Mp/+ mice, which are heterozygous for the micropinna microphthalmia mutation, and identified several abnormalities, which implied that corneal epithelial maintenance was abnormal. The Mp/+ corneal epithelium was thin, loosely packed and contained goblet cells in older mice. Evidence also suggested that the barrier function was compromised. However, there was no major effect on corneal epithelial cell turnover and mosaic patterns of radial stripes indicated that radial cell movement was normal. Limbal blood vessels formed an abnormally wide limbal vasculature ring, K19-positive cells were distributed more widely than normal and K12 was weakly expressed in the peripheral cornea. This raises the possibilities that the limbal-corneal boundary was poorly defined or the limbus was wider than normal. BrdU label-retaining cell numbers and quantitative clonal analysis suggested that limbal epithelial stem cell numbers were not depleted and might be higher than normal. However, as corneal epithelial homeostasis was abnormal, it is possible that Mp/+ stem cell function was impaired. It has been shown recently that the Mp mutation involves a chromosome 18 inversion that disrupts the Fbn2 and Isoc1 genes and produces an abnormal, truncated fibrillin-2MP protein. This abnormal protein accumulates in the endoplasmic reticulum (ER) of cells that normally express Fbn2 and causes ER stress. It was also shown that Fbn2 is expressed in the corneal stroma but not the corneal epithelium, suggesting that the presence of truncated fibrillin-2MP protein in the corneal stroma disrupts corneal epithelial homeostasis in Mp/+ mice.We investigated the corneal morphology of adult  •Mp/+ mice have small, abnormal eyes.Heterozygous mutant •The corneal epithelium is thin, loosely packed and has goblet cells.•Corneal epithelial cell turnover and radial cell movement appear normal.•The cornea-limbal border is poorly defined and the limbus appears wider than normal.•Indirect tests suggest stem cells are not depleted and numbers might be increased. The maiPAX6+/− heterozygous individuals with aniridia, show progressive corneal deterioration with encroachment of conjunctival cells, including goblet cells, onto the cornea, and evidence suggests that this is at least partly caused by LESC deficiency  and Dstncorn1/corn1. LRIG1 gene expression was upregulated in holoclone-type corneal cultures (putative stem cells). They then showed that corneal homeostasis was impaired in Lrig1−/− knockout mice and, after multiple debridements, wound healing was delayed and incomplete. As LESCs are induced to proliferate in order to repair large wounds  mutation  and Isoc1 (isochorismatase domain containing-1) genes. The Mp inversion, produces an abnormal, truncated fibrillin-2Mp (Fbn2Mp) protein and this is thought to cause the abnormal Mp/Mp and Mp/+ ocular phenotypes, which are more severe than either Fbn2−/− homozygotes or Fbn2+/− heterozygotes, for which no ocular defects have been reported , reduced the secretion of other proteins and perturbed ER homeostasis. This would lead to ER stress and trigger the unfolded protein response, which can also cause cell death. The authors, therefore, proposed that this mechanism explained the worse-than-null phenotypes of both Mp/Mp and Mp/+ mice  and crossed to the inbred CBA/Ca strain. Mp/+ heterozygotes were distinguished from WT littermates by their small ears and eyes (illustrated in Pax6+/Sey-Neu (Pax6+/−) mice were maintained on a CBA/Ca genetic background and distinguished from Pax6+/+, wild-type (WT) littermates by PCR and eye size (Tg(Hmgcr-lacZ)H253Sest, X-linked nLacZ transgene (abbreviated to XLacZ), were maintained on a mixed C57BL/6 and CBA/Ca genetic background. Hemizygous males, hemizygous females and homozygous females are designated respectively XLacZTg/Y, XLacZTg/- and XLacZTg/Tg. Both Mp/+, XLacZTg/- and WT, XLacZTg/- X-inactivation mosaics were produced by Mp/+ female × XLacZTg/Y male crosses. Pax6+/−, XLacZTg/- mosaics were produced by equivalent crosses between Pax6+/− females and XLacZTg/Y males.Animal work was approved by the University of Edinburgh Ethical Review Committee and performed in accordance with UK Home Office regulations under project license number PPL 60/3635. Mice of both sexes were used except for the analysis of X-inactivation mosaics when only mutation  has now mutation  and is deye size . H253 steye size , ubiquit2.2For acute labelling with BrdU , 15-week old mice were given single intraperitoneal (i.p.) injections of BrdU  at 10:00 a.m. and killed by cervical dislocation following inhalation of gaseous anaesthetic 4, 28 or 52 h later (at 2:00 p.m.). Eyes were removed, fixed and processed for immunohistochemistry as described below. For BrdU pulse-chase identification of label-retaining cells (LRCs), Alzet mini-osmotic pumps , containing 0.1 ml BrdU solution  were surgically implanted under general anaesthetic at 15 or 30 weeks of age. BrdU was delivered continuously (0.5 μl/h) and the pumps were surgically removed after 7 days. Mice were killed 10 weeks later (i.e. when mice were 26 or 41 weeks old) and corneal buttons were processed for immunofluorescence as described below.2.3Eyes were fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) and some lenses were removed via a posterior incision to facilitate sectioning. Eyes were dehydrated in ethanol, embedded in paraffin wax and 7 μm longitudinal sections were cut and mounted on Polysine slides . The cut surface of the wax block was kept wet during sectioning of eyes with lenses to help preserve the lens morphology. Slides were de-waxed in Histo-Clear , rehydrated to 70% ethanol, washed in PBS, stained in haematoxylin, haematoxylin and eosin (H & E) or neutral red and eosin and mounted in DPX  under coverslips. For periodic acid-Schiff (PAS) staining, de-waxed sections were treated with 0.5% periodic acid (Sigma-Aldrich) for 15 min, washed in water, stained with Schiff’s reagent (Merck Millipore) for 5 min and washed in lukewarm water for 5 min. Slides were then counterstained with haematoxylin, dehydrated, cleared in xylene and mounted on glass slides with DPX and coverslips.For BrdU and keratin 12 (K12) immunohistochemistry, sections were dewaxed, rehydrated and incubated with 3% hydrogen peroxide for 20 min to quench endogenous peroxidase activity . To avoiTwo protocols were used for BrdU immunohistochemistry on sections. For BrdU protocol 1 , the pri2.4Mid-sections of eyes  were used for all the measurements and counts. Sections were photographed using a calibrated Zeiss Axiovision 4.8 digital camera system on a Zeiss Axioplan 2 microscope. The Zeiss measuring tool was used to measure the thickness of the whole cornea, corneal stroma (plus endothelium) and corneal epithelium at peripheral, intermediate and central corneal regions (two locations for each region) in sections close to the mid-section in one eye per mouse. The number of corneal epithelial cell layers was also counted. For estimates of basal cell packing density, the numbers of nuclei were counted in measured lengths of basal corneal epithelium in digital photographs using Adobe Photoshop CS6 and the results were expressed as the mean epithelial length per basal cell. All measurements and counts were made by a single investigator (NJD).Mp/+ mice without ciliary bodies). Each basal corneal epithelial cell was scored as positive or negative across the entire corneal diameter, which was retrospectively divided into two peripheral regions (P), two intermediate regions (I) and two central regions (C). The basal labelling index (LI) was calculated as the percentage BrdU positive cells in the basal layer for the whole diameter and separate P, I and C regions. The percentage BrdU-positive cells in the combined suprabasal layers  was scored in an equivalent way. To compensate for the thinner corneal epithelium in Mp/+ mice, an adjusted suprabasal LI was also calculated, by expressing the number of suprabasal BrdU-positive cells as a percentage of the total cell number in the underlying basal layer rather than the total suprabasal cell number. All counts were made by a single investigator (EJL).To analyse cell proliferation and loss in BrdU immunostained sections, the junction of the cornea and limbus was identified near the ciliary body . The MosaicJ plug-in (http://bigwww.epfl.ch/thevenaz/mosaicj) was used to construct composite images.Whole-mount corneal immunofluorescence was based on the method described by Confocal Z-stack images of whole-mount BrdU immunofluorescence were obtained for each sector and BrdU label-retaining cells were identified as BrdU-positive nuclei in maximum projection images of the Z-stacks. ImageJ was used to superimpose a calibrated 340 × 200 μm sampling box on the image across the width of the limbal region in each sector. The number of BrdU-positive nuclei within each sampling box was counted and the mean number of LRCs per sampling box (one sampling box per sector) was calculated for each cornea. To correct for differences in corneal diameters, the mean number of LRCs per sampling box was multiplied by the corneal circumference  and divided by the length of the sampling box (340 μm) to provide an estimate of the number of LRCs in a 200 μm wide ring around the whole limbal circumference. All counts of LRCs were made by a single investigator (PD).2.6XLacZTg/-, X-inactivation mosaic mice were lightly fixed in gluteraldehyde, stained with X-gal, post-fixed in 4% paraformaldehyde then either viewed as whole-mounts or embedded in wax, sectioned, counterstained with neutral red and eosin and mounted in DPX under coverslips . To compare patch sizes and evaluate cell mixing, the mean patch length was corrected for effects of different proportions of β-gal-positive cells by dividing it by the function 1/(1-p), where p is the proportion of β-gal-positive cells  was used for statistical tests. More than two groups were compared by 1-way analysis of variance (ANOVA), followed by Tukey’s post tests or 2-way ANOVA, followed by Bonferroni’s post tests. Data that were clearly not normally distributed were analysed by Kruskal-Wallis followed by Dunn’s post tests. Two groups were compared by Student’s 33.1Mp/+ mice, on a C57BL/6 genetic background had no ciliary body, no vitreous chamber, the anterior chamber was greatly reduced, the lens had cataracts, the retina was folded and contained rosettes but iris development appeared relatively normal. We confirmed that eyes of our adult Mp/+ mice on a CBA/Ca genetic background were small, had retinal abnormalities and no ciliary body and that the vitreous chamber was either absent or very small. However, the lens and iris were more significantly affected than previously described  but not Mp/+ eyes (P = 0.3693), indicating that Mp/+ corneas were smaller in diameter by 3 weeks and failed to grow normally between 3 and 10 weeks.Sections of WT, each age H. Separa3.3Mp/+ mice could indicate that the rate of corneal epithelial cell turnover is abnormal so we examined incorporation of BrdU by immunohistochemistry. Preliminary immunohistochemistry with a mouse monoclonal anti-BrdU and a biotinylated goat anti-mouse IgG secondary antibody (protocol 1) worked well with WT eyes, as reported previously . However, BrdU labelled cells were identified successfully in both WT and Mp/+ corneas by immunohistochemistry using a sheep anti-BrdU antibody (protocol 2), as shown in The thinner, more loosely packed corneal epithelium of eviously , but proMp/+ and WT corneas and then declined by 52 h  increased between 4 and 28 h in both  by 52 h A. There  by 52 h A or sepa by 52 h . At fourMp/+ than WT eyes at both 28 h and 52 h  but this difference was not significant by student’s t-test (P = 0.1712). Thus, despite some trends for a small increase in suprabasal labelling, there was no evidence that the Mp/+ genotype had a major effect on corneal epithelial cell proliferation or loss. Although, this does not exclude the possibility that cell turnover is increased marginally in Mp/+ corneas, a larger experiment would be required to investigate this possibility.Only basal cells divide and incorporate BrdU so suprabasal cells only become labelled when basal cells move to the suprabasal layers. This is irreversible and is the first step towards cell loss, so more rapid accumulation of BrdU-positive cells in the suprabasal layers in one group would suggest a higher rate of cell loss, unless the total number of corneal epithelial cells (and thus the thickness of the epithelium) changed progressively over time. By 28 h BrdU-positive cells appeared in the suprabasal layers of both genotypes. The suprabasal LI was slightly higher in and 52 h B but thee cornea C or for e cornea . At 28 h3.4XLacZTg/-, X-inactivation mosaics reflect this movement of corneal epithelial cells. Mp/+, XLacZTg/- mosaics corneas showed similar patterns to WT, XLacZTg/- eyes with radial stripes extending between the limbal region and central cornea  deficiency. First, we compared striped patterns quantitatively in Mp/+, XLacZTg/- and WT, XLacZTg/- X-inactivation mosaics, as described elsewhere . These were expected to be predominantly putative slow cycling or quiescent stem cells . LRCs were identified by confocal microscopy in each sector of flat mounted cornea buttons  around the whole limbal circumference. The mean number of LRCs remained greater in Mp/+ eyes when expressed as LRCs per limbal circumference for both age groups but this was only significant for those labelled at 15-weeks  and both genotypes showed strong K12 staining throughout the central corneal epithelium  reagent. Eyes from two mice at each of 5 ages  for each genotype were stained for PAS. No PAS-positive goblet cells were detected in the WT corneal epithelium at any age but some PAS-positive goblet cells were present in the peripheral corneal epithelium in sections of eyes from 3 of the Mp/+ mice (both 6-month eyes and one of the two 12-month eyes), as shown in To determine whether these isolated K12-negative cells in the peripheral corneal epithelium of 3.8Mp/+ ocular surface  and the blood vessel marker, CD31. Blood vessels are usually absent from the cornea and restricted to a characteristic limbal vascular ring in the limbal stroma , which h surface A-C exten surface D and thelar ring A-C. Thislar ring .44.1Mp mutation results in the accumulation of abnormal Fbn2Mp protein, which causes ER stress in Mp/+ cell types that normally express Fbn2  the blood vessels and K19-positive cells enter the peripheral cornea, (ii) the corneal-limbal boundary is poorly defined, perhaps with an abnormally differentiated zone that includes both K12-positive and K19-positive cells, or (iii) the Mp/+ limbus is wider than normal.The distributions of K12, K19 and blood vessels are consistent with the possibility that the 4.3Mp/+ and WT mosaic corneas, despite the large difference in eye size and corneal circumference. This implies that Mp/+ and WT corneal epithelia are maintained by a similar number of LESC clones, which are distributed more densely around the smaller Mp/+ limbal circumference. This might occur, for example, if similar numbers of LESCs are specified in Mp/+ and WT eyes early in development (before the corneas diverged in size) but clones of LESCs become more densely distributed around the circumference because the Mp/+ cornea stops growing. This could result in either more LESC clones per area of limbus (perhaps with fewer LESCs per clone) or the formation of a wider limbus, as discussed above, with a smaller circumference and a more normal concentration of LESC clones and LESCs per area of limbus. In either case, the number of LESC clones per mm of circumference would be greater than usual in Mp/+ eyes. Although adult stripe patterns in X-inactivation mosaics only provide indirect information about active stem cells, it is clear that these results provide no evidence for depletion of active LESCs in Mp/+ mice, even without taking into account the smaller Mp/+ limbal circumference.Quantitative studies of X-inactivation mosaic mice showed that the mean corrected stripe number per eye was similar in Mp/+ and WT mosaic corneas. This has been shown previously for WT mosaics  were used in rats, some Langerhans cells were included as LRCs  method was originally used in the ocular surface to help identify the limbus as the location of putative stem cells . Our obs as LRCs . Also, aMp/+ limbus were wider than normal, the 200 μm wide sampling box would probably include more limbal tissue when Mp/+ LRCs were counted. Thus, the higher counts for Mp/+ eyes need not imply a higher density of LRCs per area of limbus. However, as Mp/+ eyes have more LRCs per circumference (not just per mm of circumference), it is possible that the Mp/+ limbus is both wider and has a higher density of LRCs.Although not all LRCs are LESCs, they are normally present in the limbus but not the cornea . LRCs inMp/+ limbus, might suggest that the Mp/+ limbus contained a higher proportion of quiescent LESCs than WT. However, whether a greater proportion of quiescent LESCs results in more LRCs will depend on the balance of two opposite effects (a relative decrease in the number of cells that become labelled and an increase in the proportion of labelled cells that retain the label for 10 weeks). Evidence from X-inactivation mosaics suggests that the Mp/+ limbus contains more active LESC clones per mm of circumference and, overall, it seems likely that the total number of LESCs per mm of circumference is greater in Mp/+ than WT eyes. This is also consistent with the possibility, suggested earlier, that the limbus and LESCs occupy a wider region of the ocular surface in Mp/+ than WT eyes.Many types of adult stem cells are quiescent  and lineMp/+ eye does have more LESCs, they seem barely able to keep up with the demand for corneal epithelial cells because the corneal epithelium is thin and loosely packed even though the Mp/+ cornea is greatly reduced in diameter. However, we found no convincing evidence that corneal epithelial cell proliferation was reduced or cell loss was much greater in Mp/+ eyes. It remains possible, though, that corneal maintenance is compromised because the Mp/+ LESCs function poorly. Further investigations with markers, such as ABCB5, that identify LESCs and probably early TACs (loxP lineage tracing (Mp/+ limbus is abnormally wide and contains more LESCs than normal but they function poorly.If the rly TACs , or Cre- tracing  may help4.4Mp/+ mice have a novel combination of corneal phenotypes. The thin, loosely packed Mp/+ corneal epithelium and the presence of goblet cells imply that corneal epithelial maintenance is impaired. The distribution of K19-positive and K12-positive cells and limbal blood vessels suggest either that the limbal-corneal boundary is poorly defined or the limbus is wider than normal. Indirect analyses with label-retaining cells and mosaic patterns provided no evidence that Mp/+ LESC numbers were depleted but suggested that they may be more numerous than usual yet function poorly. Further investigations are required to characterise the Mp/+ limbus more fully, test the predictions about Mp/+ LESCs and clarify to what extent ER stress in the corneal and/or limbal stroma contributes to the observed abnormalities of corneal epithelial morphology and maintenance.Together, our investigations indicate that heterozygous None of the authors have any conflicts of interest.PD, NJD, RLM and EJL performed the experiments, PD, NJD, RLM, EJL and JDW analysed the data and prepared the figures, JDW and REH designed the experiment and supervised the work, JDW and PD wrote the first draft and all authors contributed to the preparation of the final manuscript.


CMR feature tracking (CMR-FT) is a method of assessing strain from routinely acquired steady- state free precession (SSFP) cine images similar to echocardiographic speckle tracking. However, its application to determine myocardial twist and untwist has never previously been explored. We sought to determine the feasibility of measuring twist and untwist from routine cine images in healthy volunteers at rest and during inotropic stimulation.Ten healthy volunteers had routine SSFP cine images acquired at rest and after inotropic stimulation (10 and 20 micrograms of dobutamine). The rotation of the basal and apical slices, both subendocardial and subepicardial, was measured and global LV twist θ was calculated as the difference between the counter-clockwise (positive) rotation at the apex (φapex) and clockwise rotation at the base (viewed from apex), θ = φapex- φbase. Peak twist and untwist-rate and the respective times to peak were calculated using MATLAB software for both the subepicardial and subendocardial layers and compared between rest and stress.The peak and time to peak myocardial twist and untwist-rate are displayed at the endocardial Table  and epicIt is feasible to derive myocardial twist and untwist and respective times to peak, both at rest and dobutamine stress using CMR-FT. Application of these new measures of deformation by CMR-FT should next be explored in disease states.German Centre for Cardiovascular Research (DZHK Partner Site Göttingen).


The ability to determine one's location is fundamental to spatial navigation. Here, it is shown that localization is theoretically possible without the use of external cues, and without knowledge of initial position or orientation. With only error-prone self-motion estimates as input, a fully disoriented agent can, in principle, determine its location in familiar spaces with 1-fold rotational symmetry. Surprisingly, localization does not require the sensing of any external cue, including the boundary. The combination of self-motion estimates and an internal map of the arena provide enough information for localization. This stands in conflict with the supposition that 2D arenas are analogous to open fields. Using a rodent error model, it is shown that the localization performance which can be achieved is enough to initiate and maintain stable firing patterns like those of grid cells, starting from full disorientation. Successful localization was achieved when the rotational asymmetry was due to the external boundary, an interior barrier or a void space within an arena. Optimal localization performance was found to depend on arena shape, arena size, local and global rotational asymmetry, and the structure of the path taken during localization. Since allothetic cues including visual and boundary contact cues were not present, localization necessarily relied on the fusion of idiothetic self-motion cues and memory of the boundary. Implications for spatial navigation mechanisms are discussed, including possible relationships with place field overdispersion and hippocampal reverse replay. Based on these results, experiments are suggested to identify if and where information fusion occurs in the mammalian spatial memory system. Spatial navigation is one of the most important functions of animal brains. Multiple regions and cell types encode the current location in mammalian brains, but the underlying interactions between sensory and memory information remain unclear. Recent experimental and theoretical evidence have been found to suggest that the presence of a boundary fundamentally alters the task of navigation. In this paper, evidence is provided that it is possible to determine the location inside any familiar arena with 1-fold rotational symmetry, while completely ignoring sensory cues from the outside world. Surprisingly, the results show that the mere knowledge of the boundary's existence is enough, without requiring direct physical contact. Localization is robust despite the presence of noise modelled from the rodent head direction system, and even inaccuracies in the navigation system's memory of the boundary or internal models of noise. In circular arenas, rotational asymmetry can arise from interior structures such as barriers or voids, also without contact information. This theoretical evidence highlights the need to distinguish arena-based navigation common to most experimental studies, from open field navigation. These findings also point to novel ways to study information fusion in mammalian brains. Accurate spatial navigation is crucial to animal survival. Localization is the process of determining current location, critical for many navigation behaviours. Starting from an unknown location and direction , the ability to localize is thought to depend on the detection of world-based  cues such as visual landmarks. In contrast, it is thought that animal-based (‘idiothetic’) cues which provide self-motion estimates, e.g., from vestibular, proprioceptive or motor command signals, can only serve to maintain localization briefly, requiring allothetic cues for error correction In the rodent brain, the firing of both place and grid cells strongly correlate with the animal's physical location in a familiar space Most experiments investigating neural correlates of spatial behaviour have been performed in either linear tracks or 2D arenas, the latter termed ‘open fields’ Contrary to the above supposition, it is demonstrated here that long-term accurate localization is possible if idiothetic self-motion cues are combined with boundary information already in memory. In particular, the previously acquired boundary map limits the growth of uncertainty due to noisy self-motion information. Surprisingly, the act of sensing the boundary or any other landmark is not necessary. Metrics based on information theoretic principles are used to quantify localization performance. Grid cell simulations are used to provide both a visual display of time-averaged localization performance, and to predict the optimal spatial selectivity that may be expected of neural firing patterns, based on published rodent neural data.As a baseline, both iPI and aPI  performance were quantified without arena memory, in a kite-shaped arena. Even for the simplified task of PI initially orientated, localization failed . The medPI alone , consist minutes . The mor minutes  further θ, was centred on the true direction. Not surprisingly, initial orientation improved position estimation but its effect on pI was no longer detectable at 96 minutes . Likewise, V(θ) remained consistently higher when initially disoriented (−4). Lastly, 90% of changes in tic cues  demonstrTo determine whether the idiothetic localization described above could sustain spatially selective firing patterns similar to those of grid cells, a stochastic spiking model of grid cells was used pI and V(θ) of Distinct grids were seen both when the animal was initially oriented , row 1 apI distributions , some are close to the true pose while others are not , left. An-fold rotational symmetry (n-RS), there are n poses which are geometrically equivalent and consistent with the boundary map, making idiothetic localization to a unique pose impossible. Therefore the combination of boundary asymmetry and an internal model of boundary crossing sufficed for idiothetic localization.Over time, estimates cluster around the true pose, plus any rotationally symmetric poses. The latter occurs because in arenas with pI value differently in arenas with the same degree of rotational symmetry, since within each of n sectors of an arena with n-RS, localization should be possible with performance depending on the sector's asymmetry. An adjusted p*I for n-RS arenas was found to be positively correlated to the average rotational asymmetry  allowed idiothetic localization when an internal barrier was present. However, localization failed when estimated movements were represented as discrete steps which ignored barrier crossings  trajectories . AccuracX, Y or both X & Y directions, by 10%  can account for a sequence of displacements, uncertainty tends to decrease. For example, a path taken from the acute to obtuse to a right angle corner of a kite arena cannot be accounted for by any particle trajectory except one which is close to the true trajectory. In contrast, there are infinite possible paths which span the diameter of a circular arena or follow its boundary, so there are always a range of particle trajectories which can account for any displacement sequence arising in a circular arena. Consequently, determining a unique location is impossible. In a given 1-RS arena, the path structure determines the relative uniqueness of the displacement sequences. Hence very few thigmotactic loops around a 1-RS arena e.g.,  are requThe magnitudes of the competing rates of increase and decrease in uncertainty also depend on the initial pose distribution e.g., , S2. ForBy itself, rotational asymmetry is not sufficient for long term localization. However, an arena map is not always necessary if allothetic cues are available. When detected, the acute and obtuse corners of a kite arena are unique landmarks which can theoretically be used by a modular navigation system for localization It was shown previously that combining self-motion cues and arena memory significantly slowed the decay of pose estimates in a circular arena, when initially oriented. When allothetic boundary contact cues were included, the residual localization achieved could account for the prolonged stability of rodent place and grid fields observed in darkness despite an unstable head direction system Using only idiothetic cues in conjunction with information already in memory potentially reduces computational load during navigation. Such a strategy allows a navigating agent to devote computational or attentional resources for processing allothetic sensory information for other tasks. Although precision is reduced, it is likely to be a low-risk strategy since occasional use of allothetic cues suffices to recover near-optimal localization e.g., , Fig. S4The rodent medial entorhinal cortex (mEC) contains both border cells and grid cells Boundaries occurring in natural environments rarely have >1-fold rotational symmetry, making it plausible that biological navigation systems may exploit this property for localization. It remains to be tested whether species which spend significant time in enclosed spaces It is worth noting that the localizing mechanism described here need not be restricted to bounded spaces with impassable physical barriers. For instance, this strategy is equally applicable if an animal's trajectory is limited by a few distinct landmarks, forming a virtual arena with 1-fold rotational symmetry in an otherwise open trajectory space. Familiar landmarks could thus be used to break rotational symmetry in the trajectory space without a physical barrier, and resulting in successful localization without knowledge of the distance or allocentric direction to those landmarks during a journey. However, the geometry of the virtual boundary must be known. In this hypothetical example, the trajectory is guided by allothetic cues, while localization uses idiothetic cues and information already in memory.One prediction of idiothetic localization was that reverse replay of past information enabled retrospective improvement in localization . That isWhile the present study examined the information and computations which may be necessary and sufficient to be used in conjunction with a known boundary map for localization, future studies will need to address the acquisition of the boundary map itself. In robotics, particle filter methods have been used successfully to build boundary maps using only self-motion and boundary detection cues, starting from full disorientation – a Simultaneous Localization and Mapping (SLAM) problem in vivo recordings are feasible, it may be possible to isolate the cells and circuits where the fusion of idiothetic self-motion and boundary memory information occurs. For instance, if stable mEC grid fields emerge from full disorientation under the cue-restricted conditions described, information fusion must occur either at or upstream of the medial entorhinal grid cells.From the findings in this study, it is proposed that spatial memory systems which can effectively combine idiothetic self-motion cues and boundary memory can determine location in familiar arenas with 1-fold rotational symmetry. If allothetic cues are stringently removed, localization necessarily demonstrates the fusion of idiothetic self-motion and memory-based boundary information. This prediction may be tested, for example, by using blindfolded human subjects passively led along random or thigmotactic trajectories. Where Together, the reported results reveal that detection of cues from the external world is not always necessary for localization, that bounded arenas are distinct from true open fields It is known from recordings of Head Direction (HD) cells in rats, that error in the estimate of head direction increase steadily as the animal moves away from the place where it was first deprived of its vision 3 random trials in each condition, using metrics developed previously to characterise instantaneous spatial accuracy and precision. Briefly, the median place stability index, A particle filter was used to approximate Bayes-optimal fusion of idiothetic self-motion and boundary information, to provide the best estimate of successive poses given noisy displacement inputs. The localisation performance, as revealed by this particle filter approach, was analysed across 10The particle filter estimate of position was then used to simulate the firing activity of grid cells in medial entorhinal cortex, using a stochastic spiking model where spike probability decreased with estimated distance from the neurons' preferred firing locations Figure S1Matched and mismatched uncertainty. Effects of matched and mismatched angular and linear uncertainty on idiothetic localization. V(θ) functions (left), p(48)I distribution (middle), simulated grid cell spikes (top right) and firing field autocorrelograms (bottom right) during 46–48 minute period in kite-shaped arenas. A, Matched angular uncertainty using 0.25× (light blue), 0.5× (dark blue), 1× (black), 2× (dark red), and 4× (light red) the standard B, Matched linear uncertainty using 0.25× (light blue), 0.5× (dark blue), 1× (black), 2× (dark red), and 4× (light red) the standard C, Mismatched angular uncertainty using 0.25× (light green), 0.5× (dark green), 1× (black), 2× (dark purple), and 4× (light purple) the standard D, Mismatched linear uncertainty using 0.25× (light green), 0.5× (dark green), 1× (black), 2× (dark purple), and 4× (light purple) the standard E, Boxplots of p(48)I showing the effects of matched and mismatched angular and linear uncertainty on idiothetic localization performance (outliers not shown). Red bars indicate comparisons of (TIF)Click here for additional data file.Figure S2Intermittent boundary contact.A, pI (median and IQR) and V(θ) functions without vision in a kite-shaped arena, switching from idiothetic cues only (orange bars), to idiothetic cues plus boundary contact (yellow bars) in 8 minute blocks, initially oriented (green) and disoriented (black). Boxplots compare the residual effects of orientation versus disorientation, and intermittent (+) versus no (−) wall contact information on B, Simulated grid cell spikes and firing field autocorrelograms from A, initially oriented (top row) and initially disoriented (bottom row), with (right) and without (left) boundary contact. Consistent with the A, grids showed higher spatial specificity using boundary contacts while initial orientation showed residual effects beyond 30 minutes.(TIF)Click here for additional data file.Figure S3Large or discrepant arenas.A, V(θ) functions (left) and p(48)I distribution (right) using a kite arena 4-fold in area (red) compared to a standard kite arena (black). B, Quadrupling the area of the kite arena resulted in no distinguishable grid modes at 46–48 minutes (first column) when the standard 30 cm grid spacing was used in spike simulation. Doubling the grid spacing  recovered grid modes, which were less distinct than in the standard kite arena (third column). The crosscorrelogram (fourth column) between the normalized firing field of the standard and the 4×-arena (double grid spacing) yielded a gridness index of 0.39. Scale = 50 cm.(TIF)Click here for additional data file.Figure S4Idiothetic localization combined with an intermittent noisy compass in circular arenas.A, pI (median and IQR) and V(θ) using a compass stochastically, averaging once every 30 s with Gaussian measurement error p(48)I distributions were similar (right), as were kinetic parameters . Although absolute differences were small, median pI was significantly higher  and V(θ) was significantly lower  following 48 minutes in the 152 cm arena. B, Simulated grid spikes (top row) and autocorrelograms (bottom row) using 30 cm grids (left and middle), and 60 cm grids (right), in 76 cm (left) and 152 cm (middle and right) diameter circular arenas.(TIF)Click here for additional data file.Figure S5Improved retrospective localization using either offline reverse replay (beta recursions) or online backward inference (gamma recursions). (A) Uncertainty (blue particle cloud) during real-time localization (row 1) and during offline reverse replay (row 2), showing the estimated pose (cyan arrow) and the true pose (red arrow) during a two-minute period initially disoriented. Row 3 shows the same path, using online backward inference to estimate the initial pose (time 0). The particle size shown is proportional to the number of parent particles with identical poses. (B) pI  and V(θ) are shown during real-time localization (black), during offline reverse replay (red), and during online backward inference (green). The latter shows the online update in estimate of the initial pose. The right panel shows the corresponding pI distributions at time 0 following the completion of the two retrospective localization strategies.(TIF)Click here for additional data file.Table S1Spatial firing properties of simulated grid cells. Information content (bits/spike) of the top three grid modes by spike count, and gridness indices of simulated grids.(DOCX)Click here for additional data file.Text S1Modelling and Analysis, Supporting Results, and Supporting References.(PDF)Click here for additional data file.Video S1Idiothetic localization in a kite-shaped arena during 0 to 8 minutes, initially oriented. The top panel shows the uncertainty (blue particle cloud) during a random trajectory, comparing the true pose (red dot) and estimated pose (cyan dot) in 3D pose space . The vertical axis (θ) ranges from 0 to 360°. The lower left panel shows the positional distribution (blue particle cloud), with direction indicated by the red (true) and cyan (estimated) pointers. The lower right panel shows the direction distribution of the particle cloud (blue polar histogram), overlaid on the true direction (red pointer).(MP4)Click here for additional data file.Video S2Idiothetic localization in a kite-shaped arena during 0 to 8 minutes, initially disoriented. Otherwise as per (MP4)Click here for additional data file.Video S3Idiothetic localization in an egg-shaped arena during 0 to 8 minutes, initially disoriented, with an elliptic body perimeter. The top panel shows the uncertainty (blue particle cloud) during a thigmotactic trajectory, comparing the true pose centre (red dot) and estimated pose centre (cyan dot) in 3D pose space . The vertical axis (θ) ranges from 0 to 360°. The lower left panel shows the positional distribution (blue particle cloud), with direction indicated by the red (true) and cyan (estimated) ellipses. The lower right panel shows the direction distribution of the particle cloud (blue polar histogram), overlaid on the true direction (red ellipse with white arrowhead).(MP4)Click here for additional data file.


Neither intravenous nor intracardiac administration of mesenchymal stromal cells derived from either rats or humans improved MRI measures of lesion volume or neurobehavioral outcome compared to saline treatment. Few mesenchymal stromal cells (<0.0005% of injected dose) were found within 3 days of last dosage at the site of injury after either delivery route, with no mesenchymal stromal cells being detectable in brain at 30 or 56 days post-injury. These findings suggest that non-autologous mesenchymal stromal cells therapy via intravenous or intracardiac administration is not a promising treatment after focal contusion traumatic brain injury in this female rodent model.Mesenchymal stromal cells secrete a variety of anti-inflammatory factors and may provide a regenerative medicine option for the treatment of traumatic brain injury. The present study investigates the efficacy of multiple intravenous or intracardiac administrations of rat mesenchymal stromal cells or human mesenchymal stromal cells in female rats after controlled cortical impact by  In the U.S. in 2010, 2.5 million people had traumatic brain injury (TBI) of enough severity to seek care at a hospital .Mesenchymal stromal cells (MSC), also known as mesenchymal stem cells, have been proposed as a possible therapy for a variety of neurological disorders, including TBI . Known tin vivo and ex vivo MRI tracking of labeled stem cells over a time course after the injury.The purpose of the present study was to determine the optimal route of peripheral vascular delivery and whether multiple doses can maximize transplanted cell survival and functional recovery in a rat TBI model of focal contusion. We also investigated whether administration of MSC labeled with superparamagnetic iron oxide nanoparticles (SPION) would permit Guide for the Care and Use of Laboratory Animals. The protocol was approved by the National Institutes of Health Clinical Center Animal Care and Use Committee (protocol numbers LDRR 09–03 and LDRR 12–02). All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize suffering through the administration of acetaminophen post-injury as detailed in the section describing the injury protocol below.All studies were performed according to the National Research Council’s ad libitum.Female Wistar rats (n = 177) of initial age 8–12 weeks and weight of 200–250 g were obtained from Charles River Laboratories . Distribution of female rats for long-term neurobehavioral and MRI studies was as follows: 34 rats for rat MSC (rMSC) versus saline intravenous (IV) cohort; 24 rats for rMSC versus saline intracardiac (IC) cohort; 24 rats for human MSC (hMSC) versus saline IV cohort; 28 rats for hMSC versus saline IC cohort; 20 rats for uninjured neurobehavioral controls; 7 rats for uninjured MRI controls. For short-term experiments, 40 additional rats were used to determine if transplanted stem cells were detectable by histology in injured brain within the first 10 days after injury. For rat bone marrow MSC isolation, male Wistar rats (n = 20) of initial age 6–12 weeks and weight of 200–300 g were obtained from Charles River Laboratories . Rats were pair-housed in temperature-controlled conditions under 12 hour light/dark cycles, and had access to standard laboratory rodent chow and water NeuroLab.com/Leica Microsystems, Richmond, IL) with a 5 mm flat impactor tip over the left motor cortex  at a velocity of 5 m/s, depth of 2.0 mm, and dwell time of 100 msec. For each cohort of rats, the same surgeon performed all CCI surgeries. After injury, animals were observed continuously until they recovered from anesthesia and were able to move freely about their recovery cages. Rats were subsequently observed a minimum of two times a day, and more frequently on days when MRI, neurobehavioral testing, or administration of stem cell versus saline doses occurred. For post-injury analgesia, acetaminophen (100 mg/kg) was administered twice daily by mouth for the first 48 hours after injury.The rat CCI model was performed as previously described  using anRat MSC were isolated aseptically from bone marrow derived from the femurs of 6 to 10 week old male Wistar rats and characterized by flow cytometry analysis for selected surface markers per protocol as previously described . rMSC ceHuman MSC were derived and characterized by the NIH Bone Marrow Stromal Cell Transplantation Center , 13 and For IV treatments, animals were anesthetized with isoflurane and temporary tail vein catheters were placed for ease of administration. To trigger vasodilation to reduce stem cell trapping within the lung vasculature, all animals in the IV treatment cohort  were treated with nitroprusside at a dose of 25 μg from a 1 mg/mL solution followed by 100 μL saline  5 minuteFor the IC cohorts, after anesthesia with isoflurane rats received an analgesic dose of buprenorphine (0.03 mg/kg subcutaneously) prior to preparation of the left chest wall and administration of local anesthesia with bupivacaine 0.5% . IC injections were performed under ultrasound guidance with an Acuson Sequoia (8–15 MHz) ultrasound unit . One million MSC in a volume of 200 μL sterile PBS or 200 μL sterile PBS alone were slowly administered into the left ventricle of the heart over a one minute period of time. MRI was not possible immediately post CCI in the IC cohort of rats because the initial injections were completed within 15 minutes of injury.In vivo MRI of the brain was performed on days 2, 9, and 30 post-CCI on a 7 T Bruker Biospec  using a 40 mm inner diameter quadrature volume coil  as previously described [In vivo MRI of healthy control age-matched female rats (n = 7) was used to determine the variability in brain volumes. Ex vivo MRI was performed on paraformaldehyde-fixed brains on a 7 T vertical bore magnet  as previously described [escribed .The CCI injuries for these studies were centered over the left frontal motor cortex. Consequently, neurobehavioral tests that primarily assessed motor and reflex functions were utilized for outcome assessments. Rats in the rMSC IV versus saline cohorts underwent neurobehavioral severity score-revised (NSS-R) evaluation at baseline and on days 1, 14, 28, and 56 after injury as previously described . RotarodMotor coordination and limb function were tested through foot fault testing on a grid , 22 modiAfter euthanasia with pentobarbital, rats were perfused with ice-cold heparinized PBS, then 4% paraformaldehyde prior to brain extraction. Following equilibration in a sucrose gradient, brains were transferred to optimum cutting temperature (OCT) media and frozen in liquid nitrogen. Cryosections (10μm thick) were cut in the coronal plane, then mounted on glass slides and stored at -20°C. Representative sections underwent hematoxylin and eosin (H&E) staining using standard methods or Prussian blue staining as previously described .Cytospin samples of cells prepared for injection and representative brain tissue sections from animals who received IC or IV MSC versus saline were stained using immunofluorescent staining techniques as previously described , 18. ForCortical and lesion volumes were determined with MEDx software  as previously described . Lesion www.graphpad.com), with a two-tailed p value of <0.05 considered significant. Nonparametric data (NSS-R) were analyzed by Kruskal-Wallis test for multiple comparisons in Prism, with a two-tailed p value of <0.05 considered significant. MRI data were reported as mean ± standard deviation (SD), while neurobehavioral data are reported as mean ± standard error of the mean (SEM), per conventions already established for data reporting in the imaging and neurobehavioral fields, respectively.For parametric data, one-way analysis of variance (ANOVA) and subsequent post-hoc Tukey was performed using Prism version 6.0d for Macintosh . Separate cohorts of animals were given IV or IC injections of rMSC or hMSC with the dosing regimens reported in in vivo MRI tracking of SPION-labeled stem cells; and 4) rat or human MSC were not detectable in CCI-injured brains at 2 months post-injury when administered by IV or IC routes.The major findings of this study were as follows: 1) administration of three IV doses of either rat or human MSC on days 3, 5, and 7 after CCI does not improve MRI or neurobehavioral outcomes over a 1–2 month time course in the female rat; 2) administration of two IC doses of either rat or human MSC on days 0 and 1 after CCI fails to improve outcomes; 3) the evolution of hemorrhage in lesion and perilesion areas between days 2 and 9 post-CCI complicates interpretation of in vivo MRI and continued to a 2 month time course post injury for neurobehavioral and ex vivo analysis. Our initial experimental design was based upon the published literature [In these investigations, the effects of IV MSC delivered on days 3, 5, and 7 post-TBI were examined over a 1 month time course for terature –8 that hterature , 24, witin vivo and ex vivo MRI [In our studies, we used MSC labeled with SPION for MRI tracking purposes. In the IV cohorts, initial MRI scans were performed on Day 2 post-injury, prior to the administration of stem cells on days 3, 5, and 7. The first follow up scans were done on Day 9. We found in a parallel study [vivo MRI –27. PostNo long-term differences between control rats and treatment groups  were observed by NSS-R or rotarod testing in the IV cohorts . Therefo6 cells [The failure of IV or IC MSC to influence neurobehavioral outcomes in the present study is in distinct contrast to several earlier studies investigating the efficacy of MSC after focal contusion injury in the rat –8, 28–326 cells , 32.6 rMSC at 24 hours after stroke injury by tail vein, yet found that only 0.0005% of the cells (fluorescently tagged prior to injection) reached the brain in a rat TBI model. The vast majority of cells were trapped in the lungs [In contrast, Harting et al  injectedhe lungs , 48. By he lungs . Investihe lungs , 50 and he lungs . This fahe lungs .MSC have also been directly administered into the brain in several studies using a different model of TBI, a weight drop model. When MSC were transplanted intracerebrally at 2 months after injury in adult male Wistar rats, treated animals with moderate but not severe TBI demonstrated functional improvement by 4 months post-injury , 41. In A major advantage of our study is that we had larger numbers of animals per cohort than in prior investigations of the efficacy of MSC after TBI. Our MRI studies had cohorts of 12 to 17 rats per arm, consistent with a sample size that would result in a power of 0.8 and alpha of 0.05 , compareThe present study aimed to investigate whether multiple intravascular doses of MSC in the subacute phase after injury would improve outcomes by MRI and neurobehavioral outcomes. Similar doses were used as in the original single dose MSC studies –8 Table, yet no Differences in techniques may contribute to the variation in efficacy of MSC. In the original TBI studies with MSC, the investigators did bilateral decompressive craniectomies prior to TBI, but only performed CCI over the left motor cortex –8, 29, wWhether the contradictory reports of transplanted cells reaching the brain and surviving is due to labeling techniques used, differences in mechanism of injury, timing of delivery, or other factors remains unclear. The early stem cell studies may also have overestimated the migration of stem cells into the perilesion area. Assessment of cell localization in the initial studies in male rats occurred via BrdU staining , 8, whicIn addition, confirmation with fluorescent labels or antibody staining can be complicated by the presence of autofluorescence of cells post TBI. We observed autofluorescence in the hemorrhagic TBI lesions in our prior studies  and thisAnother factor that may introduce variability among studies is biological sex, of both MSC donors and of recipients. The majority of stem cell transplantation experiments have occurred in male rats, with only a small subset of studies done in female rats . Half ofIn contrast to the initial reports which found that IV administration of MSC from male donor rats were efficacious in both males  and femaWhile data are conflicting as to the efficacy of peripherally administered MSC, treatment with multipotent adult progenitor cells (MAPC) cells demonstrates more promise in preclinical studies , 65, 66.The AMNC approach may be clinically preferable, as it permits a) transfusion of the patient’s own cells after enrichment in a b) timeframe that is clinically relevant (2–3 days after injury), with minimal time for isolation and reinjection (2 hours) , 67. CliIn addition to the points discussed above, there are other limitations to the current study that employed two different methods of MSC delivery at multiple time points. The IC studies delivered MSC in the acute time frame (right after injury and on Day 1), at the height of injury, while the IV studies administered MSC at three sub-acute time points . MSC administered acutely or subacutely did not persist as long-term transplants. Changes in the inflammatory milieu of the injured brain within the first week after injury  may contThe experiments described here were originally designed to test the hypothesis that multiple doses of bone marrow derived mesenchymal stromal cells would be more efficacious than a single early dose. We chose a peripheral vascular approach because we were trying to find a method of administration that would be applicable to a broader clinical population than direct intracerebral injection. We based our initial experimental design upon the early successful reports of stem cell administration, where cells were given via multiple different routes  at a variety of time points from 24 hours to 7 days in either male or female rats, all of which were reported to be successful, at least by neurobehavioral outcomes . We usedIn conclusion, the present study fails to replicate the early reports of cell homing and improved neurobehavioral outcomes after peripheral vascular administration of MSC. No difference in MRI outcome measures or neurobehavioral outcomes was observed among the various treatment groups. We conclude that non-autologous MSC infusions via peripheral vascular routes of administration are not a promising avenue for stem cell therapy after TBI in all populations. Initial studies by other laboratories , 67–69 s


These results also raise concern that the use of AIs in women with breast cancer might enhance lymphoma progression.Most lymphomas show higher incidence and poorer prognosis in males compared to females. However, the endocrine contribution to this gender difference is not entirely known. Here we show that castration accelerates lymphoma growth in C57BL6 male mice grafted with murine EG7 T cell lymphoma cells. However, the androgen receptor antagonist Bicalutamide did not affect lymphoma growth, suggesting no impact of androgen receptor signaling on lymphoma progression. In contrast, inhibition of androgen-to-estrogen conversion by the aromatase inhibitor (AI) Letrozole induced faster lymphoma growth in mice, suggesting that androgens impact lymphoma growth through its conversion to estrogens. This was supported by the inability of dihydrotestosterone, which is not converted to estrogens by aromatase, to influence lymphoma growth in castrated male mice. Lymphoma growth was also stimulated in immunocompromised mice grafted with human B cell lymphoma (Granta-519) and treated with either reversible or irreversible AIs, showing that the blockage of estrogen synthesis caused enhanced growth of both murine T and human B cell lymphomas and with different AIs. Additionally, AI-treated EG7 lymphomas showed accelerated growth not only in male but also in intact female mice. Altogether, our results demonstrate that aromatase inhibition accelerates lymphoma growth but not androgens  Most lymphomas, particularly Non-Hodgkin lymphomas (NHL), show a higher incidence and poorer prognosis in males than in females , 2. As aEstrogen action in their target tissue is largely mediated through two nuclear receptors, namely estrogen receptor α (ERα) and ERβ , 11. TheThe major estrogen, estradiol, binds to both ERα and ERβ with similar affinity and is able to act as a potent agonist on both receptors . ERα maiAlthough the above studies show a role for estrogens on NHL progression, the role of male sex hormones (androgens) on lymphoma development is uncertain. This is despite the demonstration that androgen receptor (AR) is expressed in at least some lymphoma cells . FurtherIn this study we investigated the effect of androgens on lymphoma growth and their mechanism of action. Since androgens can be converted to estrogens by aromatase, we particularly studied whether androgens had a direct effect on lymphoma growth or if it was mediated through conversion to estrogens.Previous results have shown that grafted EG7 lymphoma grows faster in male vs. female mice. This difference was attributed to female sex hormones as it was abolished following ovariectomy . FurtherAlthough castration accelerated tumor growth, it is not possible to conclude that the effect is mediated by loss of androgens or is due to loss of estrogens as estrogens are formed following conversion of androgens to estrogens by the enzyme aromatase . To inveTo further confirm that androgen-to-estrogen conversion was the cause affecting lymphoma growth, surgically castrated male C57BL6 mice grafted with EG7 lymphoma cells were treated with dihydrotestosterone (DHT), which like testosterone is an AR agonist but in contrast to testosterone cannot be converted to estrogens by aromatase . As showTo demonstrate that aromatase inhibition indeed stimulates lymphoma growth and that it is not specific for Letrozole, or that the effect on lymphoma growth is not restricted to the murine T cell lymphoma EG7 but also affects human B cell lymphoma and, finally, is not dependent on an intact immune system, we grafted immune deficient NSG mice with human lymphoma B cells (Granta-519). Aromatase inhibition by Letrozole or by other AIs, Anastrazole and Exemestane, the latter being a steroidal irreversible inhibitor , enhanceAromatase inhibition is widely used as adjuvant therapy in female patients with hormone-sensitive breast cancer . We thervs. females as seen in several lymphomas. We show that removal of sex hormones by surgical castration in male mice accelerates lymphoma growth. This could be explained by both a higher number of proliferating Ki67-positive cells and a lower number of apoptotic tumor cells, suggesting that the lack of sex hormones stimulates tumor promotion. Previously it has been shown that lymphoma grafts grow faster in male vs. female mice and that the difference disappears following OVX, suggesting an inhibitory role of estrogens on lymphoma progression [In this paper, we studied the underlying endocrine mechanisms that could explain a higher incidence and poorer prognosis in males gression . This cogression , 16, 26.gression , 34.Surgical castration of male mice is a valuable and simple way to study the effect of the absence of sex hormones. However, in this case both male and female sex hormones are removed as estrogens are synthesized by conversion of androgens to estrogens by the enzyme aromatase in both males and females. Thus, male castration cannot distinguish whether androgens have a direct effect on lymphoma growth or whether an androgen effect is mediated indirectly through androgen conversion into estrogens.per se that inhibit lymphoma progression. This conclusion was substantiated by very low AR expression observed in the EG7 lymphoma studied  and a human B cell lymphoma (Granta-519) suggesting that aromatase inhibition promotes lymphoma growth of both human and murine lymphomas as well as both T and B cell lymphomas, thus proposing a more general effect on lymphomas. The results also emphasize a general role of estrogens in inhibiting lymphoma pathogenesis and progression. Furthermore, a role of the immune system could be excluded because similar results were obtained in immune deficient NSG mice and in normal C57BL6 mice. Since AIs are used in the clinic as adjuvant therapy for female patients with hormone sensitive breast cancer , we alsoThe conversion of androgens to estrogens in the mice most likely occurred in peripheral tissues known to express aromatase and not in the lymphoma tissue as analysis of aromatase expression was not detected in the EG7 tumors (data not shown). This is in contrast to some other endocrine-related malignancies e.g. breast cancer, which have been shown to express aromatase in the tumor tissue . Furtherthe lack of estrogens can only contribute to lymphoma progression but not to lymphoma initiation, thus making the number of relevant breast cancer cases too few. Epidemiological studies of lymphoma incidence in breast cancer patients is also complicated by the fact that females with breast cancers do show an increased risk for secondary cancers including lymphomas compared to aged-mapped healthy woman [Our results raise a concern that treatment with AIs may increase the risk for lymphomas or at least accelerate lymphoma growth in cases were lymphoma initiation already has occurred. However, so far, according to our knowledge, no reports are available showing higher lymphoma incidence in AI-treated breast cancer patients. This may be explained by that AIs are relatively young drugs (introduced into the clinic in the beginning of the century) with so far to short follow up time from their start of use (less than 10 years) for such a side effect to be detected. Considering that lymphomas only constitute 4-5% of all cancers together with the limited follow up-period, very large breast cancer patient cohorts would be needed. An additional possibility is that hy woman . In thisTaken together, our results demonstrate that blocking estrogen synthesis via inhibition of the aromatase pathways and not androgen signaling causes accelerated lymphoma growth. These results highlight a protective role of estrogens in lymphoma pathogenesis and progression. As a consequence of this, physiological or therapeutically induced reduction of estrogen synthesis could be considered as a lymphoma risk.2.The murine T cell lymphoma cells (EG7)  and humascid Il2rgPrkdctm1Wjl/SzJ) mice (referred to as NSG mice) were initially from The Jackson Laboratory  and bred at the Animal Facility of Karolinska Institutet . All animal experiments were carried out according to guidelines of the Karolinska Institutet and all the study protocols were approved by the Local Animal Experimentation Committee. Mice were kept in specific pathogen free conditions in 12 hours light-dark cycles and on soy-free diet during the experimental period.C57BL6 male and female mice (6-8 weeks of age) were purchased from Charles River. Non-obese diabetic severe combined immunodeficiency NOD/SCID IL2γ-null , 1.5 μg Domitor (Orion Corporation) and 0.05 μg Fentanyl (Janssen-Cilag) in 100 μl of physiological NaCl solution per mouse. Castration , ovariec6 EG7 cells per C57BL6 mouse or with 15×106 Granta-519 cells per NGS mouse in 100 μl sterile PBS. After palpable tumors developed, treatments with Letrozole  (Sigma), Bicalutamide (100 mg/kg) (Sigma) Anastrazole  (Sigma) or Exemestane  (Sigma) given subcutaneously once a day was initiated. All the drugs were dissolved in 100% ethanol and further diluted in rapeseed oil as a vehicle. Tumor size were monitored every day with a caliper and tumor volume (TV) was calculated according to the following formula: TV (mm3) = 0.5 × length (mm) × width2 (mm2).Mice were injected subcutaneously in the right flank with 0.5×10imagej.nih.gov/ij/) and a cell counter plugin.Tumor tissue was fixed overnight in cold 4% PFA in PBS, the following day washed with 50% ethanol and saved in 70% ethanol until sectioning and paraffin embedding. 5 μm thick slides were cut from the tumor tissue and mounted on glass slides. Antigen retrieval was done using Antigen unmasking solution (Vector Laboratories). The sections were permeabilized using 0.1% Triton X-100 in PBS after which they were incubated with primary Ki67 antibody , followed by incubation with secondary anti-rabbit Cy3 antibody (Jackson Immunoresearch Ltd.), in a 0.1% BSA, 0.1% Triton X-100 PBS solution. Zeiss Axioplan2 fluorescence microscope was used for image acquisition. TUNEL-labelling was performed according to the manufacturer's instructions using an “In Situ cell death detection kit” from Sigma. Nuclei were counterstained with DAPI (Sigma). Images were obtained from 4 different non-overlapping fields and Ki67-positive and TUNEL-positive cells were counted using ImageJ (RNA was isolated from the tumor tissue using Qiazol reagent and RNAeasy Mini Kit (Qiagen). For tumor tissue disruption, a Qiashredder instrument was used. cDNA was prepared using a Reverted H Minus First Strand cDNA synthesis kit (Thermo Scientific) and oligodT method. qPCRs were run on ABIPrism 7500 instrument (Applied Biosystems) using duplicates, melting curve analysis and gene expression normalization was calculated using the ddCt method. Primer sequences used are given in Suppl Figure legends.Statistical analysis was performed using unpaired, two-tailed t-test and p<0.05 was considered statistically significant using Microsoft Excel or GraphPad Prism 5.0. Means +/− SD values are presented. Significance between various groups is indicated with the following symbols .


Circulating endothelial progenitor cells (EPCs) reflect endothelial repair capacity and may be a significant marker for the clinical outcomes of cardiovascular disease. While some high-dose statin treatments may improve endothelial function, it is not known whether different statins may have similar effects on EPCs.This study aimed to investigate the potential class effects of different statin treatment including pitavastatin and atorvastatin on circulating EPCs in clinical setting.A pilot prospective, double-blind, randomized study was conducted to evaluate the ordinary dose of pitavastatin (2 mg daily) or atorvastatin (10 mg daily) treatment for 12 weeks on circulating EPCs in patients with cardiovascular risk such as hypercholesterolemia and type 2 diabetes mellitus (T2DM). Additional in vitro study was conducted to clarify the direct effects of both statins on EPCs from the patients.+KDR+EPCs were significantly increased  only by pitavastatin treatment. Besides, plasma asymmetric dimethylarginine level was reduced  by atorvastatin, and plasma vascular endothelial growth factor (VEGF) level was increased  by pitavastatin. In the in vitro study, while both statins increased endothelial nitric oxide synthase (eNOS) expression, only pitavastatin increased the phosphorylation of eNOS in EPCs. Pitavastatin but not atorvastatin ameliorated the adhesion ability of early EPCs and the migration and tube formation capacities of late EPCs.A total of 26 patients (19 with T2DM) completed the study. While the lipid-lowering effects were similar in both treatments, the counts of circulating CD34While both statins similarly reduced plasma lipids, only pitavastatin increased plasma VEGF level and circulating EPCs in high-risk patients, which is probably related to the differential pleiotropic effects of different statins.NCT01386853.This trial is registered at ClinicalTrials.gov,  Coronary heart disease (CHD) remains the leading cause of death globally . ElevatAccumulating evidence indicates that clinical treatment of statins may not only markedly reduce plasma LDL-c levels but also significantly improve the clinical outcomes of the patients at high risk of atherosclerosis cardiovascular disease. It has been suggested that both the LDL-c lowering and the pleiotropic effects may contribute to the clinical benefits of statins ]. Th]. Th5]].in vivo endothelial repair capacity ]. R]. Rin viapacity -]. I]. I19]].viously ].]. I]. I1The MTT  was performed to evaluate the mitochondrial activity and viability of either early or late EPCs from the patients with hypercholesterolemia and T2DM. After pre-treated with statins for 24 hours, 1 mg/mL of MTT  was added to the EPC medium culture and incubated for an additional 3–4 hours. The medium was then removed and the cells were solubilized in isopropanol. The amount of the dye released from the cells was measured with a spectrophotometer at 570 nm and subtracted background at 690 nm. An increase in the number of viable cells results in an increase in the amount of MTT formed and, therefore, in absorbance.−7 M) on the early EPCs. The adhesion assay was performed with the methods as described in detail previously ,,]. In theSeveral putative mechanisms of statin-induced EPCs mobilization were illustrated via increased endothelial NO availability, serum VEGF, receptor activator of NF-kappaB ligand (RANKL) and activation of matrix metalloproteinase (MMP)-2 and −9 -25]]. T]. T25]].]. T]. T2+KDR+ EPCs. The definition of EPCs, as the biomarkers for cardiovascular disease, remains an unresolved question ]. A]. A+KDR+Serum ADMA, as a natural eNOS inhibitor, has been linked to the risk for atherosclerosis. In the current study, atorvastatin treatment significantly decreased serum ADMA level. It is in line with the previous findings that atorvastatin treatment reduced serum ADMA levels in patients with ischemic stroke . HoweveThere are some limitations of the study. Given the limited patient number and limited study duration, we can not exclude the possibility that atorvastatin treatmet may improve EPCs with a larger dose for a longer period of duration. Besides, there were predominantly patients with T2DM and there were no clinical outcomes shown in the study. Thus, the current findings should be viewed for concept proofing rather than conclusive. Future study is required to determine if the beneficial effects of statin on circulating EPCs could be translated to the benefit in some specific clinical outcomes in the general cohort.+KDR+ EPCs number. Besides, pitavastatin but not atorvastatin activated eNOS and improve in vitro function of EPCs. Though still waiting for further confirmation, our findings did support the concept that vascular pleiotropic protection effects could vary with different statins, which might not be a class effect in clinical settings. Given the increasing importance of circulating EPCs for vascular repair in patients with severe atherosclerosis and T2DM, our fiindings may also provide a potential rationale for clinical statin treatment in some particular conditions where the improvement of circulating EPCs should be taken into consideration.In conclusion, while both similarly reduced plasma lipids, pitavastatin treatment in an ordinary dose, rather than atorvastatin treatment, increased serum VFGF level and circulating CD34EPCs: Endothelial progenitor cellsCHD: Coronary heart diseaseLDL-c: Low density lipoprotein cholesterolNOx: Total nitric oxideADMA: Asymmetric dimethylarginineKDR: Kinase insert domain receptorMTT: 3--2,5-diphenyltetrazolium bromideeNOS: Endothelial nitric oxide synthaseSDF-1α: stromal cell-derived factor-1αVEGF: Vascular endothelial growth factorThe authors declared that they have no competing interests.The study concept and design was done by LYL, CCH, SJL and JWC. TCW, PHH, HBL recruited the subjects and collected the clinical data. JSC and TTC performed the laboratory assays. Analysis and interpretation of data and drafting of the paper was done by LYL and JWC. All authored approved the final version of the paper.


To the EditorFetal gallbladder stone is a rare phenomenon, which is observed by chance during third trimester ultrasonography and does not cause significant clinical symptoms. Even though the etiology of stones found in the fetal period are yet unknown; apnea of prematurity, sepsis, parenteral nutrition, motile diseases, blood group incompatibilities, metabolic diseases and dehydration of newborns are among the causes of formation of gallstones in pediatric age groups. We present a case where the mother had used high dose flaxseed oil capsules, which are widely used as sources of omega-3 and omega-6 during pregnancy. Antenatal ultrasonography determined an echogenic focus beneath the liver position of the fetus. This was found to be gallbladder mud and stone with abdominal ultrasonography on the 2nd day of life.  This term , appropriate for gestational age, male infant was born to a 33-year-old mother who used linseed (flaxseed) oil capsules 2-3 times (2000-3000 mg) a day during pregnancy for a period of 7 months. During antenatal period, when the fetus had gestanional age of 36 weeks; a hyperechogenic focus was showed beneath the liver position of the fetus. After birth, patient was evaluated and abdominal ultrasonography revealed “gallbladder mud and stone”. Biochem3 and white blodd cells 14400/mm3. C-reactive protein value and direct Coombs test were negative. A peripheral blood smear examination did not include hemolytic findings. There had been no pyruvate kinase and pyrimidine 5’ nucleotides deficiency of patient whose glucose 6 phosphate dehydrogenase level was 12 U/g HB. Tandem mass spectrometry, urine organic acid and urine blood amino acid tests were normal.In full blood count, Hemoglobin was 16.7 g/dl, reticulocytes 5.81%, platelet count 245000/mmth postnatal day, patient was found well-fed and had gained weight. During follow-up, abdominal ultrasonography revealed a normal gallbladder without gallstones at 1 month of age.  Phototherapy was administered for three days due to indirect hyperbilirubinemia. After discharge on the 7[1]. Its prevalence is not known precisely. Its occurrence rate in the literature is low. Agnifili et al reported fetal gallbladder stone incidence as 0.39%[2]. The widest series was reported by Brown and colleagues, who detected echogenic foci in 25 fetus’ gallbladder[3]. Fetal gallbladder stone was defined for the first time by Beretski and Lankin in 1983[4]. None of the many hypotheses suggested can give a possible explanation for fetal gallbladder stone formation. Fanaroff and colleagues represent the idea that bilirubin, a breakdown product of hemoglobin, causes indirect bilirubin level increase by accessing the fetus through the placenta and that this causes fetal gallbladder stone formation[5]. It has been asserted that smoking during pregnancy, hematologic diseases, blood incompatibilities between mother and fetus, and structural anomalies like choledochal cysts may cause formation of fetal gallbladder stones[3].  On the other hand, Brown and colleagues proposed that high estrogen levels increase cholesterol secretion and decrease bile acid production and that this mechanism might cause pigment stones in gallbladder Linum usitatissimum L., Linaceae) is a vegetable product, which contains high quantity of alpha linolenic acid that can be converted into omega-3. Phytoestrogens are polyphenolic nonsteroidal vegetable origin compounds which structurally and functionally resemble β-estradiol found in mammals[6]. Phytoestrogens have both agonistic and antagonistic effects on estrogen receptors. These behave like endogen estrogens as agonists and trigger estrogenic effects. Flaxseed contains high level phytoestrogen which is in lignan structure (8 mg/g secoisolariciresinol dry weight) which mimics the structure of 17 β-estradiol and synthetic estrogen “diethyl-stilbestrol”. Breakdown products of lignans also have estrogenic activities[6]. Flaxseed ([7]. This is more likely due to high omega-3 content, flaxseed is generally preferred during pregnancy. But there is controversy in consuming flaxseed during pregnancy and lactation. It has been proved that flaxseed would pass from mother to baby during pregnancy and from breast milk following birth. However it is believed that its phytoestrogen content may increase rates of breast, prostate and endometrium cancer, as well as cause truncal obesity, hypertension, anabolic activity increase and stone formation in gallbladder due to its estrogenic activity For the patient we mentioned, high consumption of flaxseed may have caused stone formation in the gallbladder because of its phytogenic lignan structure which increases estrogenic activity.


In a previous study we observed that a fluid challenge of 4 ml/Kg is adequate to raise the mean systemic filling pressure (Pmsf) and to increase venous return in fluid responsive patients.The objective of this study is to describe the changes on microcirculation after a fluid challenge and describe the relationship with the changes on Pmsf. Our hypothesis is that changes in Pmsf could induce changes on the number of perfused vessels (PVD).plus monitor . Pmsf was measured using the stop-flow arterial venous equilibrium pressure method (1). Sublingual microcirculation was recorded with CytoCam . Data was recorded at baseline, immediately after the fluid infusion, 5 and 10 minutes after the end of 4 ml/Kg of crystalloids infused in 5 minutes. At each time at least three sublingual microcirculation videos were acquired to assess total vessel density (TVD), microcirculatory flow index (MFI), proportion of perfused vessel (PPV), perfused vessel density (PVD) and microcirculatory heterogeneity index (MHI). Analysis was performed with AVA software v. 3.2 . Responders are defined by an increase in CO greater than 10%.24 post-operative cardiac patients were prospectively enrolled. Arterial pressure and central venous pressure was measured invasively. Cardiac output (CO) was measured with a non-calibrated LiDCOp= 0.59) or Pmsf .Changes on haemodynamics and microcirculatory indices are presented in table Despite the changes in Pmsf and macrohaemodynamics, a fluid challenge does not improve microcirculation in patients after cardiac surgery.


Though the use of metagenomic methods to sample below-ground fungal communities is common, the use of similar methods to sample plants from their underground structures is not. In this study we use high throughput sequencing of the ribulose-bisphosphate carboxylase large subunit (rbcL) plastid marker to study the plant community as well as the internal transcribed spacer and large subunit ribosomal DNA (rDNA) markers to investigate the fungal community from two wetland sites. Observed community richness and composition varied by marker. The two rDNA markers detected complementary sets of fungal taxa and total fungal composition clustered according to primer rather than by site. The composition of the most abundant plants, however, clustered according to sites as expected. We suggest that future studies consider using multiple genetic markers, ideally generated from different primer sets, to detect a more taxonomically diverse suite of taxa compared with what can be detected by any single marker alone. Conclusions drawn from the presence of even the most frequently observed taxa should be made with caution without corroborating lines of evidence. Fungi are important members of ecosystem functioning and play critical roles in nutrient cycling as symbionts, saprotrophs, and pathogens . Below-gDue to the recalcitrance of many fungi towards cultivation using standard methods, and an abundance of vegetatively growing fungi with a paucity of characters for morphology-based identification, mycologists were early adopters of PCR-based detection and DNA-based identification methods –12. ManyThe internal transcribed spacer (ITS) region of nuclear encoded ribosomal DNA (rDNA) has been proposed as a suitable fungal barcode . The ITSLarge subunit (LSU) rDNA contains variable domains at the 5’ end as well as highly conserved regions at the 3’ end suitable for taxonomically diverse phylogenetic analyses as well as species- to family-level classifications. LSU rDNA reference sequences are also available through the AFTOL, UNITE, and GenBank databases. LSU rDNA is particularly heavily sampled for mushroom-forming fungi –33 and hThe ribulose-bisphosphate carboxylase large subunit (rbcL) plastid gene is one of two proposed plant barcoding markers . This muMost metagenomic studies focusing on soil fungal communities involve the use of a single DNA marker. Because we knew that fungal diversity would likely be orders of magnitude higher than plant diversity in soil, we chose to use two fungal markers to increase our chances of detecting as much of this diversity as possible. To the best of our knowledge this is the first study to use two DNA markers (ITS + LSU) with largely fungal-specific primers as well as a plant-specific marker (rbcL) to monitor both the fungal and plant communities from the same soil samples simultaneously. We hypothesized that the fungal community detected by ITS and LSU rDNA would be largely similar, and that the use of the ITS + LSU + rbcL markers would together detect a richer assortment of organisms than any single marker. This study characterizes the reproducibility and taxonomic breath detected by these various markers and highlights areas of potential concern for future metagenomic and biomonitoring studies.We sampled soil cores from two key wetland areas within the Peace-Athabasca Delta in Wood Buffalo National Park in northern Alberta, Canada. Field permits were granted by Parks Canada and samples were collected by Environment Canada and Parks Canada staff. The fieldwork did not involve endangered or protected species. Site A falls within Egg Lake (N 58° 54.535’ W 111° 25.398’) and site B falls within Johnny’s Cabin Pond (N 58° 29.688’ W 111° 30.773’). These deltaic wetland sites are currently threatened by industrial hydro-electric development and potential downstream oil sands contamination . PhysicaSoil samples were collected in August 2010 using the following method: for each sampling site the top 10cm of soil was sampled at three sampling locations within the site approximately 100 to 200 meters apart. In each sampling location, three soil cores were sampled within one square meter. Each soil core was then stored in 50 ml sterile Falcon tubes. Samples were frozen on dry ice in the field and stored in a -70°C freezer until shipped to the Hajibabaei laboratory at the University of Guelph, Ontario for processing.Frozen soil core samples were homogenized and one gram of each soil core was used for total DNA extraction using a PowerSoil DNA isolation kit . Ten extractions (100 mg each) were done for each soil core and each extraction was eluted with 50 μL of molecular biology grade water. DNA extracts of each sample were pooled and used for further amplification. The ITS rDNA region (~ 600 bp) was targeted using the fungal specific ITS1F and ITS4 primers –46. The 2 (50 mM), 0.5 μL dNTP mix (10 mM), 0.5 μL forward primer (10 mM), 0.5 μL reverse primer (10 mM), and 0.5 μL Invitrogen Platinum Taq polymerase (5 U/μL) in a total volume of 25 μL. PCR conditions were 95°C for 5 min; 15 cycles of 94°C for 40 s,  for 1 min, and 72°C for 30s; and 72°C for 5 min. Amplicons were purified with Qiagen MinElute PCR purification columns and eluted in 50 μL molecular biology grade water. The purified amplicons from the first PCR round were used as template in the second PCR round using 454 fusion tailed and MID-tagged primers in a 30-cycle amplification regime. An Eppendorf Mastercycler ep gradient S thermal cycler was used for all PCR reactions. Negative controls were included in all experiments.Marker amplification was done in a two-step PCR regime, the first PCR round was done using target specific primers (without the 454 tail). The second PCR round used the same primer sets with hybrid 454 fusion-tailed primers and specifically designed multiplex identifier (MID) tag. Each PCR contained 2 μL DNA template, 17.5 μL molecular biology grade water, 2.5 μL 10x reaction buffer, 1 μl 50x MgClThe three indexed markers amplified from each soil core were purified and fluorometrically quantified. Equimolar amounts of the MID-generated amplicons were combined and sequenced on the 454 Genome Sequencer FLX System (Roche Diagnostics) following the amplicon sequencing protocol with GS Titanium chemistry. Amplicons of each soil core were bidirectionally sequenced in 2 (1/16) regions of a full sequencing run (70 x 75 pico titer plate). Further details of the 454 pyrosequencing run are available by request from the corresponding author. Raw sequence data is available through the NCBI SRA: SRP066030A semi-automated Perl pipeline was created. Raw reads were sorted by primer sequences for the ITS, LSU, and rbcL markers using AGREP version 2.04 allowing 1 mismatch. Sorted reads were quality-trimmed using SeqTrim  with a 1Quality-trimmed reads were sorted by average read quality then clustered into operational taxonomic units (OTUs) with USEARCH version 4.0.43 . ClusterTaxonomic comparisons were also performed using MEGAN using three ecological indices. The ITS, LSU, and rbcL datasets were normalized, MEGAN classifications were summarized to the order level, and comparisons were visualized using multi-dimensional scaling plots. The Bray-Curtis dissimilarity statistic measures the number of species unique to either of two sites divided by the total number of species in both sites where each species is equally-weighted . The nonDifferences among the number of observed OTUs from the ITS and LSU datasets from both sites were compared using MEGAN. The directed homogeneity ‘up’ test was used on normalized data with the Bonferroni correction for multiple comparisons.The number and average length of reads and OTUs that were sorted, filtered, and clustered for each marker is shown We assessed sampling effort by plotting rarefaction curves. We did not rarefy to a standard number of reads because we wanted to see how each marker performed individually without subsampling the data. The number of detected OTUs can still be fairly compared across markers in our plotted curves for any standard number of reads less than or equal to the smallest library size. For each primer and site combination, curves reach a plateau indicating sampling saturation . We asseIt has been previously observed that the LCA algorithm used in MEGAN may classify reads to high level taxonomic ranks and may not classify all sequences , 62. TheA comparison of the taxonomic content of the datasets using several different ecological indices in MEGAN is shown in incertae sedis, Katablepharidiophyta (heterotrophic flagellates), Rhizaria , and Rhodophyta . Only the LSU primers recovered OTUs classified as Alveolata  and stramenopiles . Only the rbcL primers recovered OTUs classified as Bacteria. This last likely represents the presence of RuBisCO or RuBisCO-like proteins in these Bacteria [A summary of the OTUs from each marker and classified by MEGAN is shown in Bacteria , 64. TheThe top ten most frequently sampled MEGAN categories summarized at the order rank are shown in Peziza badia (cup fungus) and Pterula echo , were frequently detected by both ITS and LSU. With rbcL, we were only able to classify some of the most abundant taxa to the genus level using MEGAN. The genus Typha, bulrushes or cattails, was the most frequently sampled rbcL OTU. The genus Acorus, grasslike evergreen plants, was the second most frequently sampled rbcL OTU.The top ten most frequently sampled OTUs summarized to species rank by MEGAN are shown in Though the ITS and LSU markers did not distinguish between the two wetland sites, the rbcL marker did. rbcL OTU richness differed significantly between sites. On average 76–92 rbcL OTUs were detected from Site A and 32–54 rbcL OTUs were detected from Site B. Additionally, the taxonomical composition of rbcL OTUs differed among sites. Site A is characterized by the presence of bacterial rbcL OTUs classified in the Chromatiales, Caulobacteriales, and Rhizobiales. Among the most frequently sampled rbcL OTUs, only the Rosales and Brassicales were detected from site A. Site B is characterized by the presence of mosses in the Grimmiales (moss that grows on rocks) and Pottiales.a posteriori to track taxonomic groups of taxa. Using this approach previous work in the literature was able to track genus- to phylum-level Bacterial groups using six markers [a priori based on the availability of existing reference databases. Although the ability to link data from multiple markers to specific individuals is often lost using metagenomic methods, the data can be used to provide corroborating evidence for species presence and prevalence as we have done here.Whole genome shotgun metagenomic approaches can utilize data from an array of markers selected  markers . Though We hypothesized that the ITS and LSU rDNA markers would recover similar sets of fungal taxa and that the ITS + LSU + rbcL markers together would recover a richer assortment of taxa than any marker on its own. We produced thousands of OTUs from the ITS, LSU, and rbcL markers that we directly compared showing a significant “rare biosphere” . We obseHow can we explain our inability to detect differences among sites using the ITS and LSU markers? First, fungi are significantly more diverse than plants and our fungal sampling was not exhaustive. Despite sequencing three soil sample replicates and producing saturated rarefaction curves, the use of additional primer sets for each marker would likely recover additional taxa . Second,It is possible that next-generation sequencing platforms producing longer paired-end reads up to 600 bp may be able to produce full length ITS sequences for most fungi to circumvent the problem of working with partial ITS reads. The use of paired-end approaches would also allow forward and reverse reads to be assembled, providing an additional level of quality assurance . In contThe general rules for setting up mixed-template PCRs that detect the greatest sample diversity, particularly with 16S rDNA, have been known for some time and include using a low PCR cycle number, longer elongation times, and pooling multiple PCR reactions –84. It iS1 FigAfter quality trimming and pooling all of our ITS reads, the proportion of reads in the following categories are shown in (a) ITS1 and ITS2 regions were both detected (blue); only the ITS1 region was detected (red); only the ITS2 region was detected (green); and neither the ITS1 nor the ITS2 region was detected (purple). The number of reads of various lengths is shown in (b) for the ITS1 region (red) and the ITS2 region (blue).(PDF)Click here for additional data file.S2 Fig2 scale. A coverline at 20% OTU increase is shown as a black dashed line.In (a), sequence similarity cutoff values used with USEARCH are shown on the x-axis and the relative number of recovered OTUs, with respect to the total number of OTUs recovered at 100% sequence similarity, is shown on the y-axis. The following series are shown: the ITS region , the LSU region , and the rbcL region . In (b), the increasing proportion of clustered OTUs is shown with increasing sequence similarity cutoffs. Sequence similarity increases of 1% intervals are shown on the x-axis. The resulting increase in the proportion of OTUs is shown on the y-axis using a log(PDF)Click here for additional data file.S3 FigIndividual OTUs were plotted in rank order based on abundance at site A. Data are shown for three loci  from 5’ and 3’ fragments. Blue represents site A and red represents site B.(PDF)Click here for additional data file.S4 FigData are shown for three loci  from 5’ and 3’ primers from two sites (A and B) combined: (a) distribution of classified OTUs across ranks and (b) richness at each rank.(PDF)Click here for additional data file.S5 FigITS1 analysis used 21 taxa, including four reference sequences from GenBank, and 279 aligned characters. ITS2 analysis used 16 taxa, including four reference sequences, and 242 aligned characters. Neighbor joining analysis used the Kimura two parameter model. 1000 neighbor joining bootstrap (NJB) replicates were conducted and clades supported by greater than 60% NJB are labeled at the nodes.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.