cells in layer II. Responses at ASSN synapses display paired-pulse depression whereas stimulation of afferent LOT fibers in layer Ia exhibit facilitation  of APC were prepared in the usual method. Mice were decapitated and brains removed in oxygenated artificial cerebral spinal fluid (aCSF) containing (in mM): NaCl (120), KCl (3.1), Keviously . Patch elitation . TherefoA mediated transmission was blocked by 25 μM 1(S),9(R)-(-)-bicuculline methiodide (BMI) in all experiments in order to isolate postsynaptic excitatory currents. The NMDA receptor antagonist, 3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid ) was used to isolate AMPA receptor-mediated currents. 1 μM tetrodotoxin (TTX) was added to the perfusate in order to eliminate spontaneous action potential-dependent events. Some experiments used 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to block AMPA receptors (20 μM). All drugs were taken from stock solutions dissolved in H2O and diluted in aCSF as needed.All drugs and chemicals were applied via the perfusate by a solenoid-controlled gravity-feed system . The rate of flow of all drug perfusates was equilibrated to 2 mL/min prior to the inception of experimentation. GABAIn experiments using synaptic stimulation under voltage-clamp, the following stimulation protocol was used: each trial began with a baseline recording period of 300 ms, followed by paired-pulse stimulation of the LOT (50 ms IPI), a one-second delay, and paired-pulse stimulation of the ASSN fibers (50 ms IPI). Stimulation current levels were set to yield a reliable response with the least amount of stimulation in order to avoid polysynaptic activity or synaptic population crosstalk. Stimulation intensities were the following: LOT: 3–6 months, 40.61 ± 12.12 μA; 24–28 months, 120.00 ± 47.97 μA and ASSN: 3–6 months, 26.17 ± 3.02 μA, 24–28 months, 60.89 ± 11.17 μA. Any recordings demonstrating polysynaptic activity were excluded from analysis. Measurements of averaged  current attributes were performed in Clampfit.A and NMDA receptors, respectively. Spontaneous synaptic currents were recorded for 200 s of continuous recording from every cell. mEPSCs were identified as follows: A single 50 ms variable amplitude template was constructed (I(t) = I × exp(-t/τ ), where I is the peak amplitude and τ is a the decay time constant.For analysis of mEPSCs, slices were perfused with TTX (1 μM) until whole-cell currents evoked by LOT and ASSN fiber stimulation were abolished. Cells were maintained at a holding potential of -80 mV throughout recordings and bicuculline and CPP were used to prevent activation of GABAstructed  from >20Spontaneous EPSCs (s/mEPSCs) were recorded and quantified as for mEPSCs, except that TTX was not used.Some cells used for electrophysiology were also filled with a 4% Lucifer yellow solution passively during the recordings. Brain sections were then immediately fixed in 4% paraformaldehyde overnight, whole mounted on glass slides and coverslipped. Additional physiological slices fixed in 4% paraformaldehyde were cryoprotected in 30% sucrose in PBS. These slices were then resectioned at 30 μm, processed, and stained with 0.1% cresyl violet. Filled cells were visualized under a fluorescein isothiocyanate (FITC) filter and stained slices were visualized under bright field illumination on an Axioskop 2 microscope. Images were acquired through Axiovision software  and edited in GNU image manipulation program .t-test. Analysis of variance was used for comparisons involving more than two groups.All data are based on cells as individual sample units and are presented as means ± SEM. Statistical differences between two groups were evaluated using Student’s unpaired Figure 1). Filled cells from mice at all ages showed multiple, spiny apical dendrites which projected toward the pial surface. Other slices stained with cresyl violet were examined for cytoarchitecture. There were no obvious differences in soma morphology, cortical lamination, or cell density of APC of 3–6 months old and 24–28 months old mice.A subset of the cells chosen for electrophysiological study were filled with Lucifer yellow to confirm that they were layer II pyramidal cells . These events were abolished by perfusion with CNQX, confirming that they were mediated by AMPA receptors (data not shown). Synaptic events were collected from continuous 200 s recording epochs, aligned, averaged, and analyzed. Amplitude distributions were positively skewed in both young  and old mice . The mean amplitudes of mEPSCs recorded in slices from old mice were significantly smaller than those from young mice . Since the amplitude distributions were skewed, the median mEPSC amplitude was also calculated for each cell and compared between age groups. These measures were also significantly smaller in the aged mice . The average frequency of detected mEPSCs did not significantly differ between age groups . Cumulative amplitude distributions  also illustrate the shift in amplitude toward smaller mEPSCs in the neurons from old mice. Possible age-related changes in the kinetics of AMPA receptors mediating mEPSCs were calculated by fitting a single exponential function to the decay phase of averaged mEPSCs in each cell. The decay time constants tended to be longer in cells from old mice, although this difference only approached statistical significance .As described previously , superfiFigure 3). The robust facilitation of responses to LOT stimulation was not significantly altered by age. ASSN responses showed neither facilitation nor depression at the interval tested; normalized responses were also unaffected by age.The vast majority of glutamatergic synapses on SP neurons in APC are generated by either afferents from the olfactory bulb (LOT), terminating in the outer molecular layer (Ia) or the intrinsic associational (ASSN) system generated by the SP neurons themselves and terminating in the inner molecular layer (Ib). To test whether or not changes in the potency of individual synapses (mEPSCs) with aging were accompanied by presynaptic changes in release characteristics at either or both of these systems, paired-pulse stimulation (50 ms IPI) was applied to each pathway while recording from cells held at -80 mV in slices from young and old mice. Cells were only included in analysis if responses were present from both synaptic pathways. All traces collected for a given cell which met criteria were averaged and the percent potentiation or depression of the second response was calculated relative to that of the first response  and median  s/mEPSC amplitude. The similar size of mEPSCs recorded in the presence of TTX  and s/mEPSCs recorded in the absence of TTX  suggests either (1) that action potential-dependent and -independent release events evoke similar postsynaptic responses or (2) that the action potential-dependent (TTX-sensitive) events are a small fraction of spontaneous release events in these cells. Frequency distributions of s/mEPSC amplitudes also did not reveal any obvious differences with those of mEPSCs. In any case, it bears noting that the s/mEPSC  and mEPSC  recordings were made from completely different sets of young and old animals.Spontaneous currents in the absence of stimulation were obtained from the same cells in which evoked paired-pulse responses were obtained. Experiments were performed in the absence of TTX in order to obtain evoked responses; therefore, the spontaneous currents collected are presumed to be a mixture of action potential-dependent (sEPSCs) and independent (mEPSCs) events. The manner of analysis was identical to the method of analyzing mEPSC data. Aged animals exhibited a significant reduction in mean  are thought to be evoked by stochastic release of single glutamate quanta at individual synapses. Assuming that synaptic vesicles are the physical basis for quantal release, there are two main ways to alter quantal size: a change in glutamate loading of vesicles or a change in postsynaptic receptors in the synaptic zone. There is little precedent for changes in vesicle loading but considerable evidence that postsynaptic AMPA receptor numbers can be altered in an experience-dependent manner  or in cein vitro (It is important to note that spontaneous EPSCs (sEPSCs) were also recorded from APC cells in slices from completely independent cohorts of young and old mice, in the absence of TTX. The amplitude distributions of these events were almost identical to the events (mEPSCs) recorded in slices exposed to TTX. This suggests that the sEPSCs and mEPSCs are the same events; the lack of TTX sensitivity may be attributed to the high resting membrane potentials and low spontaneous firing of piriform pyramidal cells in vitro . In any The frequency of mEPSCs was not altered in the aged mice, suggesting that either (i) no change in the number of synapses contributing to these synapses with aging or (ii) that any such changes are accompanied by compensatory changes in spontaneous release probability. The synaptic origin of the mEPSCs could not be determined in these experiments. However, it is worth noting that the paired-pulse experiments did not reveal any differences that would be indicative of changes in release probability in either the LOT or ASSN pathways in young versus old mice. It is also worth noting that paired-pulse ratios may not be very sensitive measures of release kinetics.In toto, the results of the present study point to the conclusion that aging results in a loss of functional AMPA receptors at synapses on layer II pyramidal cells in piriform cortex. These losses may occur at LOT synapses, ASSN synapses, or both. Further studies will be necessary to establish whether these AMPA receptor losses are due to alterations in receptor subunit transcription, translation, trafficking, post-translational modification, or receptor assembly, as well as whether or not synaptic NMDA receptors are similarly effecting by aging. The decrease in synaptic potency with aging observed in the present study is in contrast to studies using minimal stimulation in rat hippocampal formation where the results suggest that dentate synapses increase in potency (larger unitary EPSPs) while CA1 synapses remain unchanged in old age ( old age .A loss of synaptic AMPA receptors would not be expected to be without effect on LTP in the APC. Although the effects of aging on LTP have not been studied in piriform cortex, activation of more synapses with fewer AMPA receptors would be needed to trigger LTP in aged mice, in order to overcome the voltage-dependent block of NMDA receptors during high frequency stimulation. A disturbance of LTP might not be evident with supramaximal stimulation paradigms but may appear with near threshold stimulation, as observed in hippocampal field CA1. In this scenario, enhancement of AMPA receptor function might compensate for loss of synaptic AMPA receptors. Drugs that act in this way are known to facilitate olfactory learning in young animals  and may The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Differentiated thyroid cancer (DTC) arises from follicular epithelium and retains basic biological features like expression of sodium iodide symporter (NIS), which is the cellular basis of radio iodine (131I) concentration during thyroid ablation. Once dedifferentiation of thyroid cells occurs, cells fail to concentrate 131I, posing both diagnostic and therapeutic problems in DTC and one may have to resort to other imaging techniques for disease localization. As DTC progression is slow, patients have a relatively good prognosis. However children with thyroid malignancies need aggressive management, as initial presentation itself maybe with nodal metastases. It is well known that FDG PET CT apart from its oncological applications, is also used in the evaluation of vascular inflammation especially Takayasu’s arteritis. It is also reported in literature, that 18F-FDG uptake can be seen relatively frequently in the arterial tree of cancer patients. Dunphy et al reported the association of vascular FDG uptake in inflammation as well as in normal arteries. This study typically describes FDG uptake in a patchwork of normal vessel, focal inflammation and or calcification of vessels. The other plausible reasons for significant vascular 18F-FDG uptake are drugs such as potent non steroidal anti-inflammatory agents, dexamethasone, prednisone and tacrolimus. Our patient showed false positive 18F Fluorodeoxyglucose (FDG) uptake in spinal cord at D11/12 and D12/L1 vertebral levels in FDG PET CT imaging performed as part of raised Thyroglobulin workup. This intra spinal FDG uptake is attributed to physiological uptake and inadequate FDG clearance from artery of Adamkiewicz, which can be added as a new physiological cause of FDG uptake unreported in literature as yet.We present a paediatric case of Papillary Ca thyroid under evaluation for elevated Thyroglobulin (Tg) level with negative  Patient was treated with 925 MBq  of 131I sodium iodide solution orally. A follow up whole body 131I scan in October 2008 showed persistence of minimal residual thyroid tissue [Our patient is an 8-year-old girl who presented to the Head and Neck department with swelling in lower neck noticed by her mother 10 days back. She had no history of pain, dysphagia, dyspnea, hoarseness of voice, cough, hemoptysis or bone pain. There is no history of hyper or hypothyroidism. Local examination revealed a 2 × 2 cm well defined, mobile nodule in midline region of thyroid, moving with deglutition. There were multiple small palpable bilateral cervical nodes. FNAC of thyroid was reported as Papillary carcinoma thyroid. Preoperative Serum Tg was 91.95 ng/ml. Total thyroidectomy with bilateral neck lymph nodal dissection (level II to V) was done in 2007. Histopathology confirmed the diagnosis of papillary carcinoma thyroid with bilateral cervical lymph nodal metastases. Patient underwent a residual thyroid and whole body d tissue . Serum T131I scan findings and raised Tg, patient was taken up for second sitting of high dose 131I therapy [131I sodium iodide solutionBased on Whole body  therapy . She was131I scan in 2009, showed no abnormal uptake in neck as well as in rest of whole body survey [Follow-up Whole body y survey . Tg off 18F-FDG accumulation is seen relatively frequently in the arterial tree of cancer patients.[18F-FDG uptake. Patient is now clinically asymptomatic and has been advised “Watchful waiting”.As part of the workup, a Whole body PET CT scan was performed (off Thyroxin) which showed an abnormal linear focus of FDG uptake in intraspinal region of D11/12 and D12/L1 vertebral levels with a Standard Uptake Value (SUV) of 3.95 g/ml. There was no abnormal FDG uptake elsewhere in neck and rest of whole body survey. Subsequent MR spine showed no abnormality at lower dorsal level Figures . This li4Numerous investigators have reported the incremental value of FDG PETCT in the evaluation of large and medium sized vasculitis. OtherwisIntraspinal FDG uptake was confirmed to be physiological FDG uptake in vessel i.e. artery of Adamkiewicz based on clinical exclusion, CT and MR findings. Normal course of this artery was identified with conventional selective angiography. However, both MR angiography and CT angiography10 have tThe Adamkiewicz artery is the largest segmental medullary artery typically arising from a left posterior intercostal artery (a branch of aorta) and supplies two thirds of the spinal cord via anterior spinal artery. In 75% of people, the artery of Adamkiewicz originates on the left side of the aorta between the D9 and D11 vertebral segments.[With the increasing availability and use of PET scans in our oncological and non oncological practice, it is imperative for us to know all the physiological sites of FDG uptake for confident image interpretation. We can add this intra spinal physiological FDG uptake to the various other listed physiological sites already known.


ESPL1 gene that encodes Separase protein results in embryonic lethality in mice and Separase overexpression lead to aneuploidy and tumorigenesis. However, the effect of Separase haploinsufficiency has not been thoroughly investigated.Cohesin protease Separase plays a key role in faithful segregation of sister chromatids by cleaving the cohesin complex at the metaphase to anaphase transition. Homozygous deletion of ESPL1 heterozygosity using a hypomorphic mouse model that has reduced germline Separase activity. We report that while ESPL1 mutant (+/hypESPL1 ) mice have a normal phenotype, in the absence of p53, these mice develop spontaneous T- and B-cell lymphomas, and leukemia with a significantly shortened latency as compared to p53 null mice. The ESPL1 hypomorphic, p53 heterozygous transgenic mice  also show a significantly reduced life span with an altered tumor spectrum of carcinomas and sarcomas compared to p53−+/ mice alone. Furthermore, +/hyp, p53−/−ESPL1 mice display significantly higher levels of genetic instability and aneuploidy in normal cells, as indicated by the abnormal metaphase counts and SKY analysis of primary splenocytes.Here we examined the effect of ESPL1+/hyp, −/−p53 mice provide a new animal model for mechanistic study of aggressive lymphoma and also for preclinical evaluation of new agents for its therapy.Our results indicate that reduced levels of Separase act synergistically with loss of p53 in the initiation and progression of B- and T- cell lymphomas, which is aided by increased chromosomal missegregation and accumulation of genomic instability.  ESPL1 gene in human lung and kidney cancers . Individuals born with a single mutated copy of p53 have the cancer pre-disposing Li-Fraumeni syndrome that pre-disposes them to the development of many different cancers in their life time Human cancer progresses as a multi-step process with cooperation between loss of key tumor suppressor genes as well as activation of oncogenes Many mouse models have been established over the past several years to study the role of gain or loss of p53 function in various cell types in vivo both alone and in cooperation with p53 loss, we have developed and characterized Separase hypomorphic mice (referred to here as +/hypESPL1 mice) with reduced Separase protein level, in the absence and presence of p53. We report here that mice heterozygous for the Separase hypomorph are viable in a C57Bl/6J background but the compound Separase hypomorphic, p53 homozygous mice  in the same genetic background have a significantly increased rate of tumorigenesis, shortened life span and mortality reaching 100% within 4–6 months of life. +/hyp, p53−/−ESPL1mice develop aggressive mixed T- and B-cell lymphomas along with leukemia involving the blood and bone marrow. Interestingly, normal tissues in these mice display significantly increased aneuploidy and chromosomal aberrations suggesting aneuploidy as precursor of tumorigenesis. These findings collectively indicate that reduction in Separase levels cooperate with loss of p53 in the generation of aggressive T- and B- cell lymphomas and leukemia in mice through increased aneuploidy and accumulation of multiple chromosomal aberrations.In an effort to understand the consequences of reduction of Separase levels ESPL1 gene resulting in effective disruption of the C-terminal 105 amino acids encoding a major part of the peptidase domain of the Separase protein  using immunoblot analysis indicated a reduction in the total Separase protein level in the +/hypESPL1 mice, compared to wild type littermate controls (+/hypESPL1 mice was found to be slightly higher than that in WT mice (data not shown) again suggesting that an unstable Separase protein results from insertion of the gene cassette in the hypomorphic mice, resulting in decrease in protein but not in mRNA levels.To evaluate the effects of reduced expression of Separase, Separase hypomorphic mice were generated in a C57BL/6 background using the Baygenomics genetrap clone XL058. In this clone the gene trap insertion selectively disrupts the last but one exon (exon 30) of the  protein , A. Homocontrols , indicat+/hypESPL1) and control WT mice in identical C57Bl/6 genetic backgrounds. Animals were sacrificed when terminally ill or when they reached the age of 24 months (∼2 years). Animals sacrificed at the study end point were censored in survival data if they did not display tumor phenotype. However, upon observation of mice maintained in our colony beyond the time period shown in survival curve  compared to that reported for WT C57Bl/6 animals ESPL1 occurred in all tissues, mice were examined for all possible tumor types upon necropsy. We failed to observe any tumors or any other obvious abnormalities in the +/hypESPL1 mice during their life span though about 5% of the control cohort of littermate WT mice developed sarcomas and appeared sick (possible lymphomas) and had to be sacrificed within a 2 year time frame.To examine if a reduction in Separase levels leads to chromosome missegregation and increases the risk of tumorigenesis, survival analysis was performed using littermate cohorts of Separase heterozygous hypomorphic mice  in a C57BL/6 background. Approximately 86% of the +/hyp, p53−/−ESPL1 animals developed lymphomas with a median survival period of 118 days, compared to about 37% −/−p53 animals that developed pathologically confirmed lymphomas in the same time period. On the other hand, approximately 50% compound heterozygote  animals developed carcinomas over a period of 460 days (about 14 months), compared to +/−p53 animals, a much smaller percentage of which developed a significant tumor phenotype over the same time period. Together, these data suggest that reduced levels of Separase co-operates with the loss of p53 in the initiation and progression of tumors, with other stochastic changes occurring along the way to initiate tumor formation. The +/hyp, p53+/−ESPL1 and +/hypESPL1, +/+p53 mice do not develop lymphomas over a period of 200 days (pre-dominant phenotype in each genotype summarized in Since the −/−p53) have been reported to develop predominantly T cell thymic lymphomas with infrequent involvement of lymph nodes in multiple organs including liver, lung and kidney. Crossing the −/−p53 mice to the +/hypESPL1 mice to generate the +/hyp, p53−/−ESPL1 mice significantly altered the lymphoma profile as well as the latency for lymphoma development. The double mutant mice developed mixed T and B cell lymphomas , the +/hyp p53−/−ESPL1 mice showed a striking increase in proliferation in their lung tissue with up to 80% cells stained positive for Ki67  of the for Ki67 . The lymfor Ki67 , indicatin vivo in DNA damage repair −/−p53 mice did not show a significant rise in DNA damage in their lymphoma affected tissues compared to the wild type litter mate cohorts.To further identify causes for the shortened latency and more aggressive lymphoma development in the double mutant mice and also to investigate the possible contribution of Separase +/hyp, p53−/−, p53−/− and WTESPL1 mice at the average age of three months (not affected by lymphoma) were stained for Ki67 and gamma H2AX markers. The normal  showed significantly higher levels of proliferation compared to the −/−p53 and WT cohorts (To further investigate the levels of proliferation and DNA damage accumulation in pre-neoplastic (not affected by lymphoma) tissues in these mice, primary splenocytes and bone marrow cells from  cohorts . However+/hypESPL1 mice in various p53 backgrounds. To carryout these experiments, tissue sections from lymphoma invaded lung, liver and thymus in the terminal +/hyp, p53−/−ESPL1 mice as well as tissues affected by various carcinomas  in the +/hyp, p53+/−ESPL1 mice were stained with Separase antibody. We observed that while various tissue sections from normal wild type age matched litter mate mice exhibited very low levels of Separase protein  antibody as a mitotic marker  from these mice  mice as early as by 2.5 months of age  mice is greatly accelerated by genomic instability and aneuploidy that starts accumulating very early in these mice. We provide a mouse model for further understanding the biology and pathways disrupted in human lymphoma and leukemia.Since its discovery in yeast as an important cell cycle component that holds sister chromatids together +/hyp, p53−/−ESPL1 mice compared to −/− (ESPL1 WT)p53 animals. Surprisingly, reduction in Separase levels alone does not lead to tumorigenesis, suggesting that very low levels of Separase within the cell may be sufficient to provide the enzymatic activity to cleave Rad21 and prevent chromosomal missegregation. Also, downstream anti-proliferative and pro-apoptotic effects of a functional p53 most likely prevent the accumulation of defects to lead to lymphomagenesis in these mice.Separase is an essential protein. Previous studies indicated that conditional depletion of Separase in mouse bone marrow causes aplasia and apparent death of hematopoietic cells other than erythrocytes +/hypESPL1, −/−p53 mice suggesting that in a tumor microenvironment, complete loss of Separase is a stochastic but non-essential event. Furthermore, spleen tissue from +/hyp, p53−/−ESPL1 mice have high accumulation of genomic instability and aneuploidy as early as in 2.5 month old mice, indicative of the critical role of Separase in maintenance of genomic stability. In contrary, the double heterozygous mice  develop tumors over a longer time, most likely resulting in the gradual accumulation of stochastic events. We have previously reported +/hypESPL1) do not have a lymphoma phenotype. This may suggest that though loss of Separase is a predisposing condition for hematopoietic malignancy, the further progression of cancer is blocked by apoptotic and other cell death pathways induced by an active p53.We show here that both the combined inactivation of Separase and p53 as well as a reduction in p53 gene dosage contributes to tumorigenesis. The reduced levels of Separase expression in only some but not all the lymphomas arising in the It is a well recognized concept now that more than one biological pathway needs to be deregulated in order to transform normal cells into malignant tumor cells In vivo studies in the mouse mammary epithelium have shown that over expression of Separase results in the development of gross aneuploidy suggesting an important aneuploidy promoting role of Separase ESPL1 in the absence of p53 is also sufficient to induce significant levels of aneuploidy in normal cells in vivo, as indicated by the abnormal metaphase counts and SKY analysis of primary splenocytes isolated from +/hyp, p53−/−ESPL1 mice. The accumulation of these genetic abnormalities in these mice may also account for the increased DNA damage levels identified in the lymphomas that infiltrated the lungs and liver of these mice.in vivo evidence of the role of Separase in DNA damage repair in higher eukaryotes is still lacking. We show here that reduced levels of Separase in the absence of p53 plays an important role in the accumulation of genomic instability in lymphocytes, possibly allowing the accumulation of gross chromosomal abnormalities as well as DNA damage finally resulting in acute lymphoma and leukemia development. Also important is the observation that +/hypESPL1 mice alone in the presence of an active p53 do not develop lymphomas and also have a longer tumor-free life span compared to wild type mice, possibly suggesting that increased levels of apoptosis and p53 mediated downstream checkpoint pathways prevents the accumulation of genetic instability and DNA damage accumulation and prevent cancer progression in these mice.The accumulation of gross genomic instability as well as DNA damage in the absence of p53 induced apoptosis could be a mechanism that aids the quicker lymphomagenesis in the double mutant mice. Lymphocytes are rapidly proliferating cells that have a very quick turn over time and require extensive and efficient DNA damage repair. Separase has been implicated in having a role in DNA damage repair −p53−/ mice alone were restricted to the thymus with occasional involvement of the lymph nodes in the lung and liver. Histopathological analysis indicated no bone marrow involvement in these animals. However, the combined reduction of Separase levels with loss of p53 changed the lymphoma spectrum in these mice to a more widely disseminated phenotype, involving lymph nodes of the lung, liver, thymus and spleen and also showing gross bone marrow involvement. It is conceivable that reduction of Separase levels along with loss of p53 results in the accumulation of DNA damage and aneuploidy in an independent subset of hematopoietic cells , resulting in the appearance of a more widespread T- and B-cell lymphoma phenotype as well as bone marrow involvement. Understanding of the specific hematopoietic subsets in this model will require further investigation with the use of cell lineage specific markers.Human lymphoma and leukemia have various subtypes indicating the involvement of many different cell lineages in its initiation and progression. In our model, lymphomas that developed in the +/hyp, p53−/−ESPL1 mice provide a new animal model for aggressive lymphoma not only to study the mechanism of lymphoma development but also for preclinical evaluation of new agents for lymphoma therapy.In summary, mESPL1 gene-trapped ES cell clone (XL058) was obtained from Bay Genomics . The gene trap is produced by a splice-acceptor-β-geo cassette within Espl1 exon 30 deleting the C-terminal 105 amino acid, a critical portion of its peptidase domain. The ES cells were injected into 129/SV blastocysts, and resulting chimeras were crossed to C57Bl/6J females and screened for germline transmission by PCR. Animals used in experiments reported here were backcrossed at least ten generations to the C57Bl/6J background. The p53 mice were obtained from Jackson Labs in a C57BL6 strain. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Baylor College of Medicine. All animal studies were carried out in strict compliance with federal and local guidelines for animal care and use.Mouse tissues were flash frozen in liquid nitrogen and stored at −80°C. Proteins were extracted in lysis buffer and quantified and Western Blot analysis performed as reported before P values were determined by Kaplan-Meier and Wilcoxon's log rank analyses . Animals that were tumor free at necropsy were censored in the survival analyses. Animals that died of unknown causes were also censored in lymphoma-free survival analysis but not in overall survival analyses. Lymphoma diagnoses were based on histological confirmation of tumors. Comparison of tumor frequencies was done by Fisher's exact test with one-tailed P values. All statistical analyses were performed with Graph Pad Prizm software .Animals were monitored regularly for signs of illness up to the age of 2 years. Overall and tumor-free survival times and DNA damage assays were performed using specific anti mouse- H2AX (Millipore) and anti-rabbit Phospho Serine H2AX antibodies . Mitotic marker anti-Phospho Histone H3 (Ser 10) was used  to detect mitotic cells.All IHC and IF analysis was performed using previously published methods GCCATGGCCATCTACAAGAA, Reverse: ATCGGAGCAGCGCTCATG) and mouse ESPL1  were used for amplification. GAPDH  was selected for normalization.Total RNA was extracted from mouse tissues by grinding the tissues in liquid nitrogen followed by Trizol  RNA extraction. cDNA was synthesized by reverse transcription of mRNA using oligo-dT primers  and Superscript II reverse transcriptase (Invitrogen) as instructed by the manufacturer. Quantitative real-time PCR was performed with the Eppendorf Mastercycler Realplex4 system. Custom oligos for mouse p53 . Hybridization and detection were carried out according to the manufacturer's protocol, with slight modifications. Chromosomes were counterstained with DAPI. Images were acquired with a SD300H Spectra cube (ASI) mounted on a Zeiss Axioplan II microscope using a custom designed optical filter (SKY-1) , and analyzed using SKY View 2.1.1 software  For the histological analysis a ratio of positively stained cell counts versus total cell counts was computed for each treatment and each genotype. The staining ratios within each genotype may not be independent. As such, an average of ratios was computed for each mouse genotype. The statistical significance of differences in mean genotype ratios between treatments were evaluated using two-tailed unequal variance Student's t-tests.Figure S1+/hyp, p53−/−ESPL1 mice show increased proliferation in normal splenocytes isolated from 3 month old mice. Primary splenocytes isolated from WT, −/−p53 and +/hyp, p53−/−ESPL1 mice were analyzed for cellular proliferation. Proliferation levels are indicated by the percentage of cells in any cycling phase of the cell cycle  (B) calculated by counting Ki67 cells (red) as a percentage of total cells . . The average counts for both mice in each genotype were plotted. Significant difference was observed in proliferation levels in the +/hyp; p53−/−ESPL1 compared to all other cohorts (P = 0.01).(TIF)Click here for additional data file.Figure S2Western blot analysis of endogenous Separase protein from testis derived from the +/hypESPL1 mice in p53 heterozygous and homozygous backgrounds show lower level of Separase compared to wild type mice. Western blot from testis tissue of mutant mice and their wild type littermate controls (as indicated by the genotypes) is shown (top). Densitometric quantification of Separase expression in the +/hypESPL1 mice in p53 heterozygous and homozygous backgrounds compared to wild type after normalization to the expression of a housekeeping gene γ tubulin to compensate for loading control (bottom).(TIF)Click here for additional data file.


Human sodium iodide symporter (hNIS) gene over-expression is under active consideration worldwide as an alternative target molecule for breast cancer (BC) diagnosis and targeted radio-iodine treatment. However, the field demands better stratified analysis of endogenous hNIS expression across major BC subtypes. Therefore, we have analyzed subtype-specific variation of hNIS overexpression in breast tumor tissue samples by immunohistochemistry (IHC) and also report the development of a homogeneous, quantitative analysis method of digital IHC images.hNIS expression was analyzed from 108 BC tissue samples by IHC. Sub-cellular localization of hNIS protein was analyzed by dual immunofluorescence (IF) staining method using hNIS and HER2 antibodies. An ImageJ based two-step digital analysis method was developed and applied for the bias-free analysis of the images.Staining of the tumor samples show 70% cases are hNIS positive indicating high incidence of hNIS positive cases in BC. More importantly, a subtype specific analysis done for the first time shows that hNIS expression is overly dominated in estrogen receptor (ER) positive cases than the receptor negative cases. Further, 56% of the ER+ve, PgR+ve, HER2-ve and 36% of ER+ve, PgR+ve, HER2+ve cases show highest intensity staining equivalent to the thyroid tissue. A significant positive correlation is also observed between hNIS and estrogen receptor expression  suggesting hNIS mediated targeted radio-iodine therapy procedures may benefit both ER+ve, PgR+ve, HER2–ve as well as HER2+ve cases. Further, in a few cases, hNIS and HER2 protein localization is demonstrated by overlapping membrane co-expression. ImageJ based image analysis method shows over 70% match with manual pathological scoring method.131I radio-ablative therapy is aimed.The study indicates a positive link between hNIS and ER expression in BC. The quantitative IHC image analysis method reported here will further help in patient stratification and potentially benefit global clinical assessment where hNIS mediated targeted  Further, quantification of the immunostained sample  is often required to establish a judgment parameter in clinical decision making processes especially for cases where a targeted treatment is available against the marker. Nonetheless, due to high degree of biological variations in clinical samples, overall the scoring process is manual which relies on training human vision to the IHC color development and thus remain prone to inter- and intra-observer variability Therefore during this study, we conducted a thorough analysis of a large dataset of 108 patient tumor tissue samples representing all major subtypes of BC and find that hNIS over-expression is significantly associated with estrogen receptor (ER) positive cases. We for the first time report development of a two-step semi-automated scoring methodology using an open resource digital image analysis program (ImageJ) and demonstrate its utility in quantitative, bias-free analysis of hNIS over-expression in breast tumor samples.The clinical study protocol was reviewed and approved by the TMC-ACTREC Institutional Review Board. For all experiments paraffin embedded tissue blocks from our tumor tissue repository were used and thus patient consent waiver was obtained.The paraffin embedded breast tumor tissue blocks were collected and used for hNIS immunostaining during this study. A total of 108 randomly collected tissue samples from patients between the age group of 32–72 years were obtained from ACTREC tumor tissue repository collected during the years 2008–2009. All tumor samples were confirmed as infiltrating duct carcinoma, grade III and were classified as 25 ER+ve, PgR +ve, HER2–ve, 11 ER+ve, PgR+ve, HER2+ve, 27 ER-ve, PgR–ve, HER2+ve, and 45 ER-ve, PgR-ve, HER2-ve samples on the basis of available standard immunohistochemistry report. Out of 108, 16 samples were obtained from the patients who had received preoperative chemotherapy treatment. Among the post chemotherapy cases, number of samples from different subtypes includes 4 ER+ve, PgR+ve, HER2-ve, 2 ER+ve, PgR+ve, HER2+ve, 3 ER-ve, PgR-ve, HER2+ve and 7 ER-ve, PgR-ve and HER2-ve. Of all cases, 48 patients were premenopausal (up to age 50 years) and 60 were postmenopausal (above 50 years). The demographic characters of patient cohort are mentioned in 2O2 in methanol for 30 minutes in dark. Tissue sections were dehydrated through graded alcohols and subjected to antigen retrieval using 10 mM sodium citrate. Sections were washed with TBST  and then blocked with 5% BSA  for one hour. Slides were incubated with the mouse monoclonal antibody against hNIS diluted with TBS in 1∶50 ratio. Slides were washed for 5 minutes in TBST and incubated for 1 hour with HRP (Horse raddish peroxidase) conjugated anti mouse antibody diluted with TBS in 1∶200 ratio. After washing, slides were incubated with DAB  (Sigma) and immediately washed under tap water after the color development. Slides not incubated with anti hNIS antibody were used as secondary control (data not shown). For each batch of sample staining, a papillary thyroid carcinoma case was kept as positive control for hNIS expression. A salivary gland section was also used as positive control. All the slides were counter stained with haematoxylin. Slides were DPX mounted and observed under light microscope (Carl Zeiss).Standard IHC protocol was followed to stain the tumor tissue samples using the mouse monoclonal antibody against hNIS . In brief, 5 µm sized paraffin embedded tissue sections were de-paraffinized with xylene and endogenous peroxidase activity was quenched with 3% HFor the purpose of immunofluorescence study, tissue sections were treated in the same way as for IHC. After blocking with BSA, sections were incubated with hNIS and HER2  antibodies one by one and then stained with respective fluorescent dye labeled secondary antibodies. For hNIS staining Alexafluor 488 labeled rabbit secondary antibody was used where as HER2 was counter-stained with Dylight633 labeled goat secondary antibody. Antibody stained sections were washed with TBST and then mounted with vecta-shield (Vector Laboratories) and images were captured using a confocal microscope (Carl Zeiss).Antibody stained permanent slides were examined under light microscope and sections showing less than 10% of stained area were considered as negative where as sections showing 10–20% of stained area were considered as focal, 20–70% of stained area were considered as patchy and more than 70–80% of stained area were considered as diffused. Intensity variation of the staining was visibly examined and scored by pathologist as 0, 1+ and 2+.IHC digital images were used for developing semi-automated analysis protocol. As a first step, we used a color de-convolution technique to un-mix the pure DAB, hematoxylin stained areas leaving a complimentary image. The pixel intensities of separated DAB or hematoxylin images range from 0 to 255. Value 0 represents the darkest shade of the color while 255 represent the lightest shade of the color in the image. In-order to assign an automated score by judging the pure DAB staining pattern, histogram profile of every image i.e. the number of pixels of a specific intensity value vs. their respective intensity was raised using imageJ standard program feature. In the histogram profile, we categorized pixel intensity ranges from 0–60 for a score value of 3+, 61–120 for 2+, 121–170 for 1+ and 171–230 for 0. We excluded the pixel intensity values above 230 as the abundant fatty tissues in breast tumor tissue sections were found to contribute in the range of 230–255. A macro was developed and plugged in the ImageJ program to receive automated counting of numbers of pixels in the four different intensity zones. After determining these numbers, we applied them to a simple algebraic formula as shown below to determine the score of the image.Any image that has more than 66% of pixels in one zone is directly graded without the need to apply the formula. In line with the standard grading procedure, we assigned a 4 tire scoring system i.e. high positive (3+), positive (2+), low positive (1+) and negative (0).All the statistical analysis was performed using Graph pad and Microsoft Excel software. The significance of difference was obtained by performing the chi square test, and the level was set at P<0.05. For comparison of two grading methods kappa statistical analysis was performed.The pattern of hNIS staining is mainly diffused cytoplasmic as appeared in representative images shown Figure-1We compared the hNIS positive expression in ER–ve and ER+ve samples. As shown in the Based on the reported age in the patient registry, we categorized samples into 2 groups i.e. premenopausal (</ = 50 years) and postmenopausal (>50 years) and compared for hNIS expression. Numbers of samples in these groups are 48 and 60 respectively. In case of pre-menopausal women 81% patients show positive expression of hNIS, whereas 63% of post-menopausal women show hNIS positivity  99mTcO4 showed visible tumor by scintigraphic imaging only in 17% of breast cancer patients 123I scintigraphic or 124I PET imaging in breast cancer patients to determine sufficient radioisotope uptake by hNIS expressing breast cancer tissues.Next we envisioned that in order to play its functional role as a transporter, hNIS proteins should be localized at the plasma membrane. But in majority of the cases a diffused intracellular staining pattern is observed. Though a few studies in the past have shown membrane specific staining in breast tumor tissues Taking the study forward, we also report the development of a semi-automated digital image analysis toolbox capable of assigning scores directly by profiling of hNIS staining intensity in IHC digital images. By performing an accuracy comparison study, we see ∼70% match in scoring obtained by manual and software-based method. Considering the biological variations in human tissue samples, unsupervised grading analysis providing such high percentage match with pathological analysis seems to be a good achievement. Further, we also observed that majority of the cases where automated scoring differs by 1 or 2 degree from that of the pathological analysis, are mostly the cases where low tumor to stromal tissue ratio are present  Analysis of image score using a 20X image of a case where low tumor cells were present. After color deconvolution, the macro was used on the DAB image to plot a histogram profile and the score was determined as positive. B) When an image is captured using a 40X objective focusing on the marked area of the previous image, score was found to be high positive.(TIF)Click here for additional data file.Table S1Summary of IHC scores for hNIS expression in different subtypes of breast cancer.(DOCX)Click here for additional data file.


C, is templated to a conformationally distinct form, PrPSc. Recent studies have demonstrated that the Sprn gene encodes a GPI-linked glycoprotein Shadoo (Sho), which localizes to a similar membrane environment as PrPC and is reduced in the brains of rodents with terminal prion disease. Here, analyses of prion-infected mice revealed that down-regulation of Sho protein was not related to Sprn mRNA abundance at any stage in prion infection. Down-regulation was robust upon propagation of a variety of prion strains in Prnpa and Prnpb mice, with the exception of the mouse-adapted BSE strain 301 V. In addition, Sho encoded by a TgSprn transgene was down-regulated to the same extent as endogenous Sho. Reduced Sho levels were not seen in a tauopathy, in chemically induced spongiform degeneration or in transgenic mice expressing the extracellular ADan amyloid peptide of familial Danish dementia. Insofar as prion-infected Prnp hemizygous mice exhibited accumulation of PrPSc and down-regulation of Sho hundreds of days prior to onset of neurologic symptoms, Sho depletion can be excluded as an important trigger for clinical disease or as a simple consequence of neuronal damage. These studies instead define a disease-specific effect, and we hypothesize that membrane-associated Sho comprises a bystander substrate for processes degrading PrPSc. Thus, while protease-resistant PrP detected by in vitro digestion allows post mortem diagnosis, decreased levels of endogenous Sho may trace an early response to PrPSc accumulation that operates in the CNS in vivo. This cellular response may offer new insights into the homeostatic mechanisms involved in detection and clearance of the misfolded proteins that drive prion disease pathogenesis.During prion infections of the central nervous system (CNS) the cellular prion protein, PrP C, changes to a distinct form, PrPSc. Recent studies have demonstrated that another glycoprotein Shadoo (Sho), which occupies a similar membrane environment as PrPC, is reduced in the brains of rodents with terminal prion disease. Our analyses of prion-infected mice revealed that reduction of Sho protein was not due to reductions in the corresponding messenger RNA. Reduction in Sho was clearly evident upon propagation of a variety of prion strains, but was not seen in mice with other types of neurodegenerative disease. Also, as prion-infected mice with only one copy of the PrP gene exhibited both accumulation of PrPSc and a reduction of Sho protein hundreds of days prior to onset of neurologic symptoms, the drop in Sho protein level can be excluded as an important trigger for clinical disease, or a non-specific consequence of brain cell damage. Instead, our studies define a effect restricted to prion disease and we hypothesize that Sho protein is a “bystander” for degradative processes aimed at destroying PrPSc.In prion infections of the nervous system the cellular prion protein, PrP C) undergoes a conformational transition to a β-sheet enriched and infectivity-associated form commonly denoted PrPSc (sometimes denoted as PrPd). This transition is often marked by reduced detergent solubility and acquisition of resistance to proteinase K (PK) digestion in vitro. In strong and independent support of a central role for altered PrP in disease pathogenesis and replication of the transmissible agent, missense mutations in the murine prion protein structural gene, Prnp, that create the allelic forms Prnpa and Prnpb cause a modulation of prion disease phenotypes and differential susceptibility to prion strain isolates Prnp renders animals completely resistant to experimental prion infections in vitro has been shown to generate prion infectivity Prion diseases, including the prototypical scrapie of sheep and Creutzfeldt-Jakob Disease (CJD) of humans, are fatal and incurable neurodegenerative disorders. They are unusual in that they are often transmissible or infectious diseases. While they can be studied to great effect in a lab setting , they can also be initiated inadvertently with contaminated material. Thus, in the case of variant CJD (vCJD), occurrence is linked to the UK epidemic of Bovine Spongiform Encephalopathy (BSE) and is thought to involve infection by an oral route from BSE-contaminated food Sc that are themselves distinct from PrPCC's hydrophobic domain (HD). Interestingly, the recently discovered Shadoo (Sho) protein bears similarity to this region of PrP by containing an HD and a preceding series of tandem repeats with positively charged residues. Like the equivalent region in PrP, this part of Sho is also considered to be natively unstructured A battery of analytical techniques demonstrates that disease-associated forms of PrP are variegated. Thus, there is heterogeneity with respect to the concentration of PK needed for complete digestion, the positions of N-terminal PK cleavage sites, detergent insolubility, antibody accessibility and denaturation with guanidinium in vivo pathway with action directed against, or activated by, protease-resistant PrPSc.As Sho is decreased in prion-infected brains Prior studies of prion infection with the RML isolate demonstrated down-regulation of Sho in different cohorts of animals analyzed in the clinical phase of disease Prnpa/a mice infected intracerebrally with RML prions  or control brains  and with brain samples isolated from early time-points onwards subsequent to inoculation  culation . As can Prnpa allele . Prior studies have shown that the time to clinical disease is greatly extended in these animals-by hundreds of days-whereas accumulation of protease-resistant PrPSc is not linked to clinical symptoms of disease and occurs at an earlier stage Sc was evident in the brain of infected animals over 220 days before the endpoint when clinical signs were apparent A second resource corresponded to mice hemizygous for the wt C protein encoded by a Prnpa cDNA Sc was readily detectable by western blot and histoblot analysis (A third resource corresponded to transgenic (Tg) mice overexpressing the PrP-A allelic form of the PrPSc that may be distinguished by parameters such as guanidine denaturation and protease-sensitivity. For these studies we restricted analysis to animals in clinical disease phase. As presented in Prnpa mice infected with two other isolates of mouse-adapted scrapie prions, ME7 and 22A. Prnpb mice were also used for these analyses. These encode the PrP-B form of PrPC, which differs from the A allele at residues L108F and T189V Prnpb allele onto a C57BL6 genetic background to make a congenic inbred strain. 87 V mouse-adapted scrapie and 301 V mouse-adapted BSE agents were assessed in B6I mice  Levels of Sprn was not noted within these initial 333 DEG's, we used this resource to test the hypothesis that down-regulation of Sho protein is a simple consequence of reduced levels of the corresponding mRNA. Thus, we interrogated the Prion Disease Database Sprn mRNA levels within the brains of prion-infected animals and corrected these data versus age-matched animals inoculated with control inocula (‘background subtraction’). Sprn-specific probes were included in genome wide microarray analyses performed upon a series of mice of different Prnp genotypes infected with one of two prion strains Sprn transcripts in infected animals  that are affected by prion infection  animals Figure 5CIn a next set of experiments we sought to understand the properties and milieu of the predominant form of wt Sho protein. For example, in cultured cells, beyond internal vesicular compartments and the cell-surface, Sho protein is present in conditioned medium. This suggests “shedding” from the cell-surface, as is the case for PrPPrnp transcriptional unit, defined operationally by a ∼42 kB genomic clone comprising the cos.Tet transgene vector Sprn 5′ and 3′ mRNA flanking sequences either to translational initiation properties or to mRNA stability, the wt mouse Sprn open reading frame (ORF) was mobilized devoid of its native 5′ UTR and 3′ UTR regions  can be found in a 100,000xThe data presented in Sprn24551 line of Sho Tg mice has a) no discernable alteration in the levels of PrPC  has yet to show signs of spontaneous neurologic disease at ages up to 500 days (n  =  20), it was deemed suitable for prion challenge studies. Accordingly, cohorts of TgSprn24551 mice were inoculated with RML scrapie prions administered by two different routes. Incubation periods to onset of clinical disease for intracerebral injection were not significantly different between non-Tg and Tg groups . Disease onset was 131 ± 9 (standard deviation) days versus 136 ± 10 days (not significant) and terminal disease was 154 ± 2 days versus 155 ± 10 days . Survival curves are presented in Sprn24551 mice overexpress Sho at 2.5x higher levels than wt mice, and insofar as the transgene arrays in these mice are present on a homozygous wt (i.e. Sprn+/+) Sprn genetic background, this leads to a conclusion that the greater fraction  of the Sho protein in these mice is transgene-encoded. Further, the indistinguishable percentage reduction in Sho between Tg and non-Tg mice data suggests that transgene-encoded Sho is effectively down- regulated. Thus, the down-regulation phenomenon observed in clinical phase of disease in Tg (and wt) mice is likely determined by an intrinsic property of the wt mouse Sho protein.Since the TgSprn transgene impacted the quantity or quality of PrPSc. These analyses are presented in Sprn24551 mice.Blot analyses of protease-resistant protein species were also performed for these cohorts of animals, to ascertain whether the Sc accumulation. Monocultures of prion-infected cells were used to explore this inference. Such chronically infected cells comprise an experimental paradigm with accumulation of PrPSc but without  by lysosomal proteases .The protein samples analyzed above derive from homogenates of CNS tissue and are thus uninformative as to the possible contributions of different cell types to the down-regulation phenomenon. However, studies presented thus far suggest that Sho down-regulation is not associated with clinical signs of disease or fulminant pathology in infected animals but, instead, is associated with PrPSince prior experiments demonstrated Sho in the conditioned medium from healthy cultured cells BRI2 protein of familial Danish dementia The generation of α-Sho antibodies has allowed an appreciation that down-regulation of the mature full-length form of Sho glycoprotein is a consistent feature of prion infections using different cohorts of animals infected with the RML isolate of rodent-adapted scrapie prions , Prnp hemizygous animals  is still subject to down-regulation  do not have notably altered neurologic signs or neuropathology  PrPnments , and c ShSc, but are triggered by other host responses to prion replication, perhaps involving a protein degradation pathway distinct from that for PrPSc. Reasons why this option is less likely are presented elsewhere in the Discussion, but future experiments deploying selective protease inhibitors will be necessary to solidify our present concept.It remains possible that reductions in Sho levels are not due to a proteolytic mechanism directly targeting PrPin vivo. However, the degree of down-regulation observed in the clinical phase of disease may vary with different prion isolates Sc . In our studies 23027 mice Sc half-life has been measured at ∼1.5 days via the use of regulated PrPC-encoding transgenes Sc is already trimmed at the N-terminus without the need for PK digestion in vitro , which is fully accredited by AAALAC International, were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, U.S. Public Health Service. MRI's Animal Assurance number from the Office of Laboratory Welfare of the National Institutes of Health is A3901-01. All procedures involving animals were reviewed and approved by MRI's Institutional Animal Care and Use committee under protocol GAC-05. Intracranial inoculations were performed under isoflurane anesthesia and every effort was made to minimize discomfort to the mice.Prnp0/0 mice Prnpb) Prnpa/a, Prnp0/+ and Tg.Prnpa mice have been described previously http://prion.systemsbiology.net. Methods for formalin fixation, paraffin embedding and staining of prion-infected tissue were as described previously TgCRND8 mice Bis(cyclohexanone)oxaldihydrazone, cuprizone,  was formulated with 6% fat rodent chow  to a final concentration of 0.4% w/w. Ten week-old male C57BL/6 mice were fed cuprizone or control chow for 4 or 8 weeks, a dose at which no mortality was observed. Subsequently, animals were euthanized, brains were removed and tissue was homogenized to 10% w/v in PBS with protease inhibitors .EcoRI endonuclease and the resulting 15 kb fragment containing Sprn genomic sequences was cloned into the plasmid vector pBR322. This plasmid was then used as a template for PCR amplification using primers that contained SalI sites (5′-ttttgcgtcgacCTGCCCAGTAGGATGAAC-3′ and reverse primer 5′-ga agg gtc gac TCT AAG GCC GAA GCA GTT C-3′). The PCR products were cloned in pTOPO2.1 plasmid (Invitrogen), and sequenced to confirm the absence of PCR errors. The resulting plasmid was digested with SalI to release a fragment that was cloned into Cos.Fse.Tet FseI transgene fragments excised from this cosmid vector were separated by gel electrophoresis, extracted by the freeze-and-squeeze method A RPCI-22 mouse BAC clone of 129 mouse genome DNA  was digested with An affinity column consisting of recombinant GST-Sho(25-80) covalently attached to glutathione-Sepharose 4B  by coupling reaction with dimethyl pimelimidate  was used to capture 06rSH1 Uninfected and infected SMB cells g for 1 h at 4°C and pellets re-suspended in cold 50 mM Tris-HCl, pH 7.5, 0.5% Triton X-100, 0.5% deoxycholate, and a protease inhibitor cocktail. 50 µg of total protein sample was used for western blot analysis.10% brain homogenates prepared in 0.32 M sucrose containing complete protease inhibitor cocktail (Boehringer) were spun at 100,000xDiethylamine (DEA) extraction of APP and Sho from mouse brains was performed by a method previously described SMB cells were either directly analyzed by SDS-PAGE or digested 20 min at 37°C with Proteinase K (PK) (20 µg/ml), followed by addition of 2 mM PMSF. Infected brain homogenates were digested 1 h at 37°C with PK  followed by addition of 2 mM PMSF. Histoblots were performed as described previously Survival curves were analyzed by Log-rank (Mantel-Cox) test (Microsoft Excel) and variance in protein levels was analyzed by GraphPad for Mac version 3.1a. Alpha value for significance was set at 0.05.Figure S1Sc in chronically-infected SMB cells.N-terminal trimming of PrP To confirm that novel PrP species present in undigested samples from prion infected cells  or with (b) NH4Cl for 5 hours (30 µM). Protein samples were detected with Sha31 recognizing a C-terminal epitope in PrP helix 1. In contrast, an N-terminal antibody 8B4 did not reveal PrP species that were distinct between undigested lysates derived from control and infected cells.(TIF)Click here for additional data file.


Lutzomyia longipalpis is the most important vector of American visceral leishmaniasis (AVL), the disseminated and most serious form of the disease in Central and South America. In the natural environment, most female L. longipalpis are thought to survive for less than 10 days and will feed on blood only once or twice during their lifetime. Successful transmission of parasites occurs when a Leishmania-infected female sand fly feeds on a new host. Knowledge of factors affecting sand fly longevity that lead to a reduction in lifespan could result in a decrease in parasite transmission. Catalase has been found to play a major role in survival and fecundity in many insect species. It is a strong antioxidant enzyme that breaks down toxic reactive oxygen species (ROS). Ovarian catalase was found to accumulate in the developing sand fly oocyte from 12 to 48 hours after blood feeding. Catalase expression in ovaries as well as oocyte numbers was found to decrease with age. This reduction was not found in flies when fed on the antioxidant ascorbic acid in the sugar meal, a condition that increased mortality and activation of the prophenoloxidase cascade. RNA interference was used to silence catalase gene expression in female Lu. longipalpis. Depletion of catalase led to a significant increase of mortality and a reduction in the number of developing oocytes produced after blood feeding. These results demonstrate the central role that catalase and ROS play in the longevity and fecundity of phlebotomine sand flies.The phlebotomine sand fly  Lutzomyia longipalpis, Lutz and Neiva 1912 is the best studied and most important vector of American Visceral Leishmaniasis (AVL) Lu. longipalpis is a permissive vector to Leishmania infections Leishmania transmission.The phlebotomine sand fly Drosophila melanogaster, previous work done on this species showed that oxidative stress increases with age, while antioxidant enzyme activity decreased over time Previous studies in other blood-feeding insect vectors have shown that reactive oxygen species (ROS) play an important role in both reproductive output 2 such as superoxide anion (O2−·) and hydrogen peroxide (H2O2) are formed during cellular respiration. These partially reduced metabolites of O2 are often referred to as reactive oxygen species due to their higher reactivity in relation to molecular oxygen 2−· to H2O2), glutathione peroxidase and catalase (which reduces H2O2 to H2O) Leishmania parasites Leishmania infection ROS are regularly generated by mitochondrial electron transport Lutzomyia longipalpis.In order to investigate the biological role of ROS-scavenging in fecundity and survival, we analysed the expression of catalase in three different age groups of female sand flies. Fecundity and catalase expression decreased with age. Catalase was incriminated as an important component in the loss of fecundity using RNAi. Dietary supplementation with an exogenous ROS-scavenger was found to partially reverse the differences in fecundity and increase mortality with a concomitant activation of the phenoloxidase (PO) cascade. These results show that both fecundity and survival are affected by endogenous and exogenous ROS-scavenging in female Lu. longipalpis from the older age group showed a decrease in the number of developing oocytes dissected five days after blood feeding in comparison to younger sand flies showed no significant difference in oocyte numbers. However, sand flies bloodfed at 9 days PE showed a significant decrease in oocyte numbers after dissecting 5 days after blood feeding . The number of developing oocytes dissected 5 days after blood feeding was significantly higher  showed a decrease in expression in ovaries dissected at 48 hours after blood feeding  , showing a high level of identity (99%) and similarity (99%)  showed a dramatic decrease in oocyte number dissected 48 hours after blood feeding  . As any  feeding . A dsRNA feeding , with noLu. longipalpis were fed with an antioxidant-supplemented sugar meal upon emergence. Mortality was recorded from day 1 PE up to day 7 PE. Survival curves depict an increase in mortality due to exogenous ROS-scavenging by an exogenous antioxidant  sand flies  presenting the lowest number of oocytes  after the blood feeding . ProteinInterestingly, oral delivery of ascorbic acid seemed to stimulate catalase LlonKat1 mRNA expression in older flies to levels similar to that of younger flies . It has Catalase LlonKcat1 does not have a signal peptide or targeting sequences to mitochondria or peroxisomes. These features suggest a cytosolic location but this needs confirmation. Based on the identities with other catalases retrieved from Peroxibase Anopheles gambiae to Plasmodium infection, a similar but more drastic effect was observed with female mosquitoes et al.Drosophila. Similarly, Bayne et al.Drosophila from oxidative stress, were detrimental for lifespan and physical fitness of the insects. Kang et al.Anopheles stephensi when the mosquitoes were bloodfed with the antioxidant MnTBAP in comparison with the buffer control. It has been hypothesized that a minimal level of ROS might be required to maintain the balance of the gut microbiota and that a baseline level of ROS activity might be crucial for basic midgut physiology. Previous studies done with other dipteran species had showed that ROS release constitutes a first line of defence against pathogens in the midgut D. melanogaster have demonstrated the existence of a midgut-specific active ROS releasing system against orally delivered bacteria ROS-scavenging by dietary supplementation of ascorbic acid  led to a2O2 was found to be significantly lower compared to catalase There is evidence of other antioxidant enzymes with catalase-like functions found in the sand fly midgut, such as peroxiredoxins Anopheles stephensi  overexpressing the protein kinase AKT gene increased the insulin signalling in the mosquito midgut, significantly reducing mosquito lifespan and inhibiting P. falciparum development Plasmodium survival within the mosquito midgut was investigated by Jaramillo-Gutierrez et al. Anopheles gambiae showed that this gene regulates the basal levels of catalase and glutathione peroxidase expression and that OXR1 gene knockdown decreased Plasmodium berghei oocyst formation. The finding of a Lu. longipalpis OXR1 gene homologue (unpublished) will shed some light into the regulation of ROS production within the sand fly gut and will also help to understand how ROS production impacts Leishmania development in the sand fly midgut.Recent studies on transgenic Current vector control strategies rely on spraying of residual insecticides to control vector population. Insect transgenesis and paratransgenesis are novel strategies that aim at reducing insect vectorial capacity by using genetic manipulation of disease vectors, rendering them incapable or less efficient to transmit a given pathogen Lu. longipalpis from a colony first started with individuals caught in Jacobina, Brazil. Insects were kept under standard laboratory conditions All experiments were carried out using insectary-reared Lu. longipalpis were allowed to mate under regular rearing conditions and fed with rabbit blood at three, six and nine days post-emergence (DPE). A batch of >500 flies was released into a large (20 m3) rearing cage and groups of ∼100 individuals were transferred to medium sized cages (5 m3) at 3, 6 and 9 DPE and blood-fed as above. Fifteen fully-engorged females were then transferred to a new medium rearing cage. Insects were dissected five days later to count developing oocytes.Female Lu. longipalpis fed on a 70% sucrose solution supplemented with 20 mM ascorbic acid and blood-fed at 9 DPE. Supplemented sucrose-meal was freshly changed daily and continued after blood-feeding. A 9 DPE control group was reared under the same conditions but fed with a 70% sucrose solution. Only fully engorged insects from both groups were selected for the experiments.Fecundity assays were carried out as described above with female 2O2 consumed per minute. All assays were carried out in triplicate. Fluorescence was measured using a Varioskan fluorescence spectrometer (Thermo Electron) with an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Ovarian catalase activity was normalised using the total amount of protein in the whole body (minus dissected ovaries) using the BIORAD® Protein assay reagent following the manufacturer's protocol and using bovine serum protein as standard. Endpoint absorbance was measured at 595 nm in a 96 well plate with a microplate reader .Ovaries were collected from 5 female sand flies at 24 and 48 hrs post blood feeding (PBF). Samples were homogenised in 50 ul of 0.15 M NaCl solution, kept on ice and transferred to a −80°C freezer until needed. Before assays, samples were centrifuged at 5000 RPM for 2 minutes and 1 µl of the supernatant was diluted in 24.9 µl of 0.15 M NaCl solution. Catalase activity was determined using Amplex Red Catalase Assay Kit (Invitrogen Ltd) following the manufacture's protocol. Enzyme-specific activities were expressed as units/mg of protein. One unit of catalase activity was defined as 1 µM of HSix DPE sand flies were blood fed and ovaries from 10 sand flies (two pools of 5 flies) were dissected at 12, 24 and 48 hours PBF, homogenised in 50 µl of TRI Reagent®  and kept at −80°C until needed. RNA was extracted following the manufacturer's protocol. Total RNA was quantified using a Nanodrop® and normalised to 10 ng/µl. RT-PCR was carried out with SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase Kit  performing 19 cycles and following the manufacture's protocol  were dissected at 48 hours PBF. Additionally, to evaluate the effect of feeding a ROS-scavenger in age-related expression of ovarian catalase, a group of 9 days old sand flies was fed with ascorbic acid-supplemented sucrose solution as described above, blood fed and dissected at 48 hours. RNA was extracted and checked for catalase relative expression as above.ad libitum. Developing oocytes were dissected and counted 48 hours after blood feeding. Non-injected flies of the same age and kept under the same conditions were used as second control. Three pools of three whole sand flies were collected from each group to evaluate knockdown by RT-PCR.Sense and anti-sense catalase-specific primers flanked by the T7 promoter site  PCR amplLu. longipalpis were collected upon emergence and sugar fed on a 70% w/v sucrose solution supplemented with 20 mM ascorbic acid. Dead sand flies were counted and removed from the cage every day until day seven. A group of sand flies fed with 70% sucrose was used as a control.To assess sand fly survival mediated by ROS-scavenging related to catalase activity, RNAi-mediated catalase knock down was carried out in a group of 50 sand flies. Flies were injected with dsRNA for catalase (dsCAT) as described above. To exclude wound-related mortality, all dead flies at 24 hrs post-injections were removed and were not included in the experiment. Dead sand flies were counted and removed from the cage daily from day 2 to 7 after injection. Flies injected with dsRNA for GFP (dsGFP) and a needle-pricked group were used as controls. To assess exogenous ROS-scavenging related survival, 50 female Phenoloxidase activity was determined by measuring the production of dopachrome from 3,4 dihydroxy-DL-phenylalanine (DOPA) The PO activity was measured to ensure that activity was proportional to protein concentration and incubation time. Independent experiments showed that the PO activity was stable in the conditions above. Controls with no enzyme or no substrate were included. One unit of enzyme (U) is defined as the amount that produces 0.001 unit of absorbance/min.http://expasy.org/). Selected amino acid sequences of catalases were aligned with catalase LlonKat1 using the ClustalW Multiple Alignment tool in BioEdit Sequence Alignment Editor (http://www.mbio.ncsu.edu/BioEdit/BioEdit.html). Alignment was generated using Boxshade (http://www.ch.embnet.org/software/BOX_form.html).The coding sequence of LlonKat1 was analyzed using the algorithms pI/Mw tool 2 test. Significance was considered when P<0.05. All data were analysed with the use of the SPSS Data Editor software .Comparisons between means of two independent groups were carried put using a pair-wise t-test. Multiple comparisons were done by one-way ANOVA. Survival curves were analyzed with the Kaplan-Meier Log Rank χFigure S1Structure-based alignment of the aminoacid sequence of Lutzomyia longipalpis catalase, translated from a whole body (GeneDB NSFM-142e04.q1k ) and a midgut-specific (GenBank Accession number: EU124624.1 ) cDNA library. Sequences show a 99% identity and a 99% similarity. > Represents the targeted region for dsRNA-mediated gene silencing.(DOC)Click here for additional data file.


Drosophila mutations, including so-called Minutes, in which heterozygotes exhibited small body size and developmental abnormalities in tissues undergoing rapid cell division, and homozygotes were lethal Minute loci were no more severely affected than a single Minute mutant. This mystery—how dozens of genes could encode similar but separate proliferative functions in all cells—was solved more than 50 years later with the realization that mutations of ribosomal protein genes occur in almost all Minute loci Lewis Carroll wrote about Alice, but he might just as well have been referring to Calvin Bridges. As a student in T.H. Morgan's lab, Bridges described some of the earliest PLOS Genetics, Watkins-Chow et al. In this issue of N-Ethyl-N-Nitrosurea (ENU) mutagenesis screens, or from targeted gene deletion Belly spot and tail is caused by a splicing abnormality in Rpl24Bst/+Rpl24) are small with white hind feet, a midline belly spot (Bst), abnormal retinal development, and skeletal abnormalities that include a curly tail Rps6 causes embryonic lethality Rpl22 or of Rpl29 have no effect.  and Zuma (Zma), that were generated from an ENU mutagenesis screen. Most Mtu/+Rps7 mice die in utero (74% on a C3H/HeJ background), but the survivors show pleiotropic phenotypes including reduced body size, abnormal skeletal morphology, and mid-ventral white spotting. This phenotype cluster is reproduced in Zma/+Rps7 mice and is similar to that in Rpl24 mutant mice Watkins-Chow et al. p53 deletion  and −/−Rpl29 mouse neural tube and somites (∼45% reduced), although no concurrent phenotype was described Mtu/+Rps7 mice, a novel defect in 18S rRNA preprocessing was identified in brain and liver, without a reduction in protein synthesis. We speculate that an accompanying reduction in protein synthesis could explain the homozygous embryonic lethality in these mice. On the other hand, the abnormal homeotic transformations observed in Ts/+Rpl38 mice were attributed to the unique role of Rpl38 in translation of specific Hox mRNAs rather than to a global effect Mtu/+Rps7 mice, impaired rRNA preprocessing and p53 activation occur simultaneously, illustrating the complex roles of RPs in mammalian tissues. With this in mind, mouse RP phenotypes (heterozygous and homozygous) need to be analyzed on a p53-null as well as on a p53 wild-type genetic background. Since so little is known about how changes in ribosomal protein levels may impact cellular function in vivo, a full repertoire of mouse RP models and improved characterization are sorely needed.Mutant RP phenotypes in mice appear to be the result of three distinct mechanisms: 1) global suppression of protein synthesis; 2) specific suppression of protein synthesis; and 3) extra-ribosomal functions. Diminished global protein synthesis was identified in Due to the essential nature of RPs, it is apparent that even small perturbations to their functions can result in an increasing array of diseases, and it is clear now more than ever that the mystery of the underlying mechanistic basis for RP mutant phenotypes needs to be solved before state-of-the art translational approaches can bring effective treatments. Moreover, additional mouse models for RP disorders will provide an important preclinical resource for developing new treatments of ribosomopathies.


Lolium perenne ingression was investigated within a larger microcosm study (Microb Ecol 63:509–521).The present study explores the potential of directly linking phylogenetic identities obtained by cloning and sequencing of ITS sequences to dominant ribotypes of molecular community fingerprints to give further insight into dominant members of the communities in three Irish grassland soils. The ten most abundant bacterial ribotypes of untreated bare soils of three grassland microcosms were chosen to represent the “baseline community” of the respective soil. Identities on phylum and order level were assigned to these ribotypes on a weighted basis, by matching sequence homologies of cloned ITS sequences with ribotypes of the same fragment lengths ±5 bp. Results showed that ribotypes were represented by the phyla Acidobacteria, Actinobacteria, Proteobacteria, and Firmicutes and the distribution of the ribotype and phylotype communities was shown to be highly site-specific. Furthermore the response of dominant bacterial phylotypes to plant species composition, fertilisation and The online version of this article (doi:10.1186/2193-1801-2-522) contains supplementary material, which is available to authorized users. Lolium perenne ingression.Microbial communities are likely to be influenced by a wide range of factors, including environmental and anthropogenic factors. In order to examine those influences on the community structure, molecular fingerprinting techniques, such as terminal restriction fragment length polymorphism (TRFLP) or automated ribosomal intergenic spacer analysis (ARISA), are commonly employed Thies . While tIn order to assign phylogenetic identities to the 10 most abundant bacterial ARISA ribotypes (hereafter referred to as baseline ribotypes) of each untreated bare soil type  of the RNA-operon  contributed significantly to this effect for Firmicutes, but not for Actinobacteria, where pH and its interaction with clay were the most significantly contributing factors. The reputed preference of Actinobacteria for an alkaline pH  while phylotypes seemed not to be affected. The Wicklow community showed no response to Lolium ingression, neither on the ribotype, nor phylotype level. A fertilisation effect on the phylotype level was only observed in the Burren and Wicklow soils, where it influenced Firmicutes (F = 12.3) only, and Firmicutes (F = 26.1) and Actinobacteria (F = 10.4), respectively. Similarly 5 and 6 ribotypes were influenced in these soils, respectively , while only 1 ribotype was affected by fertilisation in the Ardgillan soil (F = 15.8).Applying the phylotypic identities of baseline-ribotypes to their occurrence in fingerprints of the complete microcosm study et al., Additional file 2: Table S2: Experimental design of complete microcosm study (Liliensiek Method details. (PDF 110 KB)Additional file 3: Additional file 4: Table S3: Identities of cloned bacterial ARISA fragments matching the top 10 abundant ribotypes of bare untreated soils. (PDF 44 KB)