We compared rotavirus detection rates in children with acute gastroenteritis (AGE) and in healthy controls using enzyme immunoassays (EIAs) and semiquantitative real-time reverse transcription PCR (qRT-PCR). We calculated rotavirus vaccine effectiveness using different laboratory-based case definitions to determine which best identified the proportion of disease that was vaccine preventable. Of 648 AGE patients, 158 (24%) were EIA positive, and 157 were also qRT-PCR positive. An additional 65 (10%) were qRT-PCR positive but EIA negative. Of 500 healthy controls, 1 was EIA positive and 24 (5%) were qRT-PCR positive. Rotavirus vaccine was highly effective (84% [95% CI 71%–91%]) in EIA-positive children but offered no significant protection (14% [95% CI −105% to 64%]) in EIA-negative children for whom virus was detected by qRT-PCR alone. Children with rotavirus detected by qRT-PCR but not by EIA were not protected by vaccination, suggesting that rotavirus detected by qRT-PCR alone might not be causally associated with AGE in all patients. In a study from the United Kingdom, specimens from patients with AGE that tested positive for rotavirus by EIA had significantly lower qRT-PCR Ct values  than did qRT-PCR–positive specimens from patients with AGE that tested negative by EIA and from healthy controls; Ct values for the latter 2 groups did not differ  traditionally have been used to detect rotavirus in children who have acute gastroenteritis (AGE). The rate of rotavirus detection is higher with EIAs than with conventional and semiquantitative real-time reverse transcription PCRs (qRT-PCRs)  are recommended for use worldwide (>3 loose stools in 24 hours] and/or vomiting [>1 episode in 24 hours]) for <10 days and who lived in 1 of the 3 study areas were enrolled, and a fecal specimen was collected. In addition, during this period, fecal specimens were collected from healthy children <5 years of age who resided in 1 of the same 3 study counties and had a well-child visit or an immunization clinic visit at a community medical practice. These healthy children had neither acute respiratory infection symptoms in the 3 days before nor AGE in the 14 days before the recruitment visit. Parents of all enrolled children were interviewed to collect demographic information and disease history.Fecal specimens were collected through active surveillance conducted at 3 New Vaccine Surveillance Network sites in the United States  year-round during October 2008–October 2009, as described  testing was done at each study site, and then specimens were frozen and shipped to the Centers for Disease Control and Prevention  for further testing. All specimens were retested by EIA  at CDC. If any EIA result, whether obtained at the study site or at CDC, was positive, then the specimen was classified as rotavirus positive. After preparation of a 10% (vol/vol) suspension of each fecal specimen in phosphate-buffered saline, suspensions were clarified by centrifugation at 3,000 rpm for 10 min. A 100-µL volume of clarified supernatant was added to 300 µL of MagNA Pure LC Total Nucleic Acid Isolation Kit Lysis/Binding Buffer  to lyse the virus and release nucleic acid. RNA was extracted by using the MagNA Pure 96 Cellular RNA Large Volume Kit (Roche Applied Science) and Cellular RNA LV protocol on the automated MagNA Pure 96 instrument (Roche Applied Science) in accordance with the manufacturer’s protocols. The extracted RNA was eluted in 100 µL of elution buffer and stored at −80°C until qRT-PCR testing. RNA was tested for rotavirus by using the NSP3 qRT-PCR designed by Freeman et al.  ×100 for children Of the 1,145 children whose illnesses met the case definition for AGE during the study period, 815 (71%) had a specimen collected and tested by EIA as part of the surveillance platform . Of thesOf the 817 children enrolled as healthy controls, 518 (63%) had a fecal specimen that was tested by EIA and qRT-PCR. Of these, 18 (3%) were enrolled at an immunization visit and had vaccine virus detected in their feces, and they were excluded from further analysis. A total of 500 healthy control children were included in the analysis. Compared with children who had AGE (rotavirus positive or negative by EIA), healthy controls were more likely to be black, fully vaccinated, and have public insurance and less likely to have been breast-fed, attend day care, and have had a specimen collected during January–June. Healthy controls were younger than children positive for rotavirus by EIA and similar in age to children negative for rotavirus by EIA .t value was 18  specimen from a child whose fecal sample tested positive by EIA. Specimens from an additional 65 (10%) children were positive by qRT-PCR alone, with a median Ct value of 36 (range 23–45), which was significantly higher than the median Ct value for EIA-positive children (p<0.001)  . RotavirNo vaccine strains were detected among children with AGE whose specimens tested negative for rotavirus by EIA but positive by qRT-PCR. Wild-type rotavirus strains were detected in 8 (12%) of the 65 specimens with any virus detected, whereas a genotype could not be determined for the remaining 57 (88%) specimens for which virus was detected by qRT-PCR.t value of 32 (range 21–44) was significantly higher than that for EIA-positive children (p<0.001) and significantly lower than that for EIA-negative children (p = 0.02)  healthy children; the median C = 0.02) .t values for children with a vaccine virus and a wild-type virus were similar . Wild-type rotavirus was detected by qRT-PCR during the traditional January–June rotavirus season (8 [3%] of 317 specimens) and outside the rotavirus season during July–December (5 [3%] of 183 specimens).Of the 24 healthy controls whose specimens had rotavirus detected by qRT-PCR, 11 (46%) had vaccine virus detected, of which 9 contained an RV5 strain and 2 contained the RV1 strain. Six of these 11 children were unvaccinated, including both children for whom the RV1 strain was detected; 3 had received 1 dose of RV5 ; and 2 had received 2 doses of RV5, with the second dose received 28 and 64 days, respectively, before enrollment. Wild-type virus was detected by qRT-PCR in 13 (3%) of the 500 healthy controls. The median C>8 months of age, we found that the 3-dose vaccine effectiveness against rotavirus disease that required emergency department care or hospitalization was 84% (95% CI 71%–91%) (Using only the EIA result to define cases and controls among AGE patients 71%–91%) . When cat values of fecal specimens from children with AGE for whom rotavirus was detected only by qRT-PCR were significantly higher  than Ct values of specimens from children for whom rotavirus was detected by EIA (36 vs. 18). Second, full genotypes could not be determined for most (88%) specimens for which virus was detected by qRT-PCR only, probably because of the low level of viral shedding. Last, rotavirus vaccine showed limited effectiveness against virus identified by qRT-PCR alone, but this result may partially be a function of the small number of cases in this group. In contrast, 3-dose vaccine effectiveness was high (83%–84%) for children whose samples were positive by EIA and comparable to vaccine effectiveness determined by prelicensure trials and other case–control studies in the United States that similarly identified rotavirus-positive cases by EIA . qRT-PCR may be useful for identifying cases and controls during vaccine effectiveness studies. However, because such assays also detect low levels of rotavirus circulating in the population but not associated with illness, further work is needed to define a cutoff Ct value below which the detected virus is likely to cause illness. This Ct value would help to identify the few cases missed by the EIA and exclude cases with low-level background shedding.The rate of rotavirus detection was higher by qRT-PCR than by EIA. Rotavirus was detected by qRT-PCR in fecal specimens from an additional 10% of children with AGE who tested negative by EIA. However, several lines of evidence suggest that rotavirus detected by qRT-PCR alone might not have been the causative agent in some patients with AGE. First, Ct values  in fecal specimens from patients with AGE that tested positive for rotavirus by EIA than in qRT-PCR–positive specimens from patients with AGE whose feces tested negative for rotavirus by EIA or from healthy controls (Previous studies have compared different methods of detecting rotavirus in fecal specimens. These studies should be directly compared with caution because they used different commercial assays and different PCR techniques; however, trends in patterns of detection can be compared. Similar to researchers in the United Kingdom, we found significantly lower CWe detected vaccine virus in 2% of healthy controls, all of whom were either unvaccinated or had not been vaccinated within 4 weeks before illness onset; the source of vaccine virus for these children is unknown. These vaccine strains were detected only by qRT-PCR because no healthy children in whom a vaccine strain was detected were positive for rotavirus by EIA. RV5 virus also was detected in the fecal specimen from 1 unvaccinated child with AGE; the source of vaccine virus for this patient was a recently vaccinated sibling, as described (Our study had some limitations. First, if children were seen for medical care late in their illness or if specimen collection was delayed, rotavirus might have been the cause of symptoms in some children whose specimens tested negative for rotavirus by EIA but showed low levels of qRT-PCR–detected virus. However, 99% of EIA-negative specimens that had low levels of qRT-PCR–detected virus were collected from children within 7 days after they were brought for treatment, and RV5 was not effective against AGE detected by qRT-PCR only, arguing against this possibility. Second, an internal positive control was not used in this study to monitor for false-negative qRT-PCR results possibly resulting from PCR inhibitors in feces that were carried over into the RNA extracts. We believe that the numbers of such samples would have been small because we detected only 1 EIA-positive, qRT-PCR-negative sample in this study. Third, the enrollment of some healthy controls during an immunization visit resulted in oversampling of children shedding vaccine virus. Although we excluded recently vaccinated children in whom vaccine virus was detected, all detected viruses had to be sequenced to identify children who were shedding vaccine virus. However, in a true random sample of healthy children, we would expect some children to be recently vaccinated, so we might have underestimated the proportion of healthy children in whom vaccine virus can be detected. Last, because these data are from an industrialized country in which rotavirus vaccination is routine, our findings might not apply to developing countries where the severity of infection, rates of asymptomatic viral shedding, and performance of the EIA may differ.t value should be further examined as a possible diagnostic tool in a range of settings, including in developing countries.In conclusion, our study, which was performed after rotavirus vaccine was introduced, supports the use of EIA for vaccine effectiveness evaluations in patients with AGE, even though EIA may fail to detect some true rotavirus shedding at lower levels. Although qRT-PCR increases the sensitivity of rotavirus detection, some of these cases may be in children with low-level viral shedding from a resolved or asymptomatic wild-type rotavirus infection and not true disease. The use of qRT-PCR with a cutoff C


Numerous studies indicate a role for the actin cytoskeleton in secretion. Here, we have used evanescent wave and widefield fluorescence microscopy to study the involvement of the actin cytoskeleton in secretion from PC12 cells. Secretion was assayed as loss of ANF-EmGFP in widefield mode. Under control conditions, depolarization induced secretion showed two phases: an initial rapid rate of loss of vesicular cargo (tau = 1.4 s), followed by a slower, sustained drop in fluorescence (tau = 34.1 s). Pretreatment with Latrunculin A changed the kinetics to a single exponential, slightly faster than the fast component of control cells (1.2 s). Evanescent wave microscopy allowed us to examine this at the level of individual events, and revealed equivalent changes in the rates of vesicular arrival at the plasma membrane immediately following and during the sustained phase of release. Co-transfection of mCherry labeled β-actin and ANF-EmGFP demonstrated that sites of exocytosis had an inverse relationship with sites of actin enrichment. Disruption of visualized actin at the membrane resulted in the loss of specificity of exocytic site. Although the core elements of the secretory machine are increasingly becoming understood at a molecular level, our understanding of their integration with the rest of the cellular architecture remains incomplete. This includes important questions such as ‘how do vesicles arrive at the plasma membrane’ and ‘how is this fusion directed to specific sites?’ Specificity of fusion is provided to some extent by the complement of cognate Q-and R-SNAREs found on the vesicular and plasma membranes, but this does not provide an explanation for the existence of exocytic hotspots found on the surface of neuroendocrine cells There is now extensive evidence that the actin cytoskeleton plays a role in exocytosis, both at fast synapsesActin dynamics have also been studied in fast synapses, where secretion is mediated by small synaptic vesicles (SSVs). Disruption of the actin cytoskeleton in these systems has been shown to result in an increase in release PC12 cells have been extensively used as a model system for the study of secretion of large dense-cored vesicles (LDCVs), because of their ease of handling and consistency (see for example ref 7). We have used PC12 cells transfected with a releasable fluorescent cargo, ANF-EmGFP In PC12 cells were transiently transfected with pre-proANF-EmGFP The ability to monitor the actin cytoskeleton in living cells with β-actin mCherry allowed us to directly measure the rate of action of latrunculin A, a commonly used actin sequestering agent derived from a Dead Sea sponge Having an indicator of the time course of latrunculin action, we next examined the effect of latrunculin on global secretion from PC12 cells. Following either 5 min incubation with 15 µM Latrunculin A, or the same period in normal ringer, cells expressing ANF-EmGFP were stimulated with 25 mM high potassium ringer. In untreated cells, release of ANF-EmGFP showed a biphasic release curve  that couIn order to gain better understanding of the interaction between the actin cytoskeleton and secretion, we took advantage of evanescent wave microscopy, also known as Total Internal Reflection Fluorescence Microscopy (TIRFM), which restricts fluorescent excitation to a small layer, approximately 100 nm deep, immediately above the cover glass. With this technique, individual vesicles labeled with ANF-EmGFP can be resolved 1/2 of decay between events before and after stimulation, in that those events occurring after stimulation showed faster expulsion of content . Pre-treatment with latrunculin A changed this relationship  which had the characteristic of being clearly separable from any nearby events . Events were matched so that pre- and post-stimulation events came from the same cells. From these events, we were able to distinguish clear fall times, which correspond to loss of vesicular content see . Events tionship , greatlytionship  gave a rIf, as has been suggested The barrier hypothesis for actin, raises the question of what happens at the membrane when the cytoskeleton is disrupted. To this end, we examined regions of the cell before and after latrunculin treatment. The role of the actin cytoskeleton in secretion has long been controversial. While there has been no shortage of evidence supporting its involvement, it is the detailed mechanism of its role that remains unclear. Presynaptic terminals are enriched in actin and experiments in neuroendocrine cells have indicated that actin plays a barrier role in preventing excessive secretion Our initial finding was that actin depolymerization by Latrunculin A perturbed the kinetics of two phases of release of secretory vesicles, leading to an overall decrease in the amount of release observed following 2 minutes of stimulation . These fWhile secretion can be followed in PC12 cells by tracking whole cell fluorescence, amperometry, or capacitance, TIRFM allows us to observe the fates of individual vesicles  were maintained in flasks in F12 medium (Invitrogen) supplemented with 15% horse serum (ATCC) and 2.5% Fetal bovine Serum (Invitrogen). Prior to experiments they were split, transfected, and seeded into 6 well dishes containing acid-washed, cover slips previously coated with GelTrex (Invitrogen). Transfections were carried out using electroporation in a Gene Pulser (Bio Rad) using 20 µg DNA.H. sapiens) was obtained as full length cDNA from Origene, and subcloned into the pmCherry plasmid from Clontech. All constructs were fully sequenced to ensure accuracy.ANFP-EmGFP was a generous gift from Dr Ed Levitan, University of Pittsburgh. β-actin  and viewed from beneath with conventional epifluorescence optics. Image acquisition was controlled by a computer running Slidebook 4.2 (Intelligent Imaging Innovations). Imaging was carried out on an Olympus ix80 inverted microscope with integrated high precision focus drive. Fluorescence excitation was provided by a rapid switching DG4 light source (Sutter Instruments) attached by liquid light guide. The cooled CCD camera was a Hammamatsu ORCA R2, communicating to the host computer via firewire interface. For widefield illumination timelapse experiments, a 40X oil immersion objective was used (Olympus UPlanSApo 40×0.95 NA), while for 3D or TIRFM experiments, we used an APO N 65×1.49 NA objective optimized for fluorescence (Olympus). For 3D imaging, the optical sections were taken at 200 nm intervals. Digital deconvolution within Slidebook was carried out using point-spread functions empirically derived on the microscope using Point Speck beads from Molecular Probes. 3D reconstructions were carried out using SoftWorX suite from Applied Precision. TIRF illumination was achieved through the objective, using 488 and 536 nm lasers (Melles Griot), with fine adjustment by micrometers to optimize TIRF illumination. For immunocytochemistry experiments, cells were fixed in 4% paraformaldehyde and permeabilized with 0.5% Igepal in sterilized Phosphate Buffered Saline. Anti-PIP2 and Anti- PIP3 mouse primary antibodies (Echelon) were applied at 10 µL/mL PBS, and a secondary Alexafluor rabbit-anti-mouse secondary antibody was applied at 1 µg/mL.For widefield experiments, we measured a background region, and subtracted this from each frame. Each cell was then outlined and fluorescence measured over time to provide a timecourse of release following addition of KCl which brought the K+ concentration to 30 mM.TIRFM experiments depend upon a near-zero background. For that reason we generated a minimum intensity projection for each cell, and subtracted this image from each frame. This subtracted any permanent features of the image from the stack, allowing arriving vesicles to be clearly seen. As this was a flat numerical subtraction, it did not affect vesicle rise and fall times. A maximum intensity projection image of this new stack was then used to identify regions of exocytosis, and each spot was surrounded by a 9 pixel region of interest (ROI). This ROI was then extracted as an average intensity over time plot, and analyzed for peaks using Microcal Origin. Number and amplitude of peaks were then compared between conditions.


Zol 1M and Zol 1W also improved jaw alveolar and basal BV/TV, although the highest dose (Zol 1W) was less efficient, particularly in Postn−/−. Zol decreased osteoclast number and bone formation indices, i.e. MAR, MPm/BPm and BFR, independently in Postn−/− and Postn+/+, both in the long bones and in deep jaw alveolar bone, without differences between Zol doses. Zol 1M and Zol 1W did not reactivate inflammation nor increase fibrous tissue in the bone marrow of the jaw, whereas the distance between the root and the enamel of the incisor (DRI) remained high in Postn−/− vs Postn+/+ confirming latent inflammation and lack of crestal alveolar bone. Zol 1W and Zol 1M decreased osteocyte numbers in Postn−/− and Postn+/+ mandible, and Zol 1W increased the number of empty lacunae in Postn−/−, however no areas of necrotic bone were observed. These results demonstrate that zoledronate improves jaw osteopenia and suggest that in Postn−/− mice, zoledronate is not sufficient to induce bone necrosis.Osteoporosis and periodontal disease (PD) are frequently associated in the elderly, both concurring to the loss of jaw alveolar bone and finally of teeth. Bisphosphonates improve alveolar bone loss but have also been associated with osteonecrosis of the jaw (ONJ), particularly using oncological doses of zoledronate. The effects and therapeutic margin of zoledronate on jaw bone therefore remain uncertain. We reappraised the efficacy and safety of Zoledronate (Zol) in ovariectomized (OVX) periostin (Postn)-deficient mice, a unique genetic model of systemic and jaw osteopenia. Compared to vehicle, Zol 1M (100 µg/kg/month) and Zol 1W (100 µg/kg/week) for 3 months both significantly improved femur BMD, trabecular bone volume on tissue volume (BV/TV) and cortical bone volume in both OVX Postn Periodontal disease (PD) is a common problem as it is estimated that 48.2% of the US population aged more than 30 years is affected, with up to 20% of subjects showing severe tooth attachment loss in the NHANES III survey On another side, the occurrence of osteonecrosis of the jaw (ONJ) in some patients receiving bisphosphonates, particularly high-dose zoledronate for the prevention of skeletal complications of malignancy, has raised important questions regarding the role of these drugs in the development of ONJ Several drug-related mechanisms have been suggested to eventually explain the contribution of bisphosphonates to the development of ONJ, all so far unproven, including an impairment of soft tissue and/or bone vascularization; a direct toxicity on the gingival epithelium; a direct toxicity on bone cells, particularly osteocytes; and of course the suppression of bone turnover −/−Postn) represent a unique opportunity to reappraise the effects of bisphosphonates on the jaw. Periostin is a matricellular protein synthesized by osteoblast/osteocytes, that is linked to type I collagen in the periodontal ligament, where it regulates fibrillogenesis −/−Postn mice do not have overt PD with inflammation, however they still present alveolar and systemic osteopenia −/−Postn mice kept on a soft diet to reappraise the intrinsic effects of high dose zoledronate on the jaw. Here we hypothesize that zoledronate will improve jaw and systemic osteopenia, without causing necrosis of alveolar bone in absence of overt PD. Our results indicate that zoledronate improves jaw and systemic osteopenia without causing alveolar bone necrosis in absence of overt PD, despite a decrease in osteocyte number.Periostin deficient mice (−/−Postn) were generated as reported previously −/−Postn mice were subsequently bred with C57BL/6J mice, and DNA analyzed by PCR was used to identify Postn heterozygous mice. We interbred mice that were heterozygous carriers of this mutation and obtained wild-type (+/+Postn) and homozygous mutant (−/−Postn) offspring with the expected Mendelian distribution. They were subsequently back-crossed for 10 generations, resulting in a genome of 99% C57BL/6J. Mice were housed five per cage, maintained under standard non barriers conditions and had access to water and a soft diet ad libitum . Under those experimental conditions, the malnutrition that is otherwise observed in the −/−Postn mice under a standard (hard) diet that causes the enamel and dentin defects of the incisors and molars −/−Postn and +/+Postn mice were OVX (8/gr) or Sham-operated (6/gr) and administered directly after the surgery subcutaneous Zol either once weekly , or once monthly , or vehicle (Veh) for 3 months, corresponding to a cumulative dose of 16 mg/60 kg/month (ZOL 1W) or 4 mg/60 kg/month (ZOL 1M), respectively. These drug exposures correspond to 4 times, and 1 time respectively, of the monthly dose of zoledronate prescribed for the prevention of skeletal-related events in cancer patients Postn Lac Z knock-in mice (2) were measured in vivo at 10, 11.5 and 13 months of age by dual-energy X-ray absorptiometry  Total body, femoral and spinal bone mineral density  was used to assess trabecular bone volume fraction and microarchitecture in the excised 5For the vertebral trabecular region, we evaluated 250 transverse CT slices between the cranial and caudal end plates, excluding 100 µm near each endplate. For the femoral and tibial trabecular region, to eliminate the primary spongiosa, we analyzed one hundred slices from the 50 slices under the distal growth plate.−1), trabecular connectivity density  and structural model index (SMI). The structure model index was measured to determine the prevalence of plate-like or rod-like trabecular structures, where 0 represents ‘plates’ and 3 ‘rods’ 3), bone volume , the marrow volume  and the average cortical width .Femoral cortical geometry was assessed using 50 continuous CT slides (600 µm) located at the femoral midshaft. Images were segmented using an adaptative-iterative thresholding approach rather than a fixed threshold. Morphometric variables were computed from binarized images using direct, three-dimensional techniques that do not rely on prior assumptions about the underlying structure For mandible alveolar and basal bone volume fraction (BV/TV) analysis, the head of the mice was oriented with the nasal cavity floor parallel to the horizontal plane and the midpalatal suture parallel to the midsagital plane to obtain axial CT slides .Distance between cementum-Enamel junction and Alveolar bone (DEA), an index of alveolar bone loss, was measured at the mesial root of M1 on coronal CT slides, which represents the mean of external and internal DEA  9 and 2 days before euthanasia.3/µm2/day) was calculated as the product of MS/BPm and MAR. Three slides per sample were analyzed for the bone formation indices. Osteocyte number and lacunae per bone area were counted on two toluidine blue stained slides of the midshaft cross-sectionnal femur per sample.Femur were embedded in methyl-methacrylate  as previously described Distance between the Root and the enamel of the Incisor (DRI) was also evaluated on the hematoxilin-eosin stained sections.The right mandible was removed and fixed in cold (4°C) 70% ethanol. After dehydratation, the bone samples were embedded without demineralization in methylmethacrylate and polymerized at – 20° for 48 h TRAP was detected by using hexazotized pararosanilin  and naphtol ASTR phosphate  to reveal osteoclasts, ; non-osteoclastic acid phosphatase was inhibited by adding 100 mMol/L L(+)-tartric acid  to the substrate solution. The following parameters were recorded: the number of TRAP+ osteoclasts in contact with trabeculae  ; the resorption surface  ; the average length of the zone of contact per osteoclast  considered as a sensitive indicator of osteoclast activity 2) and Young's modulus (MPa) were determined by the equations previously described by Turner and Burr The night before mechanical testing, bones were thawed slowly at 7°C and then maintained at room temperature. The length of the femur  was measured using callipers with an integrated electronic digital display and the midpoint of the shaft was determined. The femur then was placed on the material testing machine on two supports separated by a distance of 9.9 mm and load was applied to the midpoint of the shaft, thus creating a three-point bending test. Between each preparation step, the specimens were kept immersed in physiological solution. The mechanical resistance to failure was tested using a servo-controlled electromechanical system  with actuator displaced at 2 mm/minute. Both displacement and load were recorded. All tested samples (n = 8 per group) have been included in the analysis. Ultimate force , stiffness , and energy  were calculated. Ultimate stress (N/mm−/−Postn and +/+Postn) by unpaired t-tests. To compare the effects of genotype and the response to treatments Zol, we used a two way ANOVA. As appropriate, post hoc testing was performed using Fisher's protected Least Squares Difference (PLSD). Differences were considered significant at p<0.05. Data were presented as mean ± SEM.The power calculation to perform our study was based on our primary criteria, the improvement of bone alterations by Zol. We first tested the effects of Zol within groups . The alteration of alveolar bone was confirmed histologically by the higher distance between the molar root and the enamel of the underlying incisor, ‘DRI’  and lower bone formation indices  and cortical bone volume (CtBV) compared to +/+Postn  and Zol 1W (100 µg/kg/week) significantly improved BMD, femur BV/TV and CtBV in both OVX Postn−/− mice .−/− and Postn+/+ mice (p<0.01)  and in Postn+/+  . Moreover, Zol 1M decreased vs Veh the Distance between cement-Enamel junction and Alveolar bone (DEA) in Postn−/−  . As a coPostn+/+ . Zol 1M p<0.001) . However p<0.05, , whereas−/− and Postn+/+, both in the long bones and in deep jaw alveolar bone  and Postn−/− mice . However, we did not observe areas of adjacent empty lacunae that define necrotic bone tissue Clinical and histological examination revealed no osteonecrotic area in the mandible of either group. Zol did not influence the size of medullar area filled by fibrous tissue . There w+/+ mice, Zol 1M and 1W significantly decreased the percentage of empty lacunae vs veh  caused by pathogenic microflora, loss of connective tissue and crestal alveolar bone Despite the prominent benefits of bisphosphonates on preventing osteoporotic fractures and the improvement of alveolar bone and tooth loss in PD with these drugs, the occurrence of ONJ, particularly with oncological doses of i.v. zoledronate, has raised new concerns about their therapeutic margin, -i.e. the difference between the optimal effective dose and the dose at which unacceptable adverse effects occur-. Our results indicate that in absence of cancer, injury and/or severe inflammation, zoledronate at up to 4 fold the human oncological dose is safe and effective in improving jaw (and systemic) bone loss in rodents. They further indicate that suppressed bone turnover together with alterations of bone mass and quality, as observed in −/−Postn, no area of necrotic bone was observed. Hence, in absence of active inflammation, suppression of bone turnover (by BPs) together with alterations of alveolar bone mass and bone quality, i.e collagen fibers as in −/−PostnIn this model, even supra-pharmacological exposure to Zol, i.e 4 folds the human oncological dose, did not reactivate inflammation and/or worsen the DRI. Although, Zol decreased osteoclast and osteocyte number, and increased the percentage of empty lacunae in In wild-type OVX mice, Zol 1W still improved trabecular and compact bone in the femur and basal bone in the jaw, i.e similar to the oncologic dose (Zol 1M), as previously described −/−Postn mice receiving Zol 1W maintained a lower alveolar BV/TV compared to mice receiving Zol 1M, and its absolute value did not differ from placebo. Moreover, in these mice, Zol 1W increased the number of empty osteocytic lacunae. Basal bone turnover is lower in −/−Postn, however the decrease of bone formation indices in response to Zol was not more pronounced in absence of periostin. Hence, it is unlikely that the poor gain of alveolar BV/TV under Zol in −/−Postn mice could be explained by an over-suppression of bone turnover.It has been shown that the decrease of bone turnover by Zoledronate does not affect serum periostin levels or periostin immunostaining in mice −/−Postn mice have latent PD, and inflammatory cells produce cytokines that decrease osteoblast functions. However, −/−Postn mice did not present an increased infiltration of jaw bone by inflammatory cells in response to Zol. We previously reported that loading and PTH increases periostin expression in osteocytes In a model of fibrosis −/−Postn mice, without producing jaw bone necrosis.To conclude, zoledronate improved systemic and alveolar bone alterations in OVX


MSI2 gene on chromosome (SSC) 14 was very strongly associated with NVD. Thirty-six SNPs were located in genome regions where QTLs have previously been reported for behavior and/or feed intake traits in pigs. The regions: 64–65 Mb on SSC 1, 124–130 Mb on SSC 8, 63–68 Mb on SSC 11, 32–39 Mb and 59–60 Mb on SSC 12 harbored several signifcant SNPs. Synapse genes , dephosphorylation genes  and positive regulation of peptide secretion genes  were highly significantly associated with feeding behavior traits. This is the first GWAS to identify genetic variants and biological mechanisms for eating behavior in pigs and these results are important for genetic improvement of pig feed efficiency. We have also conducted pig-human comparative gene mapping to reveal key genomic regions and/or genes on the human genome that may influence eating behavior in human beings and consequently affect the development of obesity and metabolic syndrome. This is the first translational genomics study of its kind to report potential candidate genes for eating behavior in humans.This study was aimed at identifying genomic regions controlling feeding behavior in Danish Duroc boars and its potential implications for eating behavior in humans. Data regarding individual daily feed intake (DFI), total daily time spent in feeder (TPD), number of daily visits to feeder (NVD), average duration of each visit (TPV), mean feed intake per visit (FPV) and mean feed intake rate (FR) were available for 1130 boars. All boars were genotyped using the Illumina Porcine SNP60 BeadChip. The association analyses were performed using the GenABEL package in the R program. Sixteen SNPs were found to have moderate genome-wide significance (p<5E-05) and 76 SNPs had suggestive (p<5E-04) association with feeding behavior traits.  However, QTLs are often mapped by linkage analysis to a large interval of 20 centimorgans (cM) or more that may contain several hundreds of genetic variants, not ideal for accurate mapping of potential causal variants Feed represents a large proportion of the variable costs of breeding. Therefore, selection for reducing feed intake is a very important goal in breeding programs, at least in Danish pig breeds. Genetic improvement in feed efficiency was historically achieved as a correlated genetic change resulting from selection for growth rate and carcass lean content for animals tested in groups, where individual feed intake was too expensive to be measured on a large number of pigs. In recent years, the study of feed intake and behavior in pigs has been greatly facilitated by development of computerized systems that record the feed intake and related measures of individual animals within a group each time they enter the feeder. Several studies have shown low to moderate and positive genetic correlation between feeding behavior traits and daily feed intake. For instance, DFI had a positive genetic correlation with NVD (r = 0.27) The obesity epidemic has become one of the most important public health problems et al. ad libitum from 30 kg to approximately 100 kg live weight with the same feed composition. The time, duration and feed consumption was recorded for each individual visit. Average daily feed intake (DFI) was derived from the total amount of recorded feed intake divided by the number of corresponding days at the feeder. The following feeding behavior traits were defined and calculated for each boar: DFI: total daily feed intake (kg/d), TPD: total time spent at feeder per day (minute), NVD: number of visits to the feeder per day, TPV: average duration of each visit ( =  TPD/NVD), FPV: mean feed intake per visit (kg) and FR: mean feed intake rate (g/minute) ( = DFI/TPD) A total of 7388 Duroc pigs had phenotypic records from the period of 2008–2011 and 1909 boars had 60 k SNP genotype records. The selection of boars to be genotyped and sent to the test station (i.e. phenotyping) was primarily based on their aggregate breeding value, but feeding behavior is not directly part of the breeding goal of Danish pig breeds and some genotyped boars have no recorded phenotype. For GWAS, anmials had to have both phenotypic and genotypic information; 1130 boars that had both genotypic and phenotypic records for feeding behavior traits were used in the study. Summary statistics of phenotypes are shown in et al. et al. et al. The estimated breeding values (EBVs) for DFI and feeding behaviors were calculated by single-trait animal model with fixed effect of herd-year-season, random effect of pen and a random additive genetic effect, as in Duy The details of the genotyping method have been described previously ibs function of GenABEL which uses identity by state (IBS) genotype sharing to determine the realised pairwise kinship coefficient. Then a genome-wide association analysis was performed using a score test, a family-based association test, implemented in the mmscore function of R/GenABELy  =  Xb + Wp + Za + e (1) is implemented in two steps in GenABEL. In the equation (1), y is the vector deregressed EBVs for a given trait, X is an incidence matrix for fixed non-genetic effects b (herd–week section and pen), W is a vector with genotypic indicators  associating records to the marker effect, p is a scalar of the associated additive effect of the SNP, Z is an incidence matrix relating phenotypes to the corresponding random polygenic effect, a is a vector of the random polygenic effect with the normal distribution a ∼ N is fitted only once and all fixed, polygenic and residual components are estimated using the REML approach. In the second step, with the estimated heritability estimate and kinship coefficients for each pair of relatives, the correlation between phenotypic records of relatives are adjusted and approximate IID  phenotypes with normality are obtained.The relationship matrix used by the “polygenic” linear mixed effects model was generated by the mmscore test for family-based association is then conducted on the adjusted phenotype from the second step which takes into account pedigree structure and allows unbiased estimations of SNP allelic effect when relatedness is present between examinees This http://animalgenome.org/cgi-bin/QTLdb/indextag (significant and suggestive) SNPs with human genomic map using RH map and comparative maps provided by Mayer et al., tag SNPs was obtained using Ensembl annotation of Sscrofa10.2 genome version (http:// ensembl.org/Sus_scrofa/Info/Index). The positional candidate genes within 1 Mb bin size on either side of top SNPs peak were scanned using the function GetNeighGenes in the NCBI2R package at http://cran.r-project.org/web/packages/NCBI2R/index.html using the R program http://david.abcc.ncifcrf.gov/SNP positions were updated according to the newest release from Ensembl (Sscrofa10.2 genome version). Comparative mapping was performed by annotating significant SNP position to previously mapped QTL in pigs using the pig QTL database: Following quality control of SNP data, 23795 markers were excluded as having a low (<5%) minor allele frequency, 1836 markers were excluded because of low (<95%) call rate and 3463 markers were excluded because they were not in HWE (p<0.001). A final set of 33945 SNPs and 1130 pigs was retained for GWAS. The number of markers on each chromosome and average distances between two markers after quality control are given in Among 92 significant SNPs, 16 were found to have moderate genome-wide significance  and 76 wA total of 652 gene identities (Entrez ID) was located in 1Mb window size from SNP positions . HoweverA total of 36 SNPs were located in the genomic region where QTLs have previously been mapped for behavior and/or feed intake traits in pigs . Eight lWe indentified the five most significant QTL (contained more than 5 significant SNPs) for eating behavior traits including regions of 64–65 Mb on SSC 1 influencing DFI, 124–130 Mb on SSC 8 influencing both FR and TPD, 63–68 Mb on SSC 11 influencing TPD, 32–39 Mb and 59–60 Mb influencing NVD and TPV on SSC 12, respectively. The QTL region for DFI on SSC 1 located on p2.1 cytogenetic band  which isSince no GWAS study for feeding behavior in pigs has been previously published, we have made an attempt to overlap our association signals with those of previously reported QTLs. However, direct comparison between data obtained in this study and those from previous QTL studies is hindered by the fact that locations given in centimorgan on different genome assemblies do not necessarily reflect the same physical location on the genome et al. et al, On SSC 1, we found that eight SNPs associated with DFI are in previously mapped QTL which spanned 49–73 (cM) for feed intake in a Pietrain/Meishan F2 family 2 = 0.56 between adjacent markers) GABRR2 and SRSF12); hence, it could be interesting to further investigate the functional involvement of these two genes in relation to DFI. Adjacent to GABRR2 is the GABRR1 gene which encodes the GABA receptor γ1 subunit  gene. GABRR2 encodes for a receptor of Gamma-aminobutyric acid (GABA) which is the most important inhibitory neurotransmitter in the vertebrate central nervous system (CNS) and is involved in manifold physiological and pathological processes GABRR1 gene, which is in the same transcriptional orientation, suggesting a similar expression and regulatory pattern as GABRR2. The GABA and these receptors have a known function in controlling feed intake, as shown in different species such as rats GABRR2 was significantly changed after fasting and refeeding in the hypothalamus in mice et al. SRSF12, ANKRD6, RRAGD, PM20D2, RNGGT, MDN1, and UBE2J1 might be interesting to investigate, since these functions are related to regulation of gene expression or signaling pathway  was found to be significantly associated with DFI in the current study and it is located in the intron region of the Gamma-aminobutyric acid receptor subunit rho-2  as a key fuel sensor in hypothalamic neurons DDIT4L may involve feeding behavior via nutritional impacts.The significant loci MARC0085057 was closest to LX1 gene , whose frotensin . NeuroteMIS2) is the known gene located closest to them. This gene encodes an RNA-binding protein and play central roles in posttranscriptional gene regulation in mammals MSI2 gene and GABA receptor genes are connected in controlling feeding behavior. Moreover, we found two mutations in the TCF7L2 gene suggestively associated with NVD  was close to significant SNPs associated with TPV (GATA3 is a transcription factor of the Gata Zn-finger family which performs important functions during organogenesis The ElaC homolog 2 (with TPV . This gewith TPV . GATA3 iACO1 gene was close to significant SNPs associated with FPV (DLGAP2 gene (DLGAP2 variants were found significantly associated with autism spectrum disorders CLN8) plays a role in cell proliferation during neuronal differentiation DLGAP2 and CLN8 were located on SSC15  is the most interesting gene for FR  plays an important role in linking the actin cytoskeleton to the plasma membrane at the synaptic junction CADPS2 gene encodes a member of the calcium-dependent activator of secretion (CAPS) protein family, which are calcium-binding proteins that regulate the exocytosis of synaptic and dense-core vesicles in neurons PTPN4, PTPN18 and PTPRZ1 genes are members of the protein tyrosine phosphatase (PTP) family. A recent review described PTPs as central regulators of metabolism, specifically highlighting their interactions with the neuronal leptin and insulin signaling pathways PPM1E was located in the nucleus of the cell and it encodes a member of the PPM family of serine/threonine-protein phosphatases. The encoded protein dephosphorylates and inactivates multiple substrates such as 5′-AMP-activated protein kinase (AMPK) which is well documented to play key roles in controlling energy balance PPM1E gene on feeding behavior may be mediated by AMPK. Another significant biological process involves the nearby genes  having a positive regulation of peptide secretion  also plays a very important role in energy homeostasis by balancing food intake MMTP) gene located in HSA q24 were found as a candidate gene for obesity MTTP gene in feed intake may be due to its involvement in the gut leptin-melanocortin pathway Our pig-human comparative mapping approaches revealed key genomic regions and/or genes on the human genome that may influence eating behavior in human beings and consequently lead to obesity and metabolic syndrome. For instance, the QTL for DFI on SSC 1 was homologous with HSA 6q23–24 region  which haFeeding or eating behavior are important traits in pig production, as they are directly related to feed efficiency and hence cost of pig production, but their genetic mechanisms have not been extensively studied. This is the first GWAS study pinpointing a number of significant SNPs associated with feeding or eating behavior in pigs. This study presented a comprehensive approach by combining GWAS and post-GWAS bioinformatics as well as comparative mapping approaches to elucidate genomic regions and candidate genes associated with eating behavioral traits in pigs. Post-GWAS analyses highlighted potential candidate genes for feeding behavior. Several nearby genes have been mentioned directly or indirectly as being involved in the genetic control of eating or feeding behavior traits in either pigs or other species. Pigs are a well-known animal model for studying human obesity. We have conducted pig-human comparative gene mapping to reveal key genomic regions and/or genes on the human genome that may influence eating behavior in human beings and consequently affect the development of obesity and metabolic syndrome, both of which are key societal and public health problems. This is the first study to report results on genes that may affect human eating behavior via such translational genomics approaches.Figure S1Multidimensional scaling plot of identity by state distances. The principal component analysis fitted the genetic distances along the two components. The results showed that no population stratification in the data. Each point on the plot corresponds to a pig, and the 2D distances between points were fitted to be as close as possible to those presented in the original identity by state matrix. You can see that study subjects clearly cluster in a group.(TIF)Click here for additional data file.Figure S2A quantile-quantile plot of observed and expected p-values for feeding behavior traits. The inset shows a quantile-quantile (qq) plot with the observed plotted against the expected p-values for total daily feed intake (DFI), total time spent at feeder per day (TPD), number of visits to the feeder per day (NVD), time spent to eat per visit (TPV), mean feed intake per visit (FPV), and mean feed intake rate (FR) from top to bottom, respectively.(TIF)Click here for additional data file.Figure S3Comparative mapping between QTL on pig chromosome 8 and humanchromosome 4. (a) Cytogenetic band, approximate positions of QTL for mean of feed intake rate FR) and total time spent at feeder per day (TPD) shown in both cM and Mb, (b) linkage map, radiation hybrid mapping and human map of selected regions based on QTL database (release19). The read band indicated QTL presence.(TIF)Click here for additional data file.Figure S4Comparative mapping between QTL on pig chromosome 11 and humanchromosome 13. (a) Cytogenetic band, approximate positions of QTL for total time spent at feeder per day (TPD) shown in both cM and Mb, (b) linkage map, radiation hybrid mapping and human map of selected regions based on QTL database (release19). The red band indicates QTL presence.(TIF)Click here for additional data file.Table S1Distribution of SNPs after quality control and average distances on each chromosome.(DOC)Click here for additional data file.Table S2Suggestive SNPs associated to studied eating behavioral traits, their positions and nearest genes for feeding behavior traits.(DOC)Click here for additional data file.Table S3List of nearby genes in 1 Mb region flanking the associated SNPs.(DOC)Click here for additional data file.Table S4Haplotypes and their frequencies in the candidate region for number of visits to feeder per day on chromosome 12.(DOC)Click here for additional data file.


Although enzyme replacement therapy (ERT) is a highly effective therapy, CRIM-negative (CN) infantile Pompe disease (IPD) patients typically mount a strong immune response which abrogates the efficacy of ERT, resulting in clinical decline and death. This study was designed to demonstrate that immune tolerance induction (ITI) prevents or diminishes the development of antibody titers, resulting in a better clinical outcome compared to CN IPD patients treated with ERT monotherapy.We evaluated the safety, efficacy and feasibility of a clinical algorithm designed to accurately identify CN IPD patients and minimize delays between CRIM status determination and initiation of an ITI regimen  concurrent with ERT. Clinical and laboratory data including measures of efficacy analysis for response to ERT were analyzed and compared to CN IPD patients treated with ERT monotherapy.Seven CN IPD patients were identified and started on the ITI regimen concurrent with ERT. Median time from diagnosis of CN status to commencement of ERT and ITI was 0.5 months (range: 0.1–1.6 months). At baseline, all patients had significant cardiomyopathy and all but one required respiratory support. The ITI regimen was safely tolerated in all seven cases. Four patients never seroconverted and remained antibody-free. One patient died from respiratory failure. Two patients required another course of the ITI regimen. In addition to their clinical improvement, the antibody titers observed in these patients were much lower than those seen in ERT monotherapy treated CN patients.The ITI regimen appears safe and efficacious and holds promise in altering the natural history of CN IPD by increasing ERT efficacy. An algorithm such as this substantiates the benefits of accelerated diagnosis and management of CN IPD patients, thus, further supporting the importance of early identification and treatment initiation with newborn screening for IPD. Pompe disease  is an autosomal recessive deficiency of lysosomal acid alpha-glucosidase  that results in progressive glycogen accumulation Despite some gaps in knowledge, certain factors have been identified as having prognostic value in IPD, most prominent among them being cross-reactive immunologic material (CRIM) status. CRIM-negative (CN) patients with two deleterious mutations and no GAA protein expression experience an initial response to ERT before entering a phase of devastating clinical decline at rate that approximates that observed in untreated IPD Evidence from long-term clinical experience with four CN IPD patients has demonstrated successful immune tolerance induction (ITI) with a regimen of rituximab (RTX) and methotrexate (MTX) ± intravenous immunoglobulin (IVIG) in the treatment-naïve (n = 2) or early ERT (n = 2) setting www.clinicaltrials.gov), written informed consent was obtained by telephone from the patients’ parent(s) or legal guardian(s) for determination of CRIM status and long-term follow-up of each patient. The patient’s local physician acted as a third party witness to the telephone consent. The consent form was signed by the parent(s)/legal guardian(s) and returned via email or fax to the Duke study staff for his/her signature. A copy of the fully signed consent form was returned to the parent(s)/legal guardian(s). An algorithm for the rapid diagnosis and timely initiation of ITI was developed and implemented specifically for CN IPD patients -approved study  and methotrexate (three doses per week for three weeks subcutaneously), with or without monthly IVIG .Specific details of the ITI regimen for each patient are shown in Baseline and follow-up data pertaining to cardiac, respiratory, motor, and feeding statuses were serially evaluated by healthcare professionals at the respective institutions through October 2012, at which time the database was locked except invasive ventilator free survival which was last evaluated in January 2013. Two-dimensional, M-mode, and Doppler echocardiography were used to assess left ventricular mass index (LVMI).Anti-rhGAA antibodies: Anti-rhGAA IgG antibodies were assessed by Genzyme Corp.  at baseline and at regular intervals throughout treatment. Antibody status was ascertained using enzyme-linked immunosorbent assays and confirmed using radioimmunoprecipitation as described previously Safety was assessed by CD19%, frequency of infections, and number of infections requiring hospital admission at or around the time of ITI administration and routine blood tests. Flow cytometry was used to assess CD19%, using standard techniques at each local institution.We compared invasive ventilator free survival, antibody titers and LVMI values at different time points with the CN ERT+ITI treated patients described here to CN cases treated with ERT monotherapy Survival data were analyzed using the Kaplan–Meier method with two-tailed P values generated using the log-rank test GAA mutation analysis, and subsequently confirmed by western blot analysis of skin fibroblast cells in all seven patients. Demographic information and mutation data for each patient are shown in i.e., IRB/ethics committee approval, procurement of resources) rather than to CRIM status determination. The median duration between CN diagnosis and start of treatment was 0.5 months . Median age at start of ITI in conjunction with ERT was 3.5 months (range: 12 days-6.5 months). In all cases, the ITI regimen in Seven CN IPD patients were identified at six different institutions in three countries. Through application of this algorithm, CN status was rapidly identified through 2; range: 160–446 g/m2) (2 (range: 64–165 g/m2) after treatment with ERT for a median of 75 weeks , considerably lower than it was at baseline  , all patients had increased LVMI (Median: 317 g/m46 g/m2) . Of the , n = 7) . One of , n = 7) .Details on laboratory parameters are shown in Details on safety related data are shown in All 11 CN patients from a previous study in which they were treated with ERT monotherapy either died or became invasive ventilator dependent by 27.1 months of age 2; range: 160–446 g/m2) treated group had a higher median LVMI value at baseline . Both groups had improvement in LVMI for the first 26 weeks , after which there was progression of cardiomyopathy in the CN ERT monotherapy group. In contrast, there was steady improvement in cardiomyopathy for the CN ERT+ ITI treated group ; 75 g/m2, range: 65.1–92.7 g/m2; When comparing LVMI values over time between the two groups, CN ERT+ITI  patients who generally respond well to ERT While the majority of CN IPD patients develop HSAT, there are rare case reports of CN IPD patients who do not develop HSAT and can have a variable immunological response to ERT regs) and/or regulatory B-cells (Bregs), is important for its success in diminishing the immune response to rhGAA In this treatment algorithm, the ITI regimen included a short course of rituximab, low-dose methotrexate and IVIG. While the exact mechanism by which this regimen induces tolerance is not known, it is believed that the suppression and/or elimination of B- and T-cell populations responsible for antibody formation, with simultaneous up regulation of regulatory T-cells  has become a reality in countries such as Taiwan and in several states in the US There are potential limitations specifically related to the ITI regimen reported herein. In particular, this ITI regimen (like most other chemotherapeutic regimens) is relatively non-antigen-specific and has the potential to cause generalized and significant immune suppression. Thus, live vaccines cannot be administered until there is full CD19% recovery and this makes infection with opportunistic pathogens a potential complication that requires close monitoring. Surprisingly, even when administered recurrently over long periods of time Another limitation is the potential need for implementation of more than a single cycle of the regimen, specifically if there is a breakthrough in anti-rhGAA IgG titers. In this study, two of the seven patients required more than one administration of ITI regimen. These patients may have FcR polymorphisms that diminish the efficacy of rituximab Although the ITI treatment described here prolongs survival and improves significant overall clinical outcomes, closer examination clearly demonstrates residual muscle weakness and gross motor function below age-appropriate levels along with feeding difficulties. However, even in long term CRIM positive survivors, despite long-term treatment with ERT, similar issues are noted The data presented here add to the two previously reported cases of successful use of this ITI regimen in CN patients


The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. Basement membranes are sheet-like extracellular matrix (ECM) structures located on the basal side of polarized cells, and provide a complex nanoscale topography that offers physical support and chemical ligand binding sites for cell attachment and migration The effects of contact guidance are mainly observed as an alignment of the cytoskeleton, elongation of cell morphology, and oriented cell migration in the direction of the underlying patterns in vivo translation problematic. Furthermore, seeding of single cells for the study of epithelial contact guidance does not recapitulate the in situ environment where epithelium spreads over surfaces en massein vivo translation standpoint. The current study circumvents these issues by investigating the influence of contact guidance on more physiologically relevant cell sheets cultured on a highly biocompatible silk film substrate.Prior studies of contact guidance have primarily focused on the behavior of single cell suspensions cultured on silicon wafers or synthetic polymer surfaces. However, the use of silicone wafers or non-biocompatible materials do not integrate with the surrounding tissue environment and make in vivoDeveloping biomaterial technology is a foundational area of research in the emerging fields of tissue-engineering and regenerative medicine. Regenerated silk fibroin protein solution is considered to be a novel choice of material selection due to the protein’s inherent biocompatibility and nonimmunogenic properties when implanted It has also been shown that silk films can be produced with nanoscale surface topographies at a resolution higher than what can be achieved by other comparable biopolymers such as collagen, alginate, and hyaluronic acid The current study aims to better understand how surface topography affects epithelial cell sheet migration, which is an essential property for successful healing of the ocular surface after injury Bombyx mori silkworm cocoons  were cut into thirds and then boiled for 40-minutes in 0.02 M Na2CO3 (Sigma-Aldrich) to extract the glue-like sericin proteins from the structural fibroin proteins as previously described Silicon wafer features were designed to have a 2-µm width, 4-µm pitch, and 1.5-µm depth using L-edit software  and then imported into a DWL66 laser pattern generator and direct write machine . The finished mask was then placed within an Autostep 200 DSW i-line wafer stepper  using 100-mm diameter silicon wafers coated with 1-µm thick layer of photoresist . A 21-die array was created in a [3∶5∶5∶5∶3] design with 10-mm diameters separated by 5-mm spacing. The wafers were then placed into a Unaxis 770 ion etching device .Flat poly dimethylsiloxane (PDMS) substrates were produced by pouring 5-mL of solution  onto the patterned silicon wafer surfaces. The cast PDMS solution was then degassed for 2-hours under vacuum, and then cured in an oven at 60°C overnight. The following day the cured PDMS was removed from the silicon substrate and then punched to form round 14-mm circles centered on the patterned regions.Water insoluble silk films 40-µms in thickness were cast using 75-uL of 8% silk fibroin solution cast upon the round PDMS surfaces as previously described 2, 0.45 vol.% bovine pituitary extract (BPE), and 0.2 ng/mL of epithelial growth factor . In order to create a collective cell sheet, 1-uL droplet of cell suspension was seeded upon the silk films and glass cover slip controls for 30-minutes before adding media.A previously established immortalized human corneal-limbal epithelium (HCLE) cell line was kindly provided by Dr. Ilene Gipson  Cell droplets seeded upon the various surfaces were incubated for 1-day. Phase contrast images were taken to record the shape of the collective cell sheets (n = 4) using a 10×objective lens . A mosaic of several images were combined together using the MozaiX program within the Zeiss AxioVision software package in order to view the entire culture. The aspect ratio of the seeded culture was the measured.The CyQuant NF  assay was used after 1 and 5 days of cell culture by adding 500-uL of CyQuant NF solution to each sample (n = 3 for each surface) for 2-hours at 37°C. Sample fluorescence intensity was then measured using a SpectraMax M2 fluorimeter microplate reader  with excitation wavelength at 485-nm and emission wavelength collected at 530-nm.Collective cell sheet migration was monitored 1-day post-seeding upon the silk film and glass surfaces. Cell migration was monitored using the microscope’s 24-well plate micro-incubator . Time-lapse phase contrast imaging was utilized to record a frame every 10-minutes over a 10-hour period. A subset of confluent HCLE cultures were incubated for 2-hours in media containing 4-ug/ml of the mitotic inhibitor Miomycin-C . Another subset of samples received mouse anti-E cadherin (AEC) at a concentration of 5-ug/ml . As a comparative measure for leader cell formation upon the various surfaces tortuosity values (τ) for the cell sheet border was assessed by measuring the arc-chord ratio from the extracted images of the time-lapse videos, which is defined as:Cell sheet migration rate was quantified by exporting phase contrast images from the time-lapse movies at initial 0-hour and 10-hour time points in culture. Quantification was accomplished by using the ‘Distance between Polylines’ plugin to measure the average distance and respective standard deviation between cell sheet borders (n = 4). Single cell migration was analyzed using the ‘Tracking’ package in AxioVision software for individual time-lapse movies. Randomly sampled cells from various cell sheet cultures  were tracked from representative locations of the cell sheet on silk and glass surfaces. The software compiled measurements for total distance, straight distance, tortuosity, and migration rate. In addition, the cell sheet migration efficiency (η) was calculated as:Samples were fixed with 4% paraformaldehyde in PBS for 15-minutes, permeabilized with 0.4% Triton in PBS for 5-minutes, and blocked with 2% BSA in PBS for 30-minutes at room temperature. Focal adhesions and GTPase proteins were visualized by immunostaining with a 1∶600 dilution of primary antibodies for vinculin , RhoA , phosphorylated RhoA , and Rac1+Cdc42  for 1-hour at room temperature, followed by incubation with Oregon Green 488 goat anti-mouse secondary antibody  at a 1∶800 dilution for 1-hour. F-actin and nuclei were then stained by incubating cells in 1∶100 dilution of Alexa Fluor 568 Phalloidin  and 1∶10,000 dilution of DAPI  for 20 and 5-minutes respectively, while protected from light. Samples were placed on a glass slide and covered with a glass cover slip and mounting media . Fluorescent images were taken using 63×objective lens . The number of vinculin-positive focal adhesions was quantified by using ImageJ, and a linescan of actin fibers was obtained by using MetaMorph software .HCLE cell sheets were cultured as described above upon the various silk film surfaces and glass substrates for 2-days. The cell media was removed, and each sample was washed 2× with PBS at 4°C. Following PBS removal, a 200-uL sample of RIPA buffer with protease and phosphatase inhibitors  was added to each sample to release intracellular protein. The cell protein was collected using a 1-cm cell scraper device , and the cell lysate solution was then collected and added to the next sample well to increase the concentration of protein. The resulting protein lysate was mixed on ice for 15-minutes and then centrifuged at 14,000-RCF for 15-minutes .The protein concentration of each sample was determined using the bicinchroninic acid (BCA) assay  as previously described Micro-patterned silk films were successfully cast from PDMS molds with both flat and patterned surfaces. SEM imaging revealed that flat silk film surfaces were free from significant roughness , and thaHigh-density cell suspension droplets were successfully seeded upon the silk film surfaces and glass controls within 2-hours post-seeding. Whole cell sheet dispersion was then characterized upon the various surfaces by measuring the length and width dimensions of each droplet over time . After 1A dynamic study of collective cell migration was performed using phase-contrast time-lapse imaging of the epithelial cell sheets. Based on the dispersion trends witnessed between flat  and pattMigration rate was assessed over a 10-hour period by comparing cell sheet borders at both 0 and 10-hour time points. The distance traveled between these two time points was quantified upon silk culture surfaces and glass controls to determine the affect of surface topography on migration rate. In addition, cell sheets were cultured in the presence of the mitotic inhibitor Mitomycin-C (MMC) or anti-E-cadherin (AEC) antibodies to evaluate the effect of cell proliferation and cell-to-cell interaction respectively. It was found that untreated cells had the highest rate of migration on flat silk surfaces, which was almost twice as fast on average when compared to glass control surfaces  . In addiSheet migration rate in a direction parallel or perpendicular to the patterned feature edge orientation was also assessed on pattered silk film substrates. It was found that cell sheet movement parallel to the feature edge direction was over 2-fold higher on average  when compared to cell sheet movement perpendicular to the feature edge direction for untreated cultures . In addiThe polarity of individual cells upon each surface was characterized through actin staining of the cell sheet cultures upon patterned silk surfaces. The deflected angles from the major length of the cell to the surface pattern feature edge axis were quantified respectively . ImagingMigratory paths of individual HCLE cells within the previously observed migrating cell sheets were analyzed from the time-lapse videos for the various surfaces . In addiIn order to better understand how cell-surface interactions influence collective cell migration, further investigation into the distribution of actin and vinculin protein formation was assessed. Initial observations revealed that cell cultures on both glass  and flatTo better understand the mechanistic basis of guided actin fibril alignment along the patterned silk film feature edge GTPase protein localization was visualized using immunofluorescent microscopy on HCLEs grown on flat and pattern silk films and glass control substrates. Imaging revealed that RhoA, phosphorylated RhoA (P.RhoA), Rac1 and Cdc42 proteins were distributed throughout the cytoplasm for cultures on glass control surfaces  and flatin vitro. This work demonstrates that the presence of edge topography plays a significant role in directing entire cell culture movement through what appears to be an inhibitory effect on individual cell movement. Previous work studying the effects of surface topography on cell migration has primarily focused on signle cell migration. A variety of patterned biopolymer surfaces like collagen, hyaluronic acid, and poly-lactic acid do in indeed appear to direct cell migration For the first time patterned silk film surfaces were successfully used to evaluate collective corneal epithelial cell migration The above results indicate that silk film surface topography may be utilized to influence whole cell sheet properties, such as culture migration rate and direction. Therefore, by designing a surface that is specified to influence individual cell movement entire culture growth may be directed. In addition, due to the transparency and accessibility of the silk film culture system The micro-to-macro effects of epithelial cell sheet directional alignment appeared to be accomplished through changes in migration rate as dictated by cellular orientation to the patterned feature edge. Collective sheets migrating parallel to the topography edge axis move at a rate 2-3 fold faster than cells moving in a perpendicular approach. Similar results have been shown for NIH 3T3 cells as well in which migratory direction is dictated by the presence of the surface topography, and that feature topography aspect ratios influence migration speed significantly en masse movement is slowed in the perpendicular direction of the feature edge. In more descriptive terms, cell migration efficiency (η) can be taken to be the cell sheet migration rate, or useful energy output, over the average individual cell migration rate, or total energy input, for the collective sheet. Thus, η in a given direction can be controlled through the use of edge topography by modulating the degree of uniformity for directed individual cell movement.The overall effect that each individual cell has on moving the collective sheet is significantly decreased for migration perpendicular to the edge topography. The edge acts similar to a guiding track for individual cell migration for parallel movement. Previous work has demonstrated that fibroblast cell orientation in culture can be readily directed using patterned silk film surfaces In regions where edge topography is located there was an increased preference for actin fibril and FA formation. The observed localization of protein at the feature edge surface has been previously observed in other studies and is attributed to a contact guidance effect from the surface topography The results above demonstrate a new understanding into the role of contact guidance in directing individual cell behavior within a collective epithelial cell sheet to produce a concerted tissue-level response. Directed localization of cytoskeleton components occurs in response to the presence of silk film surface topography by contact guidance. The topography directs individual cell movement parallel to the feature’s edge axis, which creates a collective cell response resulting in the reduction of migration rate for epithelial sheets moving perpendicular to the feature edge axis. Individual cells appear to prefer traveling in directions where edge topography is less prevalent, which increases cell sheet migration efficiency. Future work may build off these results to design cell surfaces that may more effectively direct cell sheet migration, with the hope of utilizing such surfaces to enhance cell responses such as reepithelialization rate post trauma.Video S1Time-lapse phase-contrast microscopy video of a representative HCLE cell sheet migrating on a flat silk film surface over a 10-hour period.(MP4)Click here for additional data file.Video S2Time-lapse phase-contrast microscopy video of a representative HCLE cell sheet migrating on a patterned silk film surface in the perpendicular direction of the topography edge axis over a 10-hour culture period.(MP4)Click here for additional data file.Video S3Time-lapse phase-contrast microscopy video of a representative HCLE cell sheet migrating on a patterned silk film surface in the parallel direction of the topography edge axis over a 10-hour culture period.(MP4)Click here for additional data file.


Dipodomys spectabilis) mounds and rough harvester ant (Pogonomyrmex rugosus) colonies. The presence of a scale-dependent interaction between mounds and colonies was tested by comparing fitted spatial point process models with and without interspecific effects. Also, the effect of proximity to a mound on colony mortality and spatial patterns of surviving colonies was examined. The spatial pattern of kangaroo rat mounds and harvester ant colonies was consistent with a positive interspecific interaction at small scales (<10 m). Mortality risk of vulnerable, recently founded harvester ant colonies was lower when located close to a kangaroo rat mound and proximity to a mound partly predicted the spatial pattern of surviving colonies. My findings support localized facilitation of harvester ants by kangaroo rats, likely mediated through ecosystem engineering and foraging effects on plant cover and composition. The scale-dependent effect of kangaroo rats on abiotic and biotic factors appears to result in greater founding and survivorship of young colonies near mounds. These results suggest that soil disturbance and foraging by rodents can have subtle impacts on the distribution and demography of other species.Facilitation, when one species enhances the environment or performance of another species, can be highly localized in space. While facilitation in plant communities has been intensely studied, the role of facilitation in shaping animal communities is less well understood. In the Chihuahuan Desert, both kangaroo rats and harvester ants depend on the abundant seeds of annual plants. Kangaroo rats, however, are hypothesized to facilitate harvester ants through soil disturbance and selective seed predation rather than competing with them. I used a spatially explicit approach to examine whether a positive or negative interaction exists between banner-tailed kangaroo rat ( Facilitation occurs when one species enhances the environment or performance of another species Dipodomys spectabilis) have keystone effects on community structure of plants, mammals, arthropods, and reptiles through their ecosystem engineering and selective seed predation In the Chihuahuan Desert, banner-tailed kangaroo rats (Pogonomyrmex rugosus), a dominant species of the Chihuahuan Desert ant community Banner-tailed kangaroo rats are expected to strongly interact through exploitative resource competition with another granivore, the rough harvester ant . Studying the spatial relationship between these species allows inference of whether and at what scale a positive or negative interaction occurs Bouteloua eriopoda and B. gracilis), burrograss (Scleropogon brevifolius), and sand dropseed grass (Sporobolus cryptandrus). This study was conducted under a research permit from the Sevilleta National Wildlife Refuge (Permit No. 07-020 and 08-032).The study area was located at the Sevilleta National Wildlife Refuge, near Socorro, New Mexico, USA . The site encompassed an 8.7-ha rectangular plot (397×220 m) of Chihuahuan Desert and short grass steppe vegetation dominated by grama grass  was used to map all banner-tailed kangaroo rat mounds and rough harvester ant colonies on the study site. Coordinates of structures were real-time differentially corrected using a local base station allowing for sub-meter accuracy of mapped locations.P. rugosus colonies in June–September 2007 and again in August–September 2008. Colonies were located by walking 3-m transects across the study area and visually searching for signs of colonies. New colonies found in 2008 (classified as recently founded colonies) were assumed to have first been established by dispersing queens in summer 2007 and were not large enough to be detected until the following year P. rugosus. In 2007 and 2008, inactive colonies were verified by disturbing the nest entrance to elicit an alarm response from workers The entire site was surveyed for n = 2) were included in the 2009 occupied group. Only occupied mounds were used in analyses because cessation of kangaroo rat activity leads to changes in plant communities around unoccupied mounds Mounds were located by walking adjacent 5-m transects throughout the study area during March 2005. From March 2005–February 2009 (excluding January 2007), a monthly mark-recapture census of the banner-tailed kangaroo rat population was performed on the study area to determine whether a mound was occupied. For a complete description of census methods and monthly occupancy criteria see I used fitted spatial point process models to conduct formal hypothesis tests for the existence of an interspecific interaction between mounds and colonies λ, which determines the conditional probability of finding a point at a given location based on information provided about the rest of the point process  All statistical models used were based on the Gibbs point process model, a flexible class of parametric statistical models that can include spatial interactions, spatial trends, and dependence on covariates Intraspecific spatial interactions were included in the full and reduced MSHC models to control for the strong within-species competition of both species which results in uniform distributions of mounds and colonies through repulsion of conspecifics at short distances (≤30 m for kangaroo rats and ≤20 m for harvester ants) K and A respectively. All MSHC models included parameters for intraspecific spatial interactions and spatial covariates  were only included in the full models. The hard-core distance, KAh, specifies the radius around a location in which structures of different species cannot occur. The interaction distance, KAr, determines the radius around structures in which an interspecific interaction occurs and must be KA>h. The interaction parameter, KAγ, specifies the strength and direction of the interspecific interaction. For distances between KAh and KAr the interaction parameter is biologically interpreted as a positive interaction when KAγ>1 (i.e. attraction), no interaction if KAγ = 1, and a negative interaction if 0≤KA<γ1 (i.e. repulsion). The hard-core distances, interaction distances, and interaction parameters are all symmetric  spatstat version 1.22-0 to fit MSHC models with conditional intensity estimated as a log-linear function. The model-fitting algorithm used a maximum pseudolikelihood method with a translation edge correction All locations were marked as either kangaroo rat mounds or harvester ant colonies, denoted as c) and performed a Monte Carlo test with the log-pseudolikelihood ratio, Δ, as the test statistic to determine whether to reject the reduced model in favor of the full model (See Δ) c and P<0.05 in Monte Carlo test), I then examined the interspecific parameters of the full model to interpret the biological significance of the spatial interaction between mounds and colonies . The goodness-of-fit of all models for each dataset was examined by comparing two complementary point pattern statistics, the bivariate modified K function (KA(r)L) and the refined nearest neighbor distances (KA(r)G and AK(r)G) of the observed spatial pattern to 95% critical envelopes based on 999 Monte Carlo simulations of the selected model. Translation and Kaplan-Meier edge corrections were used in calculation of the K function and refined nearest neighbor distances, respectively, at intervals of 0.1 m up to 20 m. Strong goodness-of-fit was characterized as a close match between spatial statistics of the observed spatial patterns and the mean of simulated patterns KA(r)L). The refined nearest neighbor distance is the cumulative frequency distribution of nearest neighbor distances and was calculated between sites of one species to the nearest neighboring opposing species sites KA(r)G, whereas nearest neighboring mound distances to colonies was expressed as AK(r)G. The refined nearest neighbor distances allowed detection of asymmetrical differences in goodness-of-fit between species. In a biological sense, KA(r)L, KA(r)G, and AK(r)G values above or below the 95% critical envelopes indicate that at those distances the observed locations were more aggregated or segregated, respectively, then predicted by the fitted model. I also used a modified version of the curvewise Cramer von Mises (CvM) statistic, the sum of the squared deviation of the observed spatial statistic from the expected value across all distances, to compare the goodness-of-fit between the full and reduced models. Because the expected value is unknown for MSHC models, I used the mean of the Monte Carlo simulations I examined the models' Akaike information criterion with second-order correction for small sample sizes ) between observed and 95% critical envelopes generated by 999 Monte Carlo simulations of the logistic and random models. The univariate modified K function is calculated and interpreted similarly to the bivariate version except it only characterized the spatial relationship between colonies K function was calculated at intervals of 0.1 m up to 20 m and a translation edge correction was used. Each simulation randomly thinned the 2008 point pattern based on an assigned probability of deletion. The resulting number of surviving colonies was the same as the number observed in 2009. Probability of deletion for the random mortality model was equal to the proportion of dead colonies in 2009 and the same for all colonies, whereas in the logistic mortality model probability of deletion for each colony was the fitted value of the logistic regression model L(r) of the observed data was considered the better predictor of mortality risk. I also used the curvewise CvM statistic based on the mean of the Monte Carlo simulations to assess goodness-of-fit between mortality models. I used spatstat to perform spatial analyses and the R package ecespa version 1.1-04 to compute the critical envelopes of L(r) from mortality models I tested whether the logistic mortality model or a random mortality model (i.e. null model) was a better predictor of the spatial patterns of surviving colonies in 2009. Each model was tested by comparing the univariate version of the homogeneous modified 2χ = 24.7, P<0.0001). Established colonies had a significantly larger disc diameter than recently founded colonies during both 2008 and 2009 . Disc diameter increased with age  for both established  and recently founded colonies (mean ± SE = 13.1±2.0 cm). The number of occupied kangaroo rat mounds varied slightly between years: 48 in 2007, 44 in 2008, and 42 in 2009. In total, 56 different mounds were occupied from 2007–2009. The majority of mounds were occupied all three years (65%) and at least two out of three years (84%).Colony size and mortality rate varied between years and colony type . The numc = 3469.0, wi = 1) was a significantly better fit than the reduced model  for the spatial pattern of mounds and established colonies even after controlling for intraspecific interactions . Based on the full model's interspecific parameters, mounds and established colonies were 3.7 times more likely to occur at scales of 1–5.1 m around opposing species sites than at larger scales  was also a better fit than the reduced model  for the spatial pattern of mounds and recently founded colonies even after controlling for effects of intraspecific interactions . Mounds and recently founded colonies were 2.3 times more likely to occur at scales of 2.3–8.1 m around opposing species sites L, KA(r)G, and AK(r)G) to 95% critical envelopes for both 2007 (KA(r)L and KA(r)G values remained well within the 95% critical envelopes and exhibited similar interspecific aggregation (KA(r)L>0) at small scales as the mean full model simulations values. Only observed AK(r)G values in 2007 showed any significant deviation from the full model simulations with lower values than the 95% critical envelopes between 5–14 m. The curvewise CvM statistics also supported that the full model was generally a better fit to the observed spatial patterns than the reduced model. In both years, the CvM statistics of the full model were lower than the reduced model for both KA(r)L  and KA(r)G . The CvM statistics for AK(r)G in 2007  and 2008  indicated that the reduced model was a better fit.Best-fitting spatial models supported the presence of a small-scale positive interaction between occupied mounds and both existing and recently founded colonies. In 2007, the full model with a positive interspecific interaction included . Mortality risk of recently founded colonies decreased as disc diameter increased and distance to nearest mound and influence index decreased. The observed locations of surviving recently founded colonies in 2009 were most consistent with the logistic mortality model . The observed L(r) function was completely enclosed by the 95% critical envelopes generated by the logistic mortality model (L(r)<0) as the observed spatial pattern. In addition, simulations of the logistic mortality model resulted in a small-scale hard-core property (i.e. minimum distance between colonies) between approximately 4–6 m, similar to the observed spatial pattern. The random mortality model was a poorer predictor of the observed spatial pattern of surviving recently founded colonies in 2009, particularly at small scales (L(r) function exceeded the 95% critical envelopes generated by the random mortality model several times between 5–7 m and failed to develop a consistent hard-core property. The L(r) function of the random mortality model simulations also tended to produce weaker regularity between colonies than the observed spatial pattern.Recently founded colonies had lower mortality risk near kangaroo rat mounds. Three parameters, including one based on spatial relationship to neighboring mounds, were selected for inclusion in the final logistic regression model of recently founded colony mortality risk from 2008–2009 (i = 0.48). Mortality risk of established colonies decreased as disc diameter increased and influence index decreased. The logistic mortality model was a better predictor of the spatial patterns of surviving established colonies in 2009 than the random mortality model (L(r) function was completely enclosed by the 95% critical envelopes of the logistic mortality model over all distances  at colonies <10 m from occupied mounds  than colonies located >20 m away  . Whether ant colonies near mounds actually benefit from access to greater seed resources is unclear and requires further study. Preliminary research suggests that P. rugosus workers are more common around mounds. Abundance of P. rugosus workers captured in pitfall traps on my study area (two traps per site opened 20 days during June–July 2007) was 4 times greater  at occupied mounds  than at random sites >20 m from a mound  .The most parsimonious mechanism for the observed spatial patterns is the localized facilitation of rough harvester ants by banner-tailed kangaroo rats, perhaps mediated via effects on plant cover and composition. Kangaroo rats actively reduce total plant cover on and immediately adjacent to their mounds KA(r)G) closely matched the simulation mean (AK(r)G; Any interspecific interaction is likely to be asymmetric, with kangaroo rats affecting the spatial pattern of harvester ant colonies, but not vice versa. In the Chihuahuan Desert, no dramatic changes in plant communities or rodent populations were detected when harvester ants were removed ion mean , the cumPogonomyrmex colonies often have a mortality rate from colony establishment to 2 years of age near 99% The potential effect of kangaroo rats on harvester ants appears to be greatest when colonies are young and highly vulnerable to mortality. Young P. barbatus, no more than 10% of colonies relocate per year The potential interaction between kangaroo rats and harvester ants is highly localized on the landscape. Only an estimated 17% of the study area was affected by this interaction (based on 48 occupied mounds with a 10-m radius of influence). Intraspecific competition among colonies and mounds is undoubtedly a stronger interaction than facilitation and results in the uniform distribution of these structures across the landscape KAγ<1) as expected if strong competition existed between species. Furthermore, the logistic mortality model indicated a positive rather than negative spatial influence of mounds on recently founded colony survival.The complexity and strength of interactions between harvester ants and kangaroo rats vary between desert regions in the American Southwest Spatial point pattern analysis has rarely been applied to the study of animal community structure and typically only inductive pattern analysis has been used. This study uses a model comparison approach to examine species relationships based on statistical point process modeling of interactions and mortality spatial patterns. As such, it demonstrates the usefulness of these techniques in teasing apart scale-dependent processes in animal communities. Previous experimental research failed to detect a positive interaction between kangaroo rats and harvester ants Banner-tailed kangaroo rats have well documented impacts on community structure of plants and animals in the American Southwest Information S1Spatial point process modeling methods.(DOC)Click here for additional data file.Table S1Parameters of best-fitting multi-type Strauss hard-core models for spatial point patterns of banner-tailed kangaroo rat mounds and rough harvester ant colonies (See Information S1 for descriptions of model parameters).(DOC)Click here for additional data file.