During development axons encounter a variety of choice points where they have to make appropriate pathfinding decisions. The optic chiasm is a major decision point for retinal ganglion cell (RGC) axons en route to their target in order to ensure the correct wiring of the visual system. MicroRNAs (miRNAs) belong to the class of small non-coding RNA molecules and have been identified as important regulators of a variety of processes during embryonic development. However, their involvement in axon guidance decisions is less clear.Dicer mutants a marked increase of ipsilateral projections, RGC axons extending outside the optic chiasm, the formation of a secondary optic tract and a substantial number of RGC axons projecting aberrantly into the contralateral eye. In addition, the mutant mice display a microphthalmia phenotype.We report here that the early loss of Dicer, an essential protein for the maturation of miRNAs, in all cells of the forming retina and optic chiasm leads to severe phenotypes of RGC axon pathfinding at the midline. Using a conditional deletion approach in mice, we find in homozygous Our work demonstrates an important role of Dicer controlling the extension of RGC axons to the brain proper. It indicates that miRNAs are essential regulatory elements for mechanisms that ensure correct axon guidance decisions at the midline and thus have a central function in the establishment of circuitry during the development of the nervous system. During development the correct patterning of tissues and the action of a network of guidance and signaling proteins ensure the accurate establishment of neural connectivity. The visual system is a well-studied model system to investigate these fundamental processes. Retinal ganglion cells (RGCs) are the only cells sending out axons from the eye and transmit information to a number of targets in the brain. The hierarchical interplay between different transcription factors ensures that the appropriate axon guidance molecules are expressed within RGC axons and in the target tissues Dicer made it possible to study its function in a tissue-specific manner MicroRNAs (miRNAs) are a class of small endogenous RNA molecules with important functions in gene regulation. These non-coding ∼21 nt long molecules bind to complementary sequences on protein-coding messenger RNAs and as a consequence direct their degradation and/or repress their translation Dicer in eye tissues using a Rx-cre mouse line Dicer allele Dicer in all cells of the developing visual system leads to pronounced RGC axon pathfinding defects at the optic chiasm. In addition, we find that the eyes are significantly reduced in size in homozygous Dicer mutant mice. However, both ventral retinal polarity and Zic2 expression as a marker of the ipsilaterally projecting RGC population in the VTC remain unchanged. Our findings suggest an important function of miRNAs during axon extension towards the midline, directly or indirectly, to ensure the appropriate wiring of the nervous system during development.Due to the broad extent of miRNAs functions during the development of the nervous system we speculated that they may be involved in the control of axon pathfinding decision at the midline. This could be either through indirect mechanisms, including regulation of factors important for tissue patterning, intracellular signaling or cell specification, or through direct regulation of axon guidance molecules critical for sensing the environment during axon extension. Indeed, functional RISCs have been found in developing axons Retinal homeobox gene Rx is an evolutionary conserved eye field transcription factor, essential for eye formation Dicer is conditionally deleted in these tissues, we crossed the Rx-cre mouse line with a mouse line carrying a conditional null-allele for Dicer (fl/flDicer), where the exon containing most of the essential RNaseIII domain are flanked by loxP sites  cre expression in the expected spatiotemporal pattern, we crossed the Rx-cre mouse line with the R26R-EYFP lacZ expression, respectively. As expected, we found EYFP/ß-gal positive cells at E11.5 in the developing visual system, including retina, optic stalks and at later stages at the forming optic chiasm, indicating Cre-mediated recombination in these areas . The eyes laid much deeper in the head than seen normally and, when the overlaying eyelid tissue was removed, a substantial reduction in overall eye size became apparent in homozygous Dicerfl/fl; cre+ embryos  and E16.5 (n = 6) and around 50-fold, at E13.5 (n = 6), compared to Cre-negative littermates  and cell bodies  were detected. In contrast, in wild-type and heterozygous fl/wt; cre+Dicer littermates we did not detected any labeled fibers extending into the contralateral retina , the initial retinal lamination during development was normal A recent report describes a knockdown of fl/fl;Rx-cre+Dicer mutants are missing major forebrain commissures (data not shown) and we are currently analyzing this in greater detail. In addition, functional RNA-induced silencing complexes have been found in axons and growth cones in culture Dicer in both, the retina and at the optic chiasm, the defects we detect could be based on miRNA action in either of these tissues or both. However, it is interesting to note that the extensive axon pathfinding errors we observed partially phenocopy some defects that have been previously described in studies where individual genes expressed in these regions were deleted.The perturbation of the appropriate formation of axon tracts upon Dicer loss has been reported during cortical development in mice and in trigeminal neurons in zebrafish belladonna mutation) cause mispatterning of the forebrain and expression changes in regulatory genes as well as axon guidance molecules, such as sema3d, netrin1a and slit2Increased ipsilateral projections have been found in a number of reports examining effectors at the optic chiasm. This structure is precisely patterned, creating longitudinal molecular subdivisions of the forebrain in which RGC axons run parallel to cross from one side to the other Dicer in the eye has no effect on the overall ventral identity of the retina or on the expression of Zic2 in the VTC. This suggests that the specification of ipsilaterally projecting RGCs and the patterning along the DV axis occurs independent of miRNAs. Additional marker analysis is needed to confirm if this holds true also for the nasotemporal axis of the eye.The retina is patterned along both its axes, in addition to be subdivided into a binocular (the VTC) and monocular (the remainder of the retina) region. This is achieved through the combinatorial action of morphogens and transcription factors that subsequently control the expression of axon guidance molecules in RGCs Dicer mutant mice, has been reported in Slit1/Slit2 double mutant mice Slit1 and 2 are both expressed at the region of the chiasm and create a non-permissive boundary for RGC axons, therefore channeling the fibers correctly along their path across the midline Slit1/2 double mutant mice allows axons to extend aberrantly outside their normal trajectories Dicer mutant mice. Similarly, the mutation of the Slit receptor, Robo, leads also to aberrant projection patterns at the ventral hypothalamus Dicer mutants and the Slit/Robo mutants is not 100%, since we do not observe any premature midline crossing or any intraretinal guidance defects of RGC axons Aberrant axon growth into the contralateral optic nerve similar to what we detected in our fl/fl; cre+Dicer mutant mice, suggest that the expression of multiple genes in retina, chiasm or both has been perturbed.The similarities between our results and the phenotypes caused by these individual gene alterations described above imply that miRNAs control the same or at least related pathways influencing RGC axon guidance decisions at the midline. The extent however and the range of phenotypes that we detect in the Several studies have investigated the expression patterns of miRNAs in the retina and identified a substantial number of retina-specific members of these non-coding RNA molecules Our work demonstrates in vivo the important role of miRNAs for the correct navigation of RGC axons through the optic chiasm. Although the exact mechanism how these short RNA molecules affect axon pathfinding is not yet known, we show here that they are critical for the establishment of the circuitry during the development of the nervous system. Furthermore, the defasciculation of RGC axons and their aberrant appearance within the retina suggest, at least in part, a cell-autonomous function for miRNAs during axon extension and pathfinding. Now it will be important to extend investigations in order to further identify not only the particular miRNAs involved in this complex regulatory network, but also the target genes that are affected.All animal procedures were carried out according to institutional guidelines and protocols approved by the KCL Ethics Committee and the Home Office.fl/flDicer mice lacZ reporter line Dicer mice was performed by PCR as described GTTGGGAGAATGCTCCGTAA) and MeRxgenoR (GTATCCCACAATTCCTTGCG) to amplify a 362 bp product detecting presence of cre sequences. DNA was isolated from the tail tissue of each mouse embryo, using 50 mM NaOH for 30 minutes at 95°C followed by 1 M Tris buffer, pH 8 at room temperature. 1 µl samples of DNA were used for PCR genotyping. Amplification conditions were: 2 min at 94°C, 94°C 30 sec, followed by 32 cycles of 94°C for 30 sec, 60°C for 1 min, then 72°C for 1 min, followed by a final 72°C extension for 7 min.Staged mouse embryos were dissected and fixed in 4% paraformaldehyde/PBS overnight at 4°C. After infiltration with 20% sucrose the samples were embedded and frozen in OCT medium (Tissue-Tek). For histochemistry and immunolabeling, 16–18 µm tissue sections were cut on a cryostat and processed according to standard protocols. The working dilutions and sources of antibodies used in this study were: rabbit anti Caspase-3 antibody , rat anti-L1 monoclonal antibody , mouse anti-Neurofilament 2H3 , rabbit anti Zic2  and a Leica Stereomicroscope M165FC with a cooled color CCD camera (QICAM) in combination with Volocity Acquisition software (Perkin Elmer).Total RNA was purified from tissue using TRIZOL (Invitrogen) according to manufacturers instructions and 8 µg for each genotype were subsequently resolved by 15% TBE-Urea polyacrylamide gel electrophoresis (PAGE). Gels were stained with Ethidium Bromide and photographed under UV illumination. RNA was transferred by electroblotting onto Nylon + membranes (Invitrogen) and then crosslinked by UV  and baking at 80°C for 30 minutes. Filters were pre-hybridised at 45°C for 30 minutes in Ultrahyb-Oligo-Hybridisation buffer (Ambion) before ready DIG-labeled miRCURY LNA probes  were added and hybridized overnight. Filters were then washed at 45°C 2×10 min with 2xSSC, 0.1% SDS and 1×10 min with 1xSSC, 0.1%SDS. Subsequent washes and chemiluminescent DIG signal detection was carried out using the DIG detection starter Kit (Roche) according to manufacturers instructions. Blots were stripped by incubation in 1%SDS at 85°C for 30 min for reprobing.Figure S1Cre-expression pattern in Rx-cre mice. Serial coronal sections through E17.5 heads of Rx-cre; R26R-lacZ mice after x-gal staining. The blue signal indicates expression of cre. (A) Schematic showing the level of sections along the anterior-posterior axis depicted in the other panels. (B–F) Cre expression is strongest in the retina  but also visible in more broadly in structures within the forebrain.(4.49 MB TIF)Click here for additional data file.Figure S2Dicer mutant mice. Horizontal sections through the optic chiasm of E17.5 wild-type (A–E) and Dicer mutant (F–J) embryos, followed by immunohistochemistry using L1 antibody (brown staining) and counterstaining with haematoxylin (blue). (A–E) In wild-type embryos the retinal fiber bundles are crossing at the chiasm and extend further around the ventral diencephalon (arrows), leading to strong L1 staining. (F–J) In Dicer mutant embryos, L1 staining is much weaker across the entire chiasm, possibly because axons have defasciculated. L1-positive fibres can be found not only at the surface of the diencephalon, but also deeper in the tissue (arrowheads). v, ventral; d, dorsal. Scale bar = 200 µm.Disorganization of retinal fibers at the midline in (10.02 MB TIF)Click here for additional data file.


Radial glial cells play an essential role during corticogenesis through their function as neural precursors and guides of neuronal migration. Both reelin and neuregulin1 (NRG1) maintain the radial glial scaffold; they also induce expression of Brain Lipid Binding Protein (BLBP), a well known marker of radial glia. Although radial glia in normal ferrets express both vimentin and BLBP, this coexpression diverges at P3; vimentin is expressed in the radial glial processes, while BLBP appears in cells detached from the ventricular zone. Our lab developed a model of cortical dysplasia in the ferret, resulting in impaired migration of neurons into the cortical plate and disordered radial glia. This occurs after exposure to the antimitotic methylazoxymethanol (MAM) on the 24th day of development (E24). Ferrets treated with MAM on E24 result in an overall decrease of BLBP expression; radial glia that continue to express BLBP, however, show only mild disruption compared with the strongly disrupted vimentin expressing radial glia. When E24 MAM-treated organotypic slices are exposed to reelin or NRG1, the severely disrupted vimentin+ radial glial processes are repaired but the slightly disordered BLBP+ processes are not. The realignment of vimentin+ processes was linked with an increase of their BLBP expression. BLBP expressing radial glia are distinguished by being both less affected by MAM treatment and by attempts at repair. We further investigated the effects induced by reelin and found that signaling was mediated via VLDLR/Dab1/Pi3K activation while NRG1 signaling was mediated via erbB3/erbB4/Pi3K. We then tested whether radial glial repair correlated with improved neuronal migration. Repairing the radial glial scaffold is not sufficient to restore neuronal migration; although reelin improves migration of neurons toward the cortical plate signaling through ApoER2/Dab1/PI3K activation, NRG1 does not. The developing cerebral cortex contains a transient elongated population, the radial glial cells, which play an essential role through their function as guides of neuronal migration and neural precursors In rodents, neurogenesis and neuronal migration are largely complete at birth, at which time radial glial cells transform into astrocytes in utero to the antimitotic MAM on the 24th day of development (E24) disrupts early cortical development, resulting in a thin and poorly laminated cortex, where neurons migrating radially and tangentially fail to reach the cortical plate We developed a model of cortical dysplasia in the ferret, in which radial glia display severe disruption and undergo premature differentiation into astrocytes via members of the ErbB family of receptor tyrosine kinases Radial glia are a diverse population and express a number of specific markers at different times and throughout development. They also respond to different signals, which regulate their status as both neural progenitor cells and a scaffold for migration Reelin is another key protein active during cortical development as the lack of reelin results in aberrant migration of cortical neurons and misaligned radial glial cells In addition to radial process extension, reelin and NRG1 promote expression of Brain Lipid Binding Protein (BLBP) in cortical radial glia We show here that in normal newborn ferrets, vimentin and BLBP are strongly expressed in radial glia. In our ferret model of cortical dysplasia, the expression of BLBP in radial glia is decreased after MAM treatment; however the remaining BLBP+ radial glial cells are relatively spared from disruption compared with the severely disorganized vimentin+ cells. Both reelin and NRG1 realign the disorganized vimentin+ radial glial cells. Although the morphology of BLBP+ cells was not improved from their mild disruption after these treatments, the expression of BLBP was increased. This suggests that at least two distinct populations of radial glial cells exist in ferrets that respond differently to damage and attempts at repair. Exogenous reelin improves not only the radial glial scaffold but also radial migration toward the cortical plate whereas NRG1 has no effect on neuronal migration. In addition, distinct signaling elements appear to initiate movement out of the ventricular zone, but do not play a role in allowing further movement toward the cortical plate.The use of animals and the methods of this study were approved by the Institutional Animal Care and Use Committee (IACUC) at USUHS and under Animal Welfare Assurance number A3448-01. The experiments were performed at an AAALAC accredited institute.Mustella putorius) were purchased from Marshall Farms ; ferret kits are born after 41 days of gestation. Pregnant ferrets, anaesthetized with isofluorane using a mask (5%), were injected intraperitoneally (IP) with methylazoxy methanol acetate  diluted in a sterile saline buffer. Normal and MAM treated fetuses at E27, E33, or E38-E40 were obtained by caesarean section under sterile conditions using isofluorane anesthesia under the supervision of a veterinarian. We also used normal and MAM treated newborn kits , as well as normal P3, P14, and P28 normal ferrets, which were anesthetized with an IP injection of pentobarbital sodium (50 mg/kg) prior to brain removal.Timed pregnant ferrets , the organotypic slices were fixed for 2 hours by immersion in 4% phosphate buffered paraformaldehyde. In some cases, fixed slices were also cryoprotected and subsequently re-sectioned at 14 µm using a cryostat.Brains obtained from E39-E40 embryos were cut under sterile conditions into 400 µm thick coronal slices using a tissue chopper . During the dissection, brains and slices were perfused with cold, oxygenated artificial cerebrospinal fluid . Coronal cortical slices containing the somatosensory cortex A number of MAM treated slices were co-cultured with: (1) HEK 293T cells, transfected with the mouse reelin cDNA construct pCrl In some experiments, the medium was supplemented with recombinant mouse reelin  corresponding to the central fragment. We also used recombinant NRG1  obtained from the Human DNA sequence encoding the EGF domain of NRG1 β1. To further understand the effects mediated by reelin or NRG1, the culture medium was complemented with pathway inhibitors such as: LY294002 , PP2 , TDZD-8 , or SP600125 . To block ApoER2 and VLDLR, we used human recombinant RAP . NRG1 signaling was inhibited by using blocking antibodies for NRG1 receptors, erbB-3 or erbB-4  See .For fluorescence immunocytochemistry, slices were incubated overnight at 4°C with mouse IgG monoclonal antibodies against: vimentin clone V9 , MAP2abc  or rabbit polyclonal antibodies against: BLBP  and GABA . After washes in PBS, the corresponding secondary antibodies were used . The sections were washed in PBS and mounted in Mowiol.The fixed slices were placed in 70% cold ethanol for 10 minutes at 4°C, followed by 1 hour in 2N HCl at 37°C. Slices were then placed in borate buffer (pH 8.5) and washed in PBS. The following antibodies were used: anti-rat BrdU  and goat anti-rat IgG conjugated with CY2  or goat anti-rat IgG Alexa-488 .i.e., the lowest part of the intermediate zone, IZL), a region in the IZ, but closer to the cortical plate , and the region corresponding to the cortical plate (CP) , we plotted the distribution of BrdU+ cells after 2 DIC. Boundaries were drawn indicating the pia of a ferret slice and the outer edge of the VZ. This region was divided into 3 equal bins for each coculture and the number of cells per bin counted in a slab 500 µm in width in the somatosensory cortex. The bins correspond to the intermediate zone close to the VZ (ate (CP) . Histogrz-stack images , which were collapsed into a single image; the degrees of deviation and the phenotype of radial glial processes were measured in a 250 µm2 zone within the cortical plate. On average, this zone contains 57 radial glial processes.MAM treatment leads to early radial glial differentiation, which can be reversed by treatment with exogenous NRG1 or reelin A total of 115 MAM treated embryos were obtained from 16 pregnant ferrets and 27 normal ferret embryos or kits obtained from 13 pregnant ferrets. All data are obtained from at least two independent experiments from different litters. For all data, a 2 way ANOVA was conducted followed by a Holm-Sidak test for comparisons between groups. Statistical analyses were performed using SigmaStat .For the acquisition of fluorescence images, we used an Axiovert 200 microscope (Zeiss) equipped with an Apotome and Axiovision 4.7.Vimentin is an excellent marker for radial glia in ferrets, but few others have been explored in this species in vitro (in plain medium) compared with acute sections obtained from normal and E24 MAM treated ferrets  in normal and MAM treated ferrets. These percentages are reported in in vitro  and lower (IZL) parts of the intermediate zone  reelin-dependent signaling as well as reelin-independent but Pi3K-dependent signaling facilitate neuronal migration from lower IZ toward the CP  combined with HER-3 Ab-5 or HER-4 Ab-3, two antibodies blocking the binding of NRG1 to erbB3 and erbB4 respectively . Radialisee also .The number of radial glial processes co-expressing vimentin and BLBP was significantly increased compared to control when slices were incubated in the presence of NRG1 or cocultured with Ig-NRG1 cells , Table 2via an increase of intracellular cyclic AMP In MAM treated ferrets, disruption of radial glia is associated with abnormal migration; neurons are scattered in all cortical layers compared to normal ferrets where neurons accumulate in an inside-out pattern We show here that severely disrupted dyplasic brains can be repaired by specific application of reelin or NRG1. In our model of cortical dysplasia, reelin restores the radial glial scaffold as well as glia-guided migration; NRG1, however, had a more limited effect since radial glia were realigned, but neuronal migration was not improved . The difin vivo or grown in organotypic cultures. In addition, Hack et al. Corticogenesis in E24 MAM treated ferrets is severely disrupted; misaligned radial glial cells differentiate into astrocytes and neurons fail to reach the cortical plate Others report that the N-terminal portion of reelin binds to cadherin-related neuronal receptors and the integrin receptors Reelin not only repairs radial glia but also improves migration of neurons that are likely to be generated in the neocortical ventricular zone since they express MAP2 and are also GABA-negative. In the presence of exogenous reelin, neurons move into the cortical plate but only into the lower intermediate zone when Dab1 and Pi3K were inhibited, suggesting that reelin signaling is essential for neuronal migration from the intermediate zone toward the cortical plate. This is supported by Uchida et al. via erbB4 NRG1 signaling is also essential for the normal development of radial glia Time-lapse imaging studies revealed that in postnatal ferret visual cortex translocating neurons with a long pial-contacting process coexist with short-process locomoting neurons In addition to BLBP, intermediate filaments such as vimentin, nestin, and GFAP are radial glial markers. However their expression differs across species. GFAP is expressed in the radial processes of cells in the developing cerebral cortex of primates In normal newborn ferret, radial glial cells express vimentin as well as BLBP. In MAM treated animals at a similar age, these markers are not expressed homogeneously and about half of radial glia express vimentin but do not express detectable levels of BLBP. BLBP expressing radial glia are distinguished by being less affected by MAM treatment and less affected by attempts at repair using radializing factors such as reelin or neuregulin. During normal corticogenesis in ferrets, BLBP expression is developmentally regulated and coincides with the bulk of neurogenesis (up to P0-P1); it is not present in radial glia after about P3, while vimentin expression is maintained until P14. This suggests that BLBP is reduced as radial glia differentiate into astrocytes. MAM exposure at embryonic day 24 induces a premature differentiation of radial glia Figure S1Schematic view of reelin and neuregulin1 signaling pathways. The pathway inhibitors and the blocking antibodies used in our study are boxed in red.(2.25 MB TIF)Click here for additional data file.


Oprm1) or delta- (Oprd1) opioid receptor gene alters motor impulsivity in mice. Wild-type and knockout mice were examined on either a pure C57BL6/J (BL6) or a hybrid 50% C57Bl/6J–50% 129Sv/pas (HYB) background. Mice were trained to respond for sucrose in a signaled nose poke task that provides independent measures of associative learning (responses to the reward-paired cue) and motor impulsivity (premature responses). Oprm1 knockout mice displayed a remarkable decrease in motor impulsivity. This was observed on the two genetic backgrounds and did not result from impaired associative learning, as responses to the cue signaling reward did not differ across genotypes. Furthermore, mutant mice were insensitive to the effects of ethanol, which increased disinhibition and decreased conditioned responding in wild-type mice. In sharp contrast, mice lacking the Oprd1 gene were more impulsive than controls. Again, mutant animals showed no deficit in associative learning. Ethanol completely disrupted performance in these animals. Together, our results suggest that mu-opioid receptors enhance, whereas delta-opioid receptors inhibit, motor impulsivity. This reveals an unanticipated contribution of endogenous opioid receptor activity to disinhibition. In a broader context, these data suggest that alterations in mu- or delta-opioid receptor function may contribute to impulse control disorders.Impulsivity is a primary feature of many psychiatric disorders, most notably attention deficit hyperactivity disorder and drug addiction. Impulsivity includes a number of processes such as the inability to delay gratification, the inability to withhold a motor response, or acting before all of the relevant information is available. These processes are mediated by neural systems that include dopamine, serotonin, norepinephrine, glutamate and cannabinoids. We examine, for the first time, the role of opioid systems in impulsivity by testing whether inactivation of the mu- ( Impulsivity is a behavioral trait that varies across the general population. Extreme manifestations of impulsivity are revealed in a variety of pathological conditions including antisocial and borderline personality disorders, attention deficit hyperactivity disorder (ADHD), pathological gambling, eating disorders, obsessive-compulsive disorder and substance abuse Altered serotoninergic transmission has long been associated with impulse control disorders Oprm1−/−) in mice reduces or eliminates the rewarding properties of opioids as well as non-opioid drugs such as ethanol, cocaine, nicotine and cannabinoids. The consistency of behavioral effects across different classes of abused drugs led to the hypothesis that mu-opioid receptors represent a gateway to drug addiction −/−Oprd1) show intact cannabinoid-induced reward Opioid receptors have been studied extensively in relationship to drug abuse. The opioid system consists of endogenous neuropeptides that produce effects by acting at mu, delta and kappa opioid receptors. Deletion of the mu-opioid receptor gene ,  or backcrossed onto the C57Bl6J background  for more than 12 generations (BL6). A detailed description of the construction of these lines and their genotyping has been described previously ad libitum throughout the experiment; with the exceptions noted below; access to water was restricted to 2 h per day beginning 2–4 h after testing. Behavioral testing commenced at 17–20 weeks of age and finished at 26–33 weeks of age. All animals were weighed twice per week; there were no significant group differences in weight across the experiment. Animal care was conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and the experiments were approved by the Comité régional d'éthique en matière d'experimentation animale de Strasbourg, CREMEAS, 2003-10-08-[1]-58.At 112–126 days of age, mice were transferred from the breeding facility to the phenotyping centre where they were group housed on a 12-h light/dark cycle with lights on at 700 am. Food was available Impulsivity testing was conducted in operant boxes (26.5×22.0×20.0 cm) housed in a sound-attenuating chamber . Each box was fitted with two nose poke response holes, a cue light, a houselight and a liquid dipper that was accessed through a recessed magazine. Sucrose delivery was signaled by an infrared light located 4 cm above the magazine. Nose pokes and entries to the magazine were detected by infrared beams crossing each opening vertically. The control of stimuli and recording of responses were managed by an IBM-type computer using Graphic State Notation 2 software.Sucrose and water consumption were measured in 24 automated chambers (11×22×19 cm) made of wire mesh floor and Plexiglas sidewalls . Each cage was fitted with infra-red captors located 2 and 8.5 cm from the floor, allowing measurement of ambulatory locomotor activity and rears, respectively. Water and sucrose consumption were measured using an automated lickometer.Mice were trained in the signaled nose poke task as described previously One day prior to magazine training, and following 21 h of water deprivation, a sucrose solution (25% w/v) was presented in the home cage for one hour. Beginning the next day, all mice underwent magazine training (2 sessions) consisting of 30 presentations of liquid sucrose, presented at random intervals approximately every 30 seconds. During sucrose presentation, the houselight was turned off and the magazine light was turned on. Magazine entries were recorded and used as an indicator that mice had learned the location of the sucrose reward. The liquid dipper remained elevated for 5 s after a magazine entry to a maximum of 20 s per sucrose presentation.Subsequent training was conducted in 4 phases with one training session per day. In Phase 1, mice were reinforced for nose poking on a fixed-ratio (FR1) schedule of reinforcement on either the right or left side . The discriminative stimulus  was turned on throughout these sessions, except during presentation of the reinforcer (5–20 s elevation of the sucrose dipper). Training continued until 25 reinforcers were presented in one session. In Phase 2, response requirements were increased to an FR3 schedule and the elevation of the sucrose dipper was limited to 5 s. Training continued until 25 reinforcers were presented in one session. In Phase 3, the discriminative stimulus was reduced to 5 s and presented 50 times with a fixed inter-trial-interval (ITI) of 30 s; only those responses occurring on the reinforced side during the cue were reinforced (FR1) by a 5 s elevation of the sucrose dipper. Training progressed to Phase 4 when animals made 10 reinforced nose pokes  in one session. Animals that did not reach the training criterion in Phases 1, 2, or 3 after 20 sessions were removed from the experiment, and data from these sessions was not included in the statistical analyses. In Phase 4, the ITI was set at 20 s, followed by a pre-cue interval of 1–8 s. Phase 4 testing continued for 10 sessions. Nose pokes in the presence of the discriminative stimulus immediately turned off the visual cue and elevated the sucrose dipper (5 s). In one final session, the duration of the cue presentation was increased to a maximum of 30 s per trial. Thus, mice had up to 30 s to detect and respond to the visual cue before the next ITI was initiated.Following the completion of Phase 4 testing, mice were retested in the signaled nose poke task following an injection of ethanol . Doses were administered in ascending or descending order  and all animals were tested twice at each dose. A minimum of 48 h separated each test.At the end of all behavioral tests, sucrose and water intake were assessed for 24 h, beginning at 11:00 during the light cycle (lights off at 19:00). Water and sucrose (25%) were provided in two bottles at the front of the cage and the number of licks was automatically scored for each bottle and measured as a function of time. Regular chow was freely available throughout the session. Sucrose preference was calculated for each phase as a percentage of total fluid intake (sucrose and water combined). Locomotor activity was measured as horizontal beam breaks at the front and back of the cage.Oprm1−/− and Oprd1−/− groups on a HYB background. Data were then analyzed separately (Oprm1−/− and Oprd1−/−) using an analysis of variance (ANOVA) with genotype (wild-type versus knockout) and background (BL6 versus HYB) as a between-subjects factors and session as a within-subjects factor . Post-hoc analyses were conducted using Tukey's test. Whenever there were violations of sphericity, P-values from the Greenhouse-Geisser correction to the within-group degrees of freedom were reported for all ANOVA tests. During Magazine training, the total number of magazine entries, magazine entries during sucrose presentation and the % of total magazine entries that occurred during sucrose presentations were analyzed. The dependent variable in Phases 1–3 was days to criterion. In Phase 4 and during Ethanol testing, the conditioned responses  and efficiency ratio (reinforcers/nose pokes) were dependent variables. Appetitive learning is reflected in the CR measure whereas impulsivity is reflected in the efficiency ratio. Data from the ethanol sessions were analyzed with genotype and background as between-subjects factors and dose as a repeated factor. There was no effect of repeated testing on efficiency ratios or CR so data were collapsed across this factor. In all sessions, the number of responses on the non-reinforced side, the number of magazine entries during reward and non-reward periods, the latency to enter the magazine following elevation of the sucrose dipper, were recorded. Sucrose preference data were analyzed using a two-way ANOVA (genotype x background).The same wild-type mice on a BL6 background were used the control group for both mu and delta opioid receptor knockout mice; separate groups of HYB wild-type animals were used as controls of Mice from the six groups progressed through the training phases of the signaled nose poke task at the same rate . All ani−/−Oprm1  mice on both genetic backgrounds performed significantly better than wild-type controls during Phase 4 testing. Efficiency ratios (rewards/nose pokes) increased across 10 sessions  verifying that all groups were capable of learning the task  = 8.2, p<.01] and background  reflected lower overall responses in the −/−Oprm1 groups and in the HYB strain.Responses on the rewarded side during the 20-s inter-trial interval (ITI) showed a similar pattern , but the rate of decrease differed across groups  . RespondFinally, conditioned responding  increased across sessions , but there were no genotype or background differences on this measure and no statistically significant interactions .−/−Oprd1 groups achieving lower efficiency ratios  compared to their respective control groups (post-hoc tests p<0.05). In addition, as in the −/−Oprm1 experiment, there was a significant difference in the efficiency ratios of the two wild-type strains, with the HYB animals exhibiting significantly higher efficiency ratios in sessions 3–10. The session x background and session x background x genotype interactions were not statistically significant.−/−Oprd1  mice was confined to the pre-CS period indicating that these mice are incapable of refraining from making an anticipated response.Both the genotype  and background  differences in responding were apparent during the 1–8 s pre-CS period when animals must learn to inhibit their responses . Pre-CS Oprd1 gene.The proportion of CRs also increased across sessions  , but theIn sum, mice lacking mu-opioid receptors were significantly better at inhibiting a motor response, whereas mice lacking the delta-opioid receptor were significantly worse.−/−Oprm1  mice or the increased impulsivity in −/−Oprd1  mice reflects alterations in attentional processes. Finally, sucrose preference was similar in wild-type and knockout mice, and there were no strain differences in this measure, ruling out the possibility that differential responses to the reward influenced our findings (mean sucrose preference across groups = 94.7%).It is unlikely that these changes in efficiency ratios simply reflect a general reduction in responding because there were no genotypic differences in: 1) responses on the non-reinforced side throughout training and testing  = 32.45, p<.01] , p<.01]  [F. In light of these dramatic effects of ethanol in wild-type and Oprd−/− mice, the most striking finding is that the drug was completely ineffective in altering the behavior of mice lacking the −/−Oprm gene.Higher activity under the influence of ethanol probably accounts for the increased responses during both pre-CS and ITI periods, producing lower efficiency ratios in both wild-type and Oprm1−/− mice could be mediated through an interaction with the subthalamic nucleus (STN), dopamine (DA) D2 receptors in the ventral striatum (VS), and/or excitatory projections from the prefrontal cortex (PFC) to the striatum. Lesions of either the PFC Oprm1 gene may increase activity in either structure. With regards to the STN, this hypothesis is plausible as activation of mu-opioid receptors inhibits excitatory inputs to the STN Oprm1 gene could increase PFC activity by directly removing this inhibitory input. Finally, decreased availability of DA D2-like receptors in the VS is associated with high levels of trait impulsivity Oprm1 gene show increased DA D2 receptor binding in the striatum and increased activity levels when the receptor is stimulated Oprm1 and Oprd1 genes may, together, alter inhibitory mechanisms through one or more of these neural systems.Mechanisms by which mu- and delta-opioid receptors regulate motor impulsivity remain to be elucidated: there are several potential neural substrates for opioid-controlled disinhibition. The decreased impulsivity we observed in Oprm1 or Oprd1 genes affect other measures of impulsivity, such as delay-discounting or reflection impulsivity. The latter is particularly interesting because it represents a cognitive marker for substance dependence that does not recover with prolonged abstinence and is associated with multiple drugs of abuse Oprm1 and Oprd1 genes in impulsivity has implications for understanding and treating attention deficit hyperactivity disorder, eating disorders, gambling and other disorders of impulse control.An important remaining question is whether deletion of the


Pseudomonas aeruginosa was produced, and under these conditions diaphragmatic force-generating capacity is selectively reduced in Cftr−/− mice. This is associated with exaggerated pro-inflammatory cytokine expression as well as upregulation of the E3 ubiquitin ligases (MuRF1 and atrogin-1) involved in muscle atrophy. We conclude that an intrinsic alteration of function is linked to the absence of CFTR from skeletal muscle, leading to dysregulated calcium homeostasis, augmented inflammatory/atrophic gene expression signatures, and increased diaphragmatic weakness during pulmonary infection. These findings reveal a previously unrecognized role for CFTR in skeletal muscle function that may have major implications for the pathogenesis of cachexia and respiratory muscle pump failure in CF patients.Cystic fibrosis (CF) patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR) plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and co-localized with sarcoplasmic reticulum-associated proteins. CFTR–deficient myotubes exhibit augmented levels of intracellular calcium after KCl-induced depolarization, and exposure to an inflammatory milieu induces excessive NF-kB translocation and cytokine/chemokine gene upregulation. To determine the effects of an inflammatory environment in vivo, sustained pulmonary infection with  Cystic fibrosis is an autosomal recessive disorder caused by mutations of the CF transmembrane conductance regulator (CFTR), which acts as a chloride channel and also participates in the regulation of other ions and proteins. In most CF patients, the clinical course is dominated by lung disease and recurrent pulmonary bacterial infections. Many CF patients also have significant skeletal muscle wasting and weakness, and this can affect the most essential breathing muscle, the diaphragm. Although muscle wasting in CF has generally been attributed to factors such as reduced physical activity and poor nutrition, our study reveals an intrinsic defect of skeletal muscle function caused by the lack of CFTR. Hence, we show that CFTR is normally found in skeletal muscle fibers of humans and mice. In CF muscle cells lacking CFTR, abnormal elevations of calcium and inflammatory gene expression are found. In addition, during pulmonary infections, diaphragm muscles lacking CFTR show greater weakness and induction of genes which cause muscle atrophy. These findings extend our understanding of the factors leading to exercise limitation and disability in CF and also have implications for the pathogenesis of respiratory muscle failure in CF patients during lung infections. Cystic fibrosis (CF) is caused by defects in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) −) channel −channel function, with several other roles described in the secretion or absorption of different ions as well as in the transport or regulation of other macromolecules CFTR is a member of the superfamily of ATP-binding cassette (ABC) transporter ATPases, which functions as a cAMP-regulated chloride  to confirm the presence, localization, and functionality of CFTR in normal skeletal muscle; and 2) to determine whether CFTR deficiency predisposes to diaphragmatic muscle inflammation, atrophy, or weakness. Here we report that CFTR protein is normally found at the level of the sarcoplasmic reticulum (SR) in skeletal muscle, where it appears to modulate intracellular calcium fluxes. We also show that in the setting of lung infection, the diaphragms of Cftr+/+) mice, and this was further confirmed in primary diaphragmatic myotube cultures  – markers of the sarcolemma and sarcoplasmic reticulum (SR), respectively – were used to localize the CFTR protein. On transverse cross-sections of human skeletal muscle, only occasional surface membrane staining for CFTR could be demonstrated . However−/− mice . Furthernstrated .2+ released via both RyRs and IP3Rs following muscle cell depolarization +/+Cftr and Cftr−/− myotubes were loaded with the Ca2+ probe Fura-2 and exposed to high potassium (60 mM KCl) to induce depolarization in vitro. Immunostaining confirmed the absence of intracellular staining for CFTR in Cftr−/− myotubes , there was a greater initial peak as well as a higher plateau value , nor were the results influenced by omission of Ca2+ from the extracellular medium (data not shown). Furthermore, in Cftr−/− muscle cells, pre-treatment with CFTR-inh172 had no effect on Ca2+ responses  and Pseudomonas LPS, MIP-2 showed greater upregulation in Cftr−/− diaphragmatic myotubes. This increased responsiveness also involved other pro-inflammatory chemokines such as MIP-1α and RANTES, and was similarly observed in limb muscle-derived myotubes -2 inhibitor SC-514 had similar effects in downregulating MIP-1α mRNA expression in Cftr−/− and +/+Cftr myotubes , but it was only effective in doing so for MIP-2  and RANTES  in Cftr−/− myotubes, suggesting a greater reliance upon this pathway in the latter. Finally, we observed that ERK1/2 MAPK, another signal transduction molecule implicated in NF-kB activation and cytokine gene upregulation Cftr−/− group  backcrossed onto the C57Bl/6 background that has been shown to develop spontaneous lung disease Cftr−/− mice (data not shown). At 2 days after the initiation of pulmonary infection, there was an upregulation of pro-inflammatory gene expression within the diaphragms of both Cftr−/− and Cftr+/+ mice. However, as was the case in vitro, the diaphragmatic mRNA levels of chemokines  and other cytokines  were significantly higher in infected Cftr−/− mice . Therefore, these findings are consistent with a greater susceptibility of infected Cftr−/− mouse skeletal muscles not only to cytokine upregulation, but also to ubiquitin-mediated proteolysis and atrophy.Because pro-inflammatory cytokines have been linked to muscle wasting via the ubiquitin-proteasome system, we proceeded to measure the diaphragmatic expression levels of two key regulators of this proteolytic pathway in skeletal muscle, MuRF-1 and atrogin-1 ruginosa . Measure+/+Cftr and Cftr−/− myotubes could be ascertained in intact perfused diaphragmatic muscle fibers. +/+Cftr (monophasic pattern) and Cftr−/− (biphasic pattern) fibers during KCl-induced contraction. Moreover, +/+Cftr diaphragms incubated with CFTR-inh172 exhibited a biphasic pattern similar to the Cftr−/− group, while CFTR-inh172 had no effect upon Cftr−/− diaphragms. In addition, when diaphragmatic fibers from Cftr−/− were electrically stimulated, a significant prolongation of relaxation and contraction were observed during tetanic (but not single twitch) stimulation as compared to +/+Cftr diaphragms  diaphragm muscles lacking CFTR exhibit an exaggerated upregulation of cytokines and cachexia-inducing factors during pulmonary infection, which is associated with a major deterioration of diaphragmatic function.The results of our study demonstrate a previously unrecognized role for CFTR in skeletal muscle physiology, and supply new insights into a potential mechanism favoring the development of respiratory muscle failure in CF. The main findings of our investigation can be summarized as follows: 1) CFTR is normally expressed at the SR of skeletal muscle fibers and is clearly functional, as indicated by abnormal intracellular CaCFTR expression has been reported in both cardiac − channel at the surface membrane plays the predominant role in maintaining the normal resting electrical potential of the cell − channels is more ill-defined. Several Cl− channels with distinct Cl− conductances, selectivities, and voltage-dependencies have been characterized in SR vesicles 2+-induced and voltage-activated Ca2+ release are enhanced by Cl− exposure, apparently via increased activation of RyRs − was also shown to inhibit caffeine-induced Ca2+ release − channels upon SR function are complex. One proposed function for SR Cl− channels is to permit Cl− to act as a counter ion during high rates of Ca2+ release and uptake from the SR − channel activity per se. For example, CFTR mediates glutathione flux in epithelial cells 2+ release In skeletal muscle, the ClC-1 Cl2+ mobilization in myotubes stimulated by potassium-induced depolarization. These findings are consistent with previous observations in Cftr−/− vascular smooth muscle, where greater KCl-induced vasoconstriction and impaired relaxation are found 2+ and enhanced vasoconstrictor responses during exposure to CFTR-inh172 2+ responses to depolarization were similarly increased irrespective of whether CFTR function was inhibited by genetic or pharmacologic methods. Our data indicate that the SR is the primary source of this increased Ca2+ influx, since the latter was not affected by the removal of extracellular calcium. The fact that inhibiting the IP3-mediated pathway with either 2-APB or U73122 (but not the RyR) led to a decrease of Ca2+ values to levels obtained in +/+Cftr myotubes under the same conditions, suggests that the IP3R could be the most important source of dysregulated Ca2+ release from the SR in CFTR-deficient muscle cells, although this remains to be determined. In addition, experiments performed with thapsigargin, an inhibitor of the ER/SR Ca2+ re-uptake pump, did not point to an expansion of SR Ca2+ stores in Cftr−/− muscle cells as an explanation for the greater Ca2+ responses In the present study, we found that defective CFTR function is associated with increased Ca2+ release from the endoplasmic reticulum Cftr−/− muscle cells.Many studies have reported that CF cell lines as well as airway epithelial cells from CF patients exhibit exaggerated cytokine production 2+ release, particularly from the IP3R, in the regulation of transcription factors controlling the expression of pro-inflammatory and other genes in normal skeletal muscle cells 2+ transients of even brief duration can activate NF-kB in myotubes Cftr−/− muscle cells could be linked to greater Ca2+ release from the SR. Indeed, an analogous phenomenon of exaggerated Ca2+ release from the ER leading to NF-kB activation was recently reported in CF airway epithelial cells stimulated with IL-1β There is also an accumulating literature implicating SR-mediated CaCftr−/− mice are more prone to a loss of muscle mass when exposed to a hyperinflammatory milieu. This is likely linked at least in part to the observed upregulation of E3 ubiquitin ligases, MuRF-1 and atrogin-1, which are centrally involved in proteasomal degradation of muscle proteins atrophy Cftr−/− mice. This suggests an alteration in the intrinsic contractile properties of the muscle above and beyond the loss of muscle mass. Recent work has suggested that caspase- and calpain-mediated breakdown of the contractile protein apparatus precedes protein degradation by the ubiquitin-proteasome system in sepsis, and may thus be responsible for the loss of specific force prior to atrophy 2+, and may therefore be particularly relevant to our findings Cftr−/− hearts are abnormally vulnerable to tissue damage caused by ischemia-reperfusion, a classical inducer of free radical-mediated injury In the present study, we demonstrated two potential causes for muscle weakness in CFTR-deficient skeletal muscles. Firstly, we found that P. aeruginosa, will place patients at greater risk for the development of respiratory failure. In our study, CFTR-deficient skeletal muscles demonstrated a pathological phenotype primarily during exposure to an inflammatory milieu. This is likely to be clinically relevant, since studies suggest a quasi-constant exposure to abnormally high serum levels of inflammatory mediators in many CF patients Patients with CF most commonly die of type II (hypercapnic) respiratory failure, where the limiting factor is not oxygen transfer but rather the presence of respiratory muscle pump failure. This occurs when the load placed on the diaphragm exceeds its capacity to deal with the increased work of breathing. In earlier phases of the disease, the increased load may actually induce a beneficial training effect on the respiratory muscles Cftr−/− mice and CF patients with early or mild disease), alterations in the function of CFTR-defective skeletal are more subtle, and may only be apparent with heavy usage such as during exercise Taken together, we believe our findings are consistent with a scenario see  in whichIn conclusion, the identification in this study of an intrinsic alteration of skeletal muscle function in CFTR deficiency extends our understanding of the factors leading to disability in CF patients. A major implication of our study is that the excessive levels of pulmonary and systemic inflammation found in patients with advanced CF can potentially initiate a positive feedback loop, in which the abnormal vulnerability of CFTR-deficient muscle to pro-inflammatory stimulation becomes an important component of the downward spiral leading to respiratory muscle failure. Our data also suggest that the chronic cachexia found in non-respiratory skeletal muscles of many CF patients could be related to similar mechanisms, and may thus be amenable to anti-inflammatory therapies.Primary muscle cell cultures from the mouse diaphragm and limb muscle  were established using single living muscle fibers as previously described in detail Standard staining procedures were applied to myotubes grown on glass coverslips and frozen muscle tissue sections. The analysis was performed on 3 animals from the CFTR+/+ group, 2 animals from the CFTR−/− group, and 3 human muscle samples (3 sections each). Primary antibodies consisted of the following: 1∶100 dilution of anti-CFTR ; 1∶50 dilution of anti-dystrophin ; 1∶100 dilution of anti-IP3R ; 1∶50 dilution of anti-RyR .2+ measurements were performed in diaphragmatic myotubes grown on coverslips using the ratiometric probe Fura-2 (5 µM), and frames were collected at 2 s intervals for a period of 100 s. A customized system was employed for KCl perfusion and pharmacologic reagents applied to myotubes as follows: 10 µM CFTR-inh172  for 30 min; 2 µM thapsigargin (Sigma) for 400 s; 100 µM ryanodine (Sigma) for 10 min; 50 µM 2-aminoethoxydiphenylborate (APB)  for 30 min; and 10 µM U73122 (Sigma) for 20 min.Intracellular free CaTCTCTGCCTTGTGGGAAATC (forward), and AGTACCCGGCATAATCCAAG (reverse); human CFTR primers consisted of the following (5′ to 3′): CTACATGGAACACATACCTTCG (forward), and GGTGATAATCACTGCATAGC (reverse); GAPDH primer sequences (5′ to 3′) were: AGCAATGCCTCCTGCACCACC (forward), and CCGGAGGGGCCATCCACAGTC (reverse). Real-time PCR of CFTR expression was performed with FAST SYBR Green, and relative quantification was determined using the cycle threshold method . Muscle Ring Finger 1 (MuRF-1) and atrogin-1 expression were quantified according to the manufacturer (TaqMan).Murine CFTR primers contained the following sequences (5′ to 3′): 32P-labelled riboprobes were synthesized using commercial mouse multiprobe kits (BD Biosciences), protected RNA fragments were separated using a 5% polyacrylamide gel, and detected by autoradiography. Bands of individual mRNAs were quantified and normalized to the L32 housekeeping gene .An immunoassay kit was used to quantify NF-kB (p65 subunit) within nuclear extracts, according to the manufacturer's instructions . To inhibit the NF-kB pathway, myotubes were treated with the IKK-2 inhibitor SC-514  at a dose of 100 µM CFTR protein expression was evaluated in lysates from human skeletal muscle tissue, E6/E7 human muscle cells, Calu3 cells, and HEK cells with stable overexpression of CFTR. CFTR was immunoprecipitated with anti-CFTR antibody (clone M3A7) and anti-mouse Ig beads. The precipitates were separated using an 8% polyacrylamide gel, and then analyzed by western blotting with a second anti-CFTR antibody (clone 23C5-2) and the TrueBlot detection system (eBioscience).5 cfu using a mucoid strain encapsulated within agar beads, and euthanized at day 2 post-infection, as we have previously described in detail Mice were infected by intratracheal inoculation at 1×10Diaphragms were mounted in an organ bath with oxygenated Krebs solution as previously described in detail tm1UNC mice and their wild-type littermates  on the C57Bl/6J background. The −/−Cftr and +/+Cftr littermate control mice used for generating primary muscle cell cultures were on the C57Bl/6J or Balb/c background. All mice were generated through breeding of heterozygous +/−Cftr mice maintained under specific pathogen-free conditions at the McGill University Research Institute animal facilities. Human muscle biopsy tissue was obtained from brain-dead organ donors after approval by the McGill University human ethics committee.Animal procedures were done in accordance with the guidelines established by the Canadian Council on Animal Care, and were approved by the McGill University Animal Care Committee. Pulmonary infection studies involved male mice at approximately 10 weeks of age, consisting of CFTRAll data are presented as mean values±SEM. Group mean differences were determined by Student's t-test or analysis of variance, with post hoc application of the Tukey test where appropriate. A statistics software package was used for all analyses . Statistical difference was defined as p<0.05.


Viruses have exerted a constant and potent selective pressure on human genes throughout evolution. We utilized the marks left by selection on allele frequency to identify viral infection-associated allelic variants. Virus diversity (the number of different viruses in a geographic region) was used to measure virus-driven selective pressure. Results showed an excess of variants correlated with virus diversity in genes involved in immune response and in the biosynthesis of glycan structures functioning as viral receptors; a significantly higher than expected number of variants was also seen in genes encoding proteins that directly interact with viral components. Genome-wide analyses identified 441 variants significantly associated with virus-diversity; these are more frequently located within gene regions than expected, and they map to 139 human genes. Analysis of functional relationships among genes subjected to virus-driven selective pressure identified a complex network enriched in viral products-interacting proteins. The novel approach to the study of infectious disease epidemiology presented herein may represent an alternative to classic genome-wide association studies and provides a large set of candidate susceptibility variants for viral infections. Viruses have represented a constant threat to human communities throughout their history, therefore, human genes involved in anti-viral response can be thought of as targets of virus-driven selective pressure. Here we utilized the marks left by selection to identify viral infection-associated allelic variants. We analyzed more than 660,000 single nucleotide polymorphisms (SNPs) genotyped in 52 human populations, and we used virus diversity (the number of different viruses in a geographic region) to measure virus-driven selective pressure. Results showed that genes involved in immune response and in the biosynthesis of glycan structures functioning as viral receptors display more variants associated with virus diversity than expected by chance. The same holds true for genes encoding proteins that directly interact with viral components. Genome-wide analysis identified 441 variants, mapping to 139 human genes, significantly associated with virus-diversity. We analyzed the functional relationships among genes subjected to virus-driven selective pressure and identified a complex interaction network enriched in viral products-interacting proteins. Therefore, we describe a novel approach for the identification of gene variants that may be involved in the susceptibility to viral infections. Infectious diseases represent one of the major threats to human populations, are still the first cause of death in developing countries Despite the relevance of viral infection for human health, only few genome-wide association studies (GWAS) have been performed in the attempt to identify variants associated with increased susceptibility to infection or faster disease progression Previous studies One simple prediction of our hypothesis whereby virus diversity is a reliable estimator of virus-driven selective pressure is that genes known to be involved in immune response are enriched in SNPs significantly associated with virus richness. In order to verify whether this is the case we analysed the InnateDB gene list which contains 2,915 genes involved in immune response and showing the presence of at least one SNP in the HGDP-CEPH panel. Correlations with virus richness were calculated using Kendall's partial rank correlation; since allele frequency spectra in human populations are known to be affected by demographic factors in addition to selective forces UNG (MIM 191525), encoding uracil DNA glycosylase, functions downstream of APOBEC3G (MIM 607113) to mediate the degradation of nascent HIV-1 DNA SERPING1 (MIM 606860), a regulator of the complement cascade, is also involved in HIV-1 infection (MIM 609423) as its expression is dysregulated in immature dendritic cells by Tat SERPING1 is cleaved by HCV and HIV-1 proteases It is worth mentioning that amongst these genes, p = 0.0138) (ST6GAL1 (MIM 109675), ST3GAL3 (MIM 606494), ST6GALNAC3 (MIM 610133), ST8SIA1 (MIM 601123), ST3GAL1 (MIM 607187) and ST8SIA6 (MIM 610139)). Notably, sialic acids represent the most prevalent terminal monosaccharides on the surface of human cells and determine the host range of different viruses including influenza A Genes involved in the biosynthesis of glycan structures have also been considered as possible modulators of infection susceptibility. Indeed, since Haldane's prediction in 1949  0.0138) . SeveralST6GAL1ST3GAL1 regulates apoptosis of CD8+ T cells ST8SIA6 is expressed in NK cells, possibly playing a role in the regulation of Siglec-7 lectin inhibitory function in these cells XYLT1 (MIM 608124), HS3ST3A1 (MIM 604057), UST (MIM 610752) and CHSY3 (MIM 609963)) carrying SNPs associated with virus diversity are involved in the biosynthesis of either heparan sulphate or chondroitin sulphate. The former is an ubiquitously expressed glycosaminoglycan serving as the cell entry route for herpesviruses LARGE (MIM 603590) among the genes subjected to virus-driven selective pressure .Since genes involved in immune response and in the biosynthesis of glycan structures are likely to be subjected to selective pressures exerted by pathogens other than viruses, we verified whether a set of genes directly involved in interaction with viral proteins also displays more SNPs significantly correlated with virus diversity. To this aim we retrieved a list of 1,916 genes known to interact with at least one viral product and displaying at least one genotyped SNP in the HGDP-CEPH panel see . In ordeof 0.017 . Notablyth among MAF-matched SNPs. A total of 441 SNPs mapping to 139 distinct genes satisfied both requirements. HLA genes (both class I and II), therefore covering a minor fraction of genetic variability at these loci; as a consequence HLA genes cannot be expected to be identified as targets of virus-driven selective pressure using the approach we describe herein.Given these results, we wished to identify SNPs significantly associated with virus richness on a genome-wide base. We therefore calculated Kendall's rank correlations between allele frequency and virus diversity for all the SNPs  typed in the HGDP-CEPH panel. We next searched for instances which withstood Bonferroni correction (with α = 0.05) and displayed a τ percentile rank higher than the 99We next verified whether the correlations detected between the SNPs we identified and virus diversity could be secondary to climatic variables. Hence, for all countries where HGDP-CEPH populations are located we obtained see  the foll2 test, p = 0.026).Previous works have reported an enrichment of selection signatures within or in close proximity to human genes p = 10−17 and p = 10−12) which were merged into a single interaction network  as 2.88 human-virus interactions would be expected for 23 genes. Analysis of the whole network indicated that a 31 of 66 genes encode proteins interacting with viral products . Thus, the interaction network we have identified is enriched in genes subjected to virus-driven selective pressure and in genes coding for proteins interacting with viral products. It is worth mentioning that, in agreement with previous findings We investigated the role and functional relationship among genes subjected to virus-driven selective pressure using the Ingenuity Pathway Analysis  and the PANTHER classification system  network . The net network . Based oproducts : again tDNMT1 (MIM 126375) and MGMT (MIM 156569) are involved in DNA methylation and repair, respectively, two processes that are often dysregulated during viral infection. In particular, altered expression of DNMT1 is induced by diverse viruses including HIV-1 DNMT1 plays a pivotal role in the expansion of effector CD8+ T cell following viral infection HSPG2 (MIM 142461), the gene coding for perlecan, a cell surface heparan sulfate proteoglycan which mediates the internalization of Tat protein In addition to proteins directly interacting with viral products, several network genes showing correlation with virus diversity might play central roles during viral infection. We next investigated the over-representation of PANTHER classification categories among genes subjected to virus-driven selective pressure. The identification of non-neutrally evolving loci with a role in immunity can be regarded as a strategy complementary to classic clinical and epidemiological studies in providing insight into the mechanisms of host defense The second issue relevant to the data we present herein is that environmental variables tend to co-vary across geographic regions: the distribution of different pathogens (e.g. parasitic worms and viruses/bacteria/protozoa) is correlated across HGDP-CEPH populations As a further control for the possible confounding effects of other environmental factors, we verified that the variants we identified at the genome-wide level do not correlate with climate (temperature) and UV radiation. This analysis was motivated by the known association of virus diversity and biodiversity in general, with temperature IL1A (MIM 147760) and B (MIM 147720), IL4 (MIM 147780), TGFB1 (MIM 190180), IL16 (MIM 603035), IFNG (MIM 147570) and TNF (MIM 191160), OAS2 (MIM 603350) encodes a protein that activates latent RNases, resulting in the degradation of viral RNA and in the inhibition of viral replication ULBP2 (MIM 605698) encodes an MHC1-related protein that binds to NKG2D (MIM 602893) E2F1 (MIM 189971) is known to have a pivotal role in the control of cell cycle and in the activation of tumour suppressor proteins and, together with TP53I3, TADA3L, and TP53BP2 mediates p53-dependent and independent apoptosis CCND3 (MIM 123834) is involved in cell cycle progression through the G2 phase, whereas RAD23A (MIM 600061) up-regulates the nucleotide excision activity of 3-methyladenine-DNA glycosylase NR4A2 (MIM 601828) encodes a nuclear orphan receptor expressed in T cells and involved in apoptosis NR4A2 is also known to play a central role in eliciting the production of inflammatory cytokines in multiple sclerosis (MS (MIM 126200)) PPP3CA (JMJD2C (MIM 605469), C20orf133 ) and CSMD1 (MIM 608397) have been associated with MS C20orf133 is unknown, JMJD2C encodes a histone demethylase expressed at very high levels in B cells and cytotoxic lymphocytes  PPP3CA  have recytes see , a patteKIAA1529 (MIM 611258), LHFPL3 (MIM 609719), LOC51149, RNF217, TMEM132B, LEPREL1 (MIM 610341), ANKFN1, MYO5C (MIM 610022), ANXA4 (MIM 106491) and SCRN3). Among these genes, MYO5C, ANXA4 and SCRN3 are involved in membrane trafficking events along exocytotic and endocytotic pathways, suggesting that they might play a role in either viral cell entry ANXA4 which is expressed at high levels in NK cells (see EYA4 (MIM 603550) (PDE2A (MIM 602658) and SCNN1A (MIM 600228) might play a role in maintaining lung epithelial barrier homoeostasis during viral infection. Indeed, both genes can be induced by TNF-alpha in lung epithelial cells SCNN1A codes for the α subunit) is affected by infection with influenza virus, severe acute respiratory syndrome coronavirus and respiratory syncitial virus.Analysis of the 30 stronger associations  indicateells see . Most in 603550)  has receTRIM5 (MIM 608487)  and IFIH1 (MIM 606951)  showed significant associations with virus-diversity, although they did not withstood genome-wide analysis. Also, it is worth mentioning that variants with a well established role in resistance to viral infections may be neutrally evolving; this is the case for the Δ32 allele of CCR5 (MIM 601373) for example, which confers protection against HIV-1 infection and possibly against other pathogens, but displays no selection signature In humans, resistance to infectious diseases is thought to be under complex, multigenic control with single loci playing a small protective role http://www.gideononline.com), a global infectious disease knowledge tool. Information in Gideon is weekly updated and derives from World Health Organization reports, National Health Ministries, PubMed searches and epidemiology meetings. The Gideon Epidemiology module follows the status of known infectious diseases globally, as well as in individual countries, with specific notes indicating the disease's history, incidence and distribution per country. We manually curated virus absence/presence matrices by extracting information from single Gideon entries. These may refer to either species, genera or families (in case data are not available for different species of a same genus/family). Following previous suggestions http://www.ngdc.noaa.gov/ecosys/cdroms/ged_iia/datasets/a04/, Legates and Willmott Average, re-gridded dataset) using the geographic coordinates reported by HGDP-CEPH website for each population (http://www.cephb.fr/en/hgdp/table.php). Similarly, net short wave radiation flux data were obtained from NCEP/NCAR ; these data were read using Grid Analysis and Display System . Daily values for four years (1948–1951) were averaged to obtain an annual mean.Virus absence/presence matrices for the 21 countries where HGDP-CEPH populations are located were derived from the Global Infectious Disease and Epidemiology Network database , the same procedure was applied to climatic variables. Therefore the values of annual temperature and radiation flux were averaged for populations located in the same country. This assures that a similar number of ties is maintained in all correlation analyses.Data concerning the HGDP-CEPH panel derive from a previous work http://www.innatedb.com/) and it contains a non-redundant list of 5,070 immune genes derived from ImmPort, IRIS, Septic Shock Group, MAPK/NFKB Network and Immunome Database; it only includes genes derived from curated immune gene lists.A SNP was ascribed to a specific gene if it was located within the transcribed region or no farther than 500 bp upstream the transcription start site. MAF for any single SNP was calculated as the average over all populations. The list of immune response genes was derived from the InnateDB website (http://pbildb1.univ-lyon1.fr/virhostnet/) and the HIV-1 Human Protein Interaction Database http://www.ncbi.nlm.nih.gov/RefSeq/HIVInteractions/).Genes involved in glycan biosynthesis were obtained by merging genes from two KEGG pathways (“Glycan structures - biosynthesis 1” and “Glycan structures - biosynthesis 2”). Additional genes were identified by searching Gene Ontology categories for genes that act as glycosyltransferases (GO:0016757) and are located in either the Golgi or the endoplasmic reticulum . The list of human genes coding for proteins interacting with viral products was derived from three sources: a previously published study http://symatlas.gnf.org/). The location of genomic elements that are highly conserved among vertebrates was derived from UCSC annotation tables .Expression data were obtained from SymAtlas , a non-parametric statistic used to measure the degree of correspondence between two rankings. The reason for using this test is that even in the presence of ties, the sampling distribution of τ satisfactorily converges to a normal distribution for values of n SNPs located within m genes and significantly associated with virus diversity, we applied a re-sampling approach: samples of m genes were randomly extracted from a list of all genes covered by at least one SNP in the HGDP-CEPH panel  and for each sample the number of SNPs significantly associated with virus diversity was counted. The empirical probability of obtaining n SNPs was then calculated from the distribution of counts deriving from 10,000 random samples. A SNP was ascribed to a gene if it was located within the transcribed region or in the 500 upstream nucleotides.In order to estimate the probability of obtaining http://www.pantherdb.org/). Briefly, gene lists are compared to the reference list using the binomial test for each molecular function, biological process, or pathway term in PANTHER.Analysis of PANTHER over-represented functional categories and pathways was performed using the “Compare Classifications of Lists” tool available at the PANTHER classification system website http://www.r-project.org/).All calculation were performed in the R environment www.ingenuity.com). IPA builds networks by querying the Ingenuity Pathways Knowledge Base for interactions between the identified genes and all other gene objects stored in the knowledge base; it then generates networks with a maximum network size of 35 genes/proteins. We used all genes showing at least one significantly associated SNP as the input set; in this case a SNP was ascribed to a gene if it was located within the transcribed region or in the 25 kb upstream. All network edges are supported by at least one published reference or from canonical information stored in the Ingenuity Pathways Knowledge Base. To determine the probability of the analysed genes to be found together in a network from Ingenuity Pathways Knowledge Base due to random chance alone, IPA applies a Fisher's exact test. The network score represents the -log .Biological network analysis was performed with Ingenuity Pathways Analysis (IPA) software using an unsupervised analysis (Table S1SNPs in InnateDB genes that significantly correlate with virus diversity.(0.05 MB PDF)Click here for additional data file.Table S2SNPs in genes involved in glycan biosynthesis that significantly correlate with virus diversity.(0.03 MB DOC)Click here for additional data file.Table S3SNPs in genes coding for proteins interacting with viral products that significantly correlate with virus diversity.(0.05 MB PDF)Click here for additional data file.Table S4th among MAF-matched SNPs, as described in the main text and in material and methods. SNPs are ranked according to the value of Tau. If the SNP is located within a genic region (or in the 500 upstream nucleotides) the gene symbol is reported. Also, the gene closest to the SNP and its distance (in bp) are indicated. The aminoacid substitution is reported for nonsynonymous variants; SNPs annotated as “phastCons element” are located within non-coding genomic regions that display high sequence conservation among mammals (as described in the text).SNPs significantly associated with virus diversity. The table reports all SNPs that withstood Bonferroni correction at the genome-wide level (with α = 0.05) and displayed a Tau percentile rank higher than the 99(0.21 MB DOC)Click here for additional data file.Table S5p values were >0.05.Correlations between SNPs associated with virus diversity and other climatic variables. The table shows correlation coefficients between each SNP associated with virus diversity and the following climatic variables: average annual maximum temperature (Tmax), average annual minimum temperature (Tmin), short wave radiation flux (Irradiation SW). After Bonferroni correction all (0.42 MB DOC)Click here for additional data file.Table S6List of viruses identified in at least one country (n = 81)(0.01 MB DOC)Click here for additional data file.


SOX2 (SRY [sex determining region Y]) box 2) and OTX2 (orthodenticle homeobox 2), in individuals with developmental eye disease. We conclude that MLGA has the potential to be a useful technique in diagnostic research for the identification of deletions or duplications of known genes due to its speed and relatively low cost.Whole gene deletions or duplications are an important cause of genetic disease and phenotypic variation. Targeted techniques for the routine testing of gross rearrangements have become essential tools for diagnostic researchers with the search for the most cost-effective and efficient tool assuming high priority. We used the new selector technique, MLGA (multiplex ligation-dependent genome amplification), to confirm deletions in two genes,  SOX2 (SRY (sex determining region Y)-box 2), OTX2 (orthodenticle homeobox 2) and BMP4 (bone morphogenetic protein 4), genes involved in severe eye abnormalities  and SRY [sex determining region Y], respectively) to allow for experimental validation in the form of sex confirmation. The five positive cases with known deletions , SOX2OT (SOX2 overlapping transcript) (GenBank RNA accession number BC041898.2), and autosomal control gene, NPM1  (GenBank accession number NM_002520.5). Second, newly designed selector probe and fragment sequences were uploaded to the BLAT alignment tool [SOX2 (SOX2OT) was selected for interrogation due to the relative ease of optimization compared with the GC-rich SOX2 itself. In addition to the control probe, NPM1, mentioned above, selector probes for control genes, MADH4 (mothers against decapentaplegic homolog 4), SRY, and AR, were used as designed by Isaksson et al. [Selector probe design was performed as described by Isaksson et al.  with two browser : OTX2  with previously identified OTX2 deletions and a proportional reduction in the peak area of the SOX2 locus fragment in individuals (cases 4 and 5) with previously identified deletions of the entire SOX2 locus (OTX2 and SOX2 locus deletions). Each result was confirmed in a second assay.The ABI GeneMapper® traces demonstrated a proportional reduction in the peak area of the OTX2 and SOX2 deletions in developmental eye anomalies. For MLGA to become widely used in a diagnostic service setting, further detailed validation work is required to prove reproducibility in a large number of positive cases and controls. However, the low running costs along with the ease and speed of the assay would be particularly attractive to human genetic diagnostic laboratories requiring regular CNV screens of known disease loci. By amplifying genomic DNA rather than probe molecules, MLGA assays offer the potential to screen at least 50 loci in one assay by using several vectors and multiple labels on the universal primers. Although the routinely used technique, MLPA, is able to analyze this number of loci, MLGA has the potential to do this faster and more economically. Furthermore, MLGA provides great flexibility in the choice of target loci through the library of restriction enzymes available to create fragments for subsequent circularization and amplification.Our results demonstrate that MLGA can identify whole gene deletions in a human diagnostic research setting as exemplified by the diagnosis of human MLGA still requires further validation when multiplexing up to 40-50 loci for it to be deemed as useful as other multiplex targeted approaches (including MLPA) in CNV detection. Moreover, significantly multiplexed assays may require more stringent selector probe design criteria and a more thorough initial optimization of reaction conditions than MLPA since genomic DNA rather than designed probe sequences are amplified. Aside from repetitive elements, most of the genome is accessible to MLGA with only high GC content (>60%) known to affect probe success rates .One significant advantage that MLGA does possess but was not noted in the initial report is the capacity to provide data on insertion-deletion polymorphisms or mutations with a target sequence of interest. For example, a two base pair deletion within a target sequence will give a shifted peak on the GeneMapper® trace and thus may indicate an avenue for further investigation, such as sequencing. This potential may prove valuable to a researcher screening for mutations as well as deletions, providing a useful ‘pre-screen’ to detect intragenic deletions or insertions.


To the Editor: During April–May 1999, 3 Bartonella isolates  were cultivated and established from the blood of 5 Macropus giganteus gray kangaroos from central coastal Queensland, Australia. We used multigene sequencing to evaluate whether these Bartonella isolates fulfill the minimum requirements for classification as a new species.Bartonella isolate was extracted by using the QIAamp tissue kit  according to the manufacturer’s instructions. Partial PCR amplification and sequencing of the genes encoding the 16S rDNA (rrs), citrate synthase (gltA), β-subunit of the RNA polymerase (rpoB), and cell division protein (ftsZ), as well as for the 16S–23S rDNA intergenic spacer (ITS) were attempted by using previously described primers and conditions  from 5 Macropus giganteus gray kangaroos, Australia, 1999*