Trabecular bone volume of the distal femoral metaphysis was determined by microCT. In young rats, vasodilation was diminished by OVX and restored with estrogen replacement , which corresponded with similar modifications in BV/TV . In old animals, vasodilation was unaffected by OVX but enhanced with estrogen replacement . Likewise, modifications in BV/TV followed the same pattern . Furthermore, in old animals with low endogenous estrogen , vasodilation was correlated with BV/TV . These data demonstrate parallel effects of estrogen on vascular endothelial function and BV/TV, and provide for a possible coupling mechanism linking endothelium-dependent vasodilation to bone remodeling.Bone loss with aging and menopause may be linked to vascular endothelial dysfunction. The purpose of the study was to determine whether putative modifications in endothelium-dependent vasodilation of the principal nutrient artery (PNA) of the femur are associated with changes in trabecular bone volume (BV/TV) with altered estrogen status in young (6 mon) and old (24 mon) female Fischer-344 rats. Animals were divided into 6 groups: 1) young intact, 2) old intact, 3) young ovariectomized (OVX), 4) old OVX, 5) young OVX plus estrogen replacement (OVX+E2), and 6) old OVX+E2. PNA endothelium-dependent vasodilation was assessed  Postmenopausal osteoporosis is characterized by pronounced bone resorption. A clear link between estrogen deficiency and reduced bone mass is evident by the increased prevalence of osteoporosis in women vs. men 2) that are known to modulate osteoclast and osteoblast activity There is increasing evidence in the literature that the osseous vasculature influences bone remodeling. For example, vascular endothelial cells release factors such as nitric oxide (NO) and prostacyclin  rats would be associated with lower femoral PNA endothelium-dependent vasodilation, and that estrogen replacement in young and old OVX rats would enhance PNA endothelium-dependent vasodilation through a NO signaling pathway. Furthermore, we hypothesized that PNA endothelial vasodilator function would be directly associated with trabecular bone volume.Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health .The procedures used in this study were approved by the University of Florida and West Virginia University School of Medicine Institutional Animal Care and Use Committees and conform to the n = 10), 2) old intact (n = 11), 3) young OVX (n = 10), 4) old OVX (n = 9), 5) young OVX+E2 (n = 11), and 6) old OVX+E2 (n = 10). After arrival all animals were housed for an additional 8 weeks in a temperature-controlled (23±2°C) room with a 12∶12 h light dark cycle. Rats in the latter four groups underwent ovariectomy surgery and were housed post-operatively for 8 weeks. Thus, at the time of sacrifice young animals were at least 6 months of age and old rats were at least 24 months of age. At 6 months of old, Fischer-344 rats are considered sexually-mature young adults, and at 24 months of age are considered senescent when mortality rates are ≥30% ad libitum. Prior to sacrifice, at least two complete estrous cycles were monitored in all female rats by daily vaginal smears. At the time of sacrifice, 3 ml of blood was collected in chilled tubes containing dipotassium EDTA. Blood samples were centrifuged at 10,000 rpm at 4°C for 5 min, and the plasma was collected and stored at −80°C for later analysis. The ovaries , uterus and cervix were dissected and trimmed of connective tissue and fat to obtain uterine mass. Plasma estrogen was determined with ELISA immunoassay . Rats were sacrificed at 6 and 24 months of age.Young (4 mon old) and old (22 mon old) virgin female Fischer-344 rats were obtained from the vendor  and divided into six groups: 1) young intact  and subjected to bi-lateral ovariectomy according to standard procedures as described previously 2 balance) and euthanized by removing the heart. To assess endothelium-dependent vasodilation of the femoral PNA, in vitro experiments were performed. The femur and surrounding musculature was removed and placed in a dissecting dish containing physiological saline buffer solution (PSS) at 4°C. The PNA was isolated from the femur and surrounding muscle tissue by use of a stereomicroscope as previously described 2O) with two hydrostatic pressure reservoirs. This intravascular pressure was selected based on previously reported intravascular pressures of 43±1.8 mmHg and 46±2.6 mmHg in skeletal muscle resistance arteries of similar size to the PNA in normotensive rats Rats were anesthetized (isoflurane 5%/O−9–10−4 M) to the bathing medium. Prior to each concentration-response protocol, the PNAs developed spontaneous tone. After completion of the experimental protocols, the maximal diameters of the PNAs were determined subsequent to a 30-minute incubation period in calcium-free PSS buffer.Endothelium-dependent vasodilation was assessed by the cumulative addition of acetylcholine , 3) PSS buffer containing l-NAME (10−5 M) plus the cyclooxygenase (COX) inhibitor indomethacin  PNAs were cannulated and allowed to develop spontaneous tone. Vasodilation to ACh was evaluated after a 20-min incubation with one of the following: 1) PSS buffer alone, 2) PSS buffer containing the nitric oxide synthase (NOS) inhibitor N−9–10−4 M) to the bathing medium. Prior to each concentration-response protocol, the PNAs developed spontaneous tone. After completion of the experimental protocols, the maximal diameters of the PNAs were determined subsequent to a 30-minute incubation period in calcium-free PSS buffer.Endothelium-independent vasodilation was assessed by the cumulative addition of DEA NONOate  as previously described Stock solutions were prepared in distilled water; whereby ACh was frozen at −20°C and serial dilutions of the ACh stock solution were prepared daily. Serial dilutions of DEA NONOate were prepared fresh as needed. Serial dilutions of the stock solutions were prepared daily. All drugs were purchased from Sigma Chemical .2O in calcium-free PSS, Ds is the steady-state inner diameter recorded after each addition of the vasodilator substance, and Db is the initial baseline inner diameter recorded immediately prior to the first addition of ACh. One-way ANOVAs were used to determine the significance of differences in body mass, uterine mass, uterine∶body mass ratio, plasma estrogen concentration, and spontaneous tone and maximal diameter of the PNAs. Repeated measures ANOVAs with pairwise comparisons were used to determine the significance of differences among age and within factors for vasodilator responses. To examine the relation between peak endothelium-dependent vasodilation and trabecular bone volume according to age and estrogen status, linear regression analyses were measured in young and old rats. In addition, regression analyses were performed examining the relations between trabecular bone volume and plasma estrogen, uterine mass, and peak vasodilation in old and old OVX rats. All data are presented as mean ± S.E. Significance was defined as P≤0.05.Vasodilator responses were expressed as a percentage of maximal relaxation according to the following formula:Body mass  was highPlasma estrogen concentration for some of the animals in the old intact (5 out of 11), young OVX (1 out of 10) and old OVX (5 out of 9) groups was below the detectable range (5 pg/ml) for the ELISA immunoassay technique used to measure estrogen levels. Therefore, the estrogen concentrations reported for these three groups are an overestimate of the actual levels because estrogen values for these animals could not be included in the analysis. For this reason, we provide median and range values, as well as report uterine mass as a surrogate marker for estrogen concentration. Plasma estrogen concentration in young intact animals  was greater than that in old intact rats . OVX decreased plasma estrogen in young rats , but did not alter estrogen levels in old rats . In young animals OVX+E2 increased plasma estrogen  above that in the young OVX group and to a level similar of that in the young intact group. In the old rats OVX+E2 increased plasma estrogen concentration  to levels above that in both the old intact and OVX groups. Uterine mass  was loweSpontaneous tone of the PNA did not differ among groups . HoweverACh-induced vasodilation of the femoral PNA was lower in old intact relative to that in young intact . InhibitOvariectomy eliminated the old age-related difference in PNA vasodilation  that wasIn young rats, the lowering of estrogen levels with OVX lowered endothelium-dependent vasodilation, whereas estrogen repletion in OVX+E2 rats restored vasodilation to levels equivalent to that in young intact rats . In PNAsVasodilation to the direct NO donor DEA NONOate was not different among groups , indicatIn young rats, the lowering of plasma estrogen concentration through OVX reduced trabecular bone volume (BV/TV) by 43% compared to young intact . Estroge2 values of 0.641 and 0.671 in young and old rats, respectively. In addition, in rats with low estrogen status , there was no relation between trabecular bone volume and plasma estrogen (Trabecular bone volume was significantly correlated to peak vasodilation in both young and old rats  and 3C westrogen  and 6A oestrogen  and 6B. estrogen  and 6C.The purpose of this study was to determine whether estrogen status affects endothelium-dependent vasodilation in the femoral diaphyseal principal nutrient artery, and whether these putative changes in vascular endothelial function are associated with alterations in trabecular bone volume. In young rats, the results demonstrate that lowering circulating estrogen through ovariectomy impairs endothelium-dependent vasodilation  and dimi2It has long been appreciated that estrogen can have positive effects on the skeletal system. For example, during adolescent growth estrogen is the critical reproductive factor for both girls and boys There is a growing body of literature that supports the notion that the bone vasculature can modulate bone cell activity Results from the present study also demonstrate an association between NO-mediated vasodilation of bone arteries and trabecular bone volume when estrogen status is altered through ovariectomy and estrogen replacement  and 3C. Intact young and old female rats regulate vasodilation of the femoral PNA primarily through a NOS signaling mechanism and to a lesser extent through a COX signaling pathway . These rThe decrement in endothelium-dependent vasodilation of the femoral PNA with OVX in young rats has also been reported to occur in other vascular beds. For example, OVX has been shown to depress endothelium-dependent vasodilation of pulmonary arteries The second study with disparate results relative to those of the current study investigated the effects of OVX in medullary arterioles from the femur of rats using the pressurized vessel technique Few studies have examined the effects of OVX on endothelium-dependent vasodilation with advanced age. Castillo et al. The lack of bone loss with ovariectomy in the aged animals was counterintuitive to anticipated results and other published reports. For example, ovariectomy in rats 18 months of age has shown volume decrements in long bones as well as the vertebrae In conclusion, the results of the present study demonstrate that lowering circulating estrogen through ovariectomy impairs endothelium-dependent vasodilation  and dimi


Prnp alleles, genetic background, prion strain and route of inoculation. Sex differences have been described in age of onset for vCJD and in disease duration for both vCJD and sporadic CJD and have also been shown in experimental models. The sex effects reported for mouse incubation times are often contradictory and detail only one strain of mice or prions, resulting in broad generalisations and a confusing picture. To clarify the effect of sex on prion disease incubation time in mice we have compared male and female transmission data from twelve different inbred lines of mice inoculated with at least two prion strains, representing both mouse-adapted scrapie and BSE. Our data show that sex can have a highly significant difference on incubation time. However, this is limited to particular mouse and prion strain combinations. No sex differences were seen in endogenous PrPC levels nor in the neuropathological markers of prion disease: PrPSc distribution, spongiosis, neuronal loss and gliosis. These data suggest that when comparing incubation times between experimental groups, such as testing the effects of modifier genes or therapeutics, single sex groups should be used.Prion disease incubation time in mice is determined by many factors including PrP expression level,  C to an abnormal form, PrPSc. They are a group of fatal neurodegenerative diseases that affect both humans and animals and include sheep scrapie, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans PRNP). Prion diseases are also infectious and are therefore transmissible (acquired). Human prion diseases have been transmitted through medical procedures (iatrogenic) such as the administration of contaminated pituitary hormones and the use of infected neurosurgical electrodes. Following human exposure to BSE contaminated material, a new acquired prion disease, variant CJD (vCJD), was recognised.Prion diseases or transmissible spongiform encephalopathies involve the conversion of the normal host PrPSc accumulation, spongiform change (vacuolation), gliosis and neuronal death. Prion disease incubation time in experimental models such as inbred mice is highly reproducible under standard conditions. However, there is also considerable variation that is determined by several factors including host genetic background, prion strain and route of inoculation. The main genetic determinant is variation in Prnp itself that results in two major isoforms of PrP: aPrnp  and bPrnp  that are associated with short and long incubation times respectively aPrnp inbred lines shows a difference of over 100 days between the shortest and longest incubation time strains suggesting the involvement of additional genetic loci Irrespective of aetiology and host, prion diseases are distinguished by long, clinically silent incubation periods and characteristic neuropathology such as PrPSc and result in different incubation times even in the same host Prion strains are associated with conformational and glycosylation differences in PrPThe most common route for the inoculation mice with prions is either intracerebral or intraperitoneal. Delivery of prions directly to the central nervous system rather than to the periphery results in shorter incubation times Sex has also been implicated in influencing prion disease incubation time in mice. This effect has been suggested by a few studies, however, these have not been comprehensive and have largely focused on only one strain of mice or prions In order to address the effect of sex on prion disease incubation time in mice we have compared transmission data for males and females gathered at the MRC Prion Unit over the last ten years. This includes data from twelve different inbred lines of mice inoculated with at least two prion strains representing both mouse adapted scrapie (Chandler/RML) and mouse adapted BSE. We show that sex can have a profound influence on incubation time, however, this is not a general effect and is not seen in all mouse strains but rather is highly specific for given combinations of mouse model and prion strain.aPrnp allele mice, however, JU/FaCt is a bPrnp strain and MAI/pas is a cPrnp strain Groups of males and females from twelve different inbred lines of mice (including two sub-strains of C57BL/6) were inoculated intra-cerebrally with various strains of prions. All mouse lines were challenged with a strain of mouse-adapted scrapie and mouse-adapted BSE. These were generally Chandler/RML scrapie prions (I856) and C57BL/6 passaged BSE (I874), however, ME7 scrapie prions (I9459) and mouse-passaged BSE  were also transmitted in some cases . The inbIncubation times for all transmissions are shown in Two lines of mice, C57BL/6 and FVB/NHsd, showed significant sex differences with multiple prion strains . For C57C expression is inversely correlated to incubation time. The shortest incubation times are seen in transgenic models over-expressing mouse PrP Prnp null mice are resistant to prion disease, one copy of Prnp restores susceptibility but with an increased incubation time relative to wild type controls C levels could explain the sex effects seen in C57BL/6 and FVB/N mice, we measured total PrPC in 10% brain homogenates for both males and females using a PrP specific ELISA In experimental mouse models, the level of PrPSc deposition, spongiosis, gliosis and neuronal loss in groups of males and females (5–14 per group) for C57BL/6 and FVB/NHsd for Chandler/RML, ME7 and MRC2 prion strains. Endocrine differences have been implicated as determinants of sex effects in prion disease To establish whether regions of the brain showed differential susceptibility between males and females we compared the pattern of PrPSc) as shown by immunoblotting. We compared the pattern of PrPSc in end stage brains from males and females (3 per group) for C57BL/6 and FVB/NHsd for Chandler/RML, ME7 and MRC2 transmissions. In all cases the characteristic PrPSc pattern for the prion strain was preserved and no differences were observed between males and females  or the Jackson Laboratory . MAI/Pas and PWK/Pas mice were a gift from Jean-Louis Guénet ; JU/FaCt were a gift from Bruce Cattanach  and CAST/Ei were obtained from the Mary Lyon Centre .All inocula were prepared as 1% (weight/volume) prion infected brain homogenate in PBS made from the brains of terminally sick mice. The mouse adapted scrapie prion strain Chandler/RML  was generated by a single passage in CD-1 Swiss mice 2 and inoculated intra-cerebrally into the right parietal lobe with 30 µl of a 1% prion infected brain homogenate as previously described Mice were anaesthetized with isofluorane/OMouse brains were fixed in 10% buffered formal saline (BFS) and prion infectivity was inactivated by incubation in 98% formic acid for one hour. Tissues were paraffin wax embedded, sectioned and stained as previously described 10% w/v brain homogenates in D-PBS were prepared by ribolysing, benzonase treated, proteinase K digested (50 µg/ml of proteinase K for 1 h at 37°C) and western blotted as described previously C were determined in 10% brain homogenates by ELISA as previously described C was captured by anti-PrP monoclonal antibody ICSM18  and detected using biotinylated anti-PrP monoclonal antibody ICSM35  and streptavidin-horseradish peroxidise conjugate . Total protein concentration was determined with a Pierce BCA Protein Assay kit (Thermo Scientific) according to the manufacturer's instructions.Endogenous levels of PrPStatistical tests were carried out using GraphPad InStat . Where the group incubation times passed a normality test (Kolmogorov-Smirnov), data were evaluated using a two-tailed t-test. Where the normality test was not passed the non-parametric Mann-Whitney test was used.bPrnp mice  have a shorter incubation time than males following challenge with the mouse adapted scrapie strain; shorter incubation time for female NZW mice with Chandler/RML but not ME7 prions et al in C57BL/6J mice inoculated with ME7 Sex effects have been implicated in prion disease incubation time for many years, however, to date, these data have been equivocal with few conclusions drawn. We have now presented a comprehensive study with 12 different inbred lines of mice all of which were challenged with a mouse-adapted scrapie strain of prions (Chandler/RML) and mouse-adapted BSE (I874). In addition, two mouse lines (C57BL/6 and FVB/N) were challenged with ME7 and the mouse-adapted BSE strain, MRC2. Our data show that sex can have a significant effect on incubation in some instances. However, this is highly specific to the mouse and prion strain combination. 8 of the 12 mouse strains used showed no sex differences with either prion strain and two mouse strains (NZW and SM) only showed a sex effect with the Chandler/RML prion strain. C57BL/6 and FVB/N were the only mouse strains to show sex differences with both mouse adapted scrapie and BSE. For these two mouse lines, females had a shorter incubation time than males when challenged with either Chandler/RML or ME7 but this was reversed following inoculation with BSE derived prions. This is broadly in agreement with previous studies showing that: female C57BL/6N have a shorter incubation time following inoculation with ME7 et al who described a highly significant increase in C57BL/6J and DBA/2J females on inoculation with BSE et al and Abiola et al respectively. The extent of the similarity between these two sub-strains has not been characterised but it is possible that this may be sufficient to explain the contradictory findings. In our present study we looked at mouse-passaged BSE and where significant differences were observed, females showed an increase in incubation times relatives to males. It may not be appropriate to compare data from BSE primary passages with data from murine BSE strains as the presence of a species barrier and subsequent strain adaptation may significantly alter the sex effects.Few studies have looked at the effect of sex on prion disease incubation time following inoculation with BSE. On primary passage of BSE to C57 and RIII mice no sex differences were observed although an X chromosome-cytoplasm interaction was observed in the F1 backcross generation et al also reported an increased incubation time in female B.10.AKM, C3H/SwSn and C3H/D1Sn mice following inoculation with mouse-passaged sporadic CJD strain K.Fu Sex differences have also been reported for age of onset and disease duration of the human prion diseases, vCJD and sporadic CJD In this study we have used the number of days from inoculation to the onset of clinical signs as our measure of incubation time. It is also possible to measure incubation period as the time from inoculation to the time of death and to measure the duration of the disease. Using these alternative data it is possible that other sex differences may be observed.C between males and females that show differences in incubation time with a range of prion strains. We have also shown that there are no detectable differences between males and females in terms of PrPSc deposition in the brain and the pattern and extent of spongiosis, gliosis and neuronal loss. No differences were seen in prion strain types between males and females as determined by PrPSc pattern by immunoblotting. Loeuillet et al investigated the effect of androgens on incubation time by using castrated mice The reason for the observed sex differences is still unresolved. In this study we have shown that there is no difference in the endogenous levels of PrPFor some mouse inbred lines and prion strain combinations there is a highly significant difference in incubation time with a mean difference of up to 7%. While this effect is not seen with all mouse lines, it is of particular importance in C57BL/6 and FVB/N strains as these are commonly used for the generation of knockout and transgenic mouse models. We therefore recommend that when testing the effect of a modifying gene or therapeutic agent on prion disease incubation time it is essential to use single sex groups.Figure S1Histological features of Chandler/RML inoculated male and female C57BL/6 mice. Comparison of histological features between male C57BL/6/ and female C57BL/6 mice  inoculated with Chandler/RML prions. Panels A and E show the PrPSc distribution in a cross section of the brain and panels B–D and F-H are higher power views of the hypothalamus (boxed area). (B and F) mild spongiosis (C and G) synaptic deposition of PrPSc (D and H) gliosis. Overall, the pattern of PrPSc distribution, spongiosis or gliosis, shows no difference between both groups. Scale bar corresponds to 2 mm , 80 µm  or 160 µm in all other panels.(JPG)Click here for additional data file.Figure S2Sc from the brains of C57BL/6 and FVB/N mice following Me7 transmission.Western blot of PrP Western blot of proteinase-K treated 10% w/v brain homogenates  immunoblotted with anti-PrP monoclonal antibody ICSM-35 . (A) C57BL/6 mice (B) FVB mice. The PrPSc from both male and female brains is characteristic of the Me7 scrapie prion strain and no sex differences are seen.(TIF)Click here for additional data file.Figure S3Sc from the brains of C57BL/6 and FVB/N mice following MRC2 transmission.Western blot of PrP Western blot of proteinase-K treated 10% w/v brain homogenates  immunoblotted with anti-PrP monoclonal antibody ICSM-35 . (A) C57BL/6 mice (B) FVB mice. The PrPSc from both male and female brains is characteristic of the MRC2 prion strain and no sex differences are seen.(TIF)Click here for additional data file.


Macaca mulatta) as they watched social and nonsocial videos. We found that social videos, as compared to nonsocial videos, captured more visual attention, especially if the social signals depicted in the videos were directed towards the subject. Subject-directed social cues and nonsocial nature documentary footage, compared to videos showing conspecifics engaging in naturalistic social interactions, generated larger pupil diameters . These findings indicate that rhesus monkeys will actively engage in watching videos of various kinds. Moreover, infrared eye tracking technology provides a mechanism for sensitively gauging the social interest of presented stimuli. Adult male rhesus monkeys' visual attention and physiological arousal do not always trend in the same direction, and are likely influenced by the content and novelty of a particular visual stimulus. This experiment creates a strong foundation for future experiments that will examine the neural network responsible for social information processing in nonhuman primates. Such studies may provide valuable information relevant to interpreting the neural deficits underlying human psychiatric illnesses such as autism, schizophrenia and social anxiety disorder.The sophisticated analysis of gestures and vocalizations, including assessment of their emotional valence, helps group-living primates efficiently navigate their social environment. Deficits in social information processing and emotion regulation are important components of many human psychiatric illnesses, such as autism, schizophrenia and social anxiety disorder. Analyzing the neurobiology of social information processing and emotion regulation requires a multidisciplinary approach that benefits from comparative studies of humans and animal models. However, many questions remain regarding the relationship between visual attention and arousal while processing social stimuli. Using noninvasive infrared eye-tracking methods, we measured the visual social attention and physiological arousal (pupil diameter) of adult male rhesus monkeys ( Social cognition describes a collection of perceptual, cognitive and regulatory processes that coordinate one's interactions with others Despite the advances brought about through research on rhesus monkeys, this work comes with several caveats. First, most neurotoxic lesion and transient inactivation studies have measured social behavior deficits in operated and control animals while freely interacting in groups In the current experiment, we measured visual social attention and sympathetic nervous system arousal in adult, male, rhesus monkeys as they watched a large library of social and nonsocial video clips. We measured visual attention using an infrared eye-tracking system and a noninvasive method of head restraint All experimental procedures were noninvasive and developed in collaboration with the veterinary, animal husbandry and environmental enrichment staff at the California National Primate Research Center (CNPRC). All data presented here were collected at the CNPRC under a protocol (13483) approved by the University of California, Davis, Institutional Animal Care and Use Committee. All attempts were made  to promote the psychological well-being of the animals in accordance with recommendations made by the Weatherall report, “The use of non-human primates in research.”ad libitum. The current study did not begin until the animals were 5.8–8.7 years old and weighed 10–14 kg.Six adult male rhesus monkeys participated in this study. Each was born at the CNPRC and lived in 1 of 24, half-acre outdoor enclosures for at least 2 years before being relocated to indoor housing. Each of these outdoor enclosures contained approximately 70 animals of various ages and sexes. Once relocated indoors, each animal was housed in a standard adult macaque laboratory cage (66 cm width×61 cm length×81 cm height) with a minimum of 6 hours of socialization permitted with a neighboring animal each weekday. Depending on the relationship, daily socialization was either full, unrestrained interactions in either of the 2 adjoining cages or restricted to mostly visual interaction through a metal grate . The housing room was maintained on a 12-hour light/dark cycle. All animals were maintained on a diet of fresh fruit, vegetables and monkey chow , with water available Training methods and noninvasive head restraint strategies have been described in detail elsewhere Visual stimuli were presented to each monkey using a PC running the Eprime 2.0 Professional software package . All gaze data were collected using the Eye-Trac 6 .NET User Interface program (Applied Science Laboratories) on a separate PC. Infrared luminance level, pupil threshold and corneal reflection threshold were set individually for each animal at the start of each session. Sampling rate for the infrared eye-tracking camera was set to 120 Hz. A standard nine-point calibration  was conducted prior to testing with each animal to ensure accuracy of gaze data collection. Calibration stimuli were videos presented in small portions of the screen  of rhesus monkeys from the outdoor housing enclosures at the CNPRC. The goal here was to attract the animal's gaze to different portions of the screen to calibrate the data acquisition software.Once reliably calibrated, each monkey was trained to fixate color animated GIF images at random spatial positions on the computer screen for juice rewards that were manually dispensed by the experimenter. Animals completed this phase of training once they fixated the GIF images consistently for 2 consecutive days. During the second phase of fixation training, animals viewed either photographs  on a 50% gray background or color video clips  on a black background. Each photograph or movie was separated by four 50% gray screens: 1) blank, 10-second duration, 2) black square target  at center, 3) same black square target positioned randomly at 1 of 8 points around the screen periphery, and 4) blank, 10-second duration. Animals were required to fixate each black square target for at least 250 ms to obtain a small juice reward and move on to the next picture or movie trial, thus ensuring accuracy of the point-of-gaze data throughout a prolonged testing session. The animals completed the final phase of training once they finished 100 picture or 50 movie trials in less than 90 minutes on 3 consecutive days.A library of 600, 30-second color videos was created for this study. Half of these videos (300) were created from raw video footage of rhesus monkeys collected at the CNPRC. A majority of these “social” videos showed rhesus macaques from the CNPRC field cages engaging in social behaviors, such as aggression (25 videos), grooming (50 videos), play (50 videos), mounting (15 videos), foraging (50 videos) or sitting in groups without overt social behavior . These stimuli will be collectively referred to as Naturalistic Social videos. In addition, a series of videos was created that depicted camera- or subject-directed facial expressions of aggression or subordination (20 videos each), as well as videos of monkeys simply looking towards the camera but not producing any social signals . These stimuli will be collectively referred to as Subject Directed Social videos. The remaining 300 videos were gathered from commercial nature documentaries such as “Planet Earth”  and “Life in the Undergrowth” . These Nature videos depicted birds (53 videos), insects/invertebrates (43 videos), land mammals (62 videos), marine mammals/fish (57 videos) and landscapes/flowers (85 videos). None of the Nature, Naturalistic Social or Subject Directed Social videos showed humans, other nonhuman primate species, snakes or other natural predators of macaques .Raw video footage was edited using the PowerDirector Express version 5.0 software  on a PC. Each video clip only included footage depicting social behavior from a single category  or animals from only one Nature category . Given that the frequency of scene changes within a video may influence visual attention in rhesus monkeys Data described here were gathered over 12 days. On each test day, the animal was transported to the eye-tracking room, placed into the testing chair and its head was restrained with a thermoplastic helmet The first phase of a daily testing session required the animal to watch 10, 30-second “Baseline” videos that were made up of typical PC or Macintosh screen savers . These same 10 Baseline videos were shown in widescreen format (16∶9) and in random sequence each test day. Requiring the animals to watch these basic video stimuli served to verify eye-tracker calibration and the animal's willingness to participate in the testing session before the animal viewed the Naturalistic Social, Subject Directed Social and Nature videos of main interest. As shown at the top of In the second testing phase, the animal viewed a pseudorandom mixture of 25 Social and 25 Nature videos , bottom,Each animal's total fixation duration, total frequency of fixations, average fixation duration, average gaze dwell duration and average pupil diameter was measured for each video using the ASL Results software package (Applied Science Laboratories). Eye tracking parameters were computed for a single rectangular area of interest (AOI) that encompassed the entire video frame. Default parameters were used to define fixations (Applied Science Laboratories). A fixation was recorded if gaze coordinates remained within 1°×1° visual angle for at least 100 ms. The duration of a given fixation ended when gaze coordinates deviated by more than 1°×1° visual angle for more than 360 ms. Total fixation frequency represented the cumulative number of discrete fixations that fell within the video AOI during each 30-second trial. Total fixation duration was the cumulative time (maximum = 30 seconds) that the animal spent fixating the video AOI. The average fixation duration was derived by dividing the total fixation duration by the total fixation frequency. Finally, the average dwell duration measures the average amount of time that gaze remained within the video AOI without leaving.We opted to normalize data within each animal to control for individual differences across test days. For each video, the animal's total fixation duration, total fixation frequency, average fixation duration or average dwell duration was divided by the average of that same variable across all 50 Social and Nature videos viewed that day. The resulting quotients were multiplied by 100 to give a metric that can be interpreted as a percent of the animal's typical total fixation duration, total fixation frequency, average fixation duration or average dwell duration on a given test day. As an example, if an animal fixated on a given video for 23 seconds, but their average fixation duration across all Social and Nature videos on that day was 18 seconds, then their normalized total fixation duration for the video in question was (23/18)×100 = 128 or 28% longer than the daily average fixation duration.t-tests of significant main effects and interactions were Bonferroni corrected for multiple comparisons. Alpha was set at p<.05, but significance levels up to p = .07 are also presented as marginally significant due to the small sample size and conservative approach to correction for multiple comparisons.For each of these 4 dependent variables, we examined values for the entire 30-second video, as well as values for each 10-second block. Data were compared across broad categories of video stimuli , as well as within each category  using ANOVAs with Category (3–6) and Block (3) as within-subjects factors with repeated measures. A Huynh-Feldt correction was used to adjust the degrees of freedom if the group variance did not remain equal across the three 10-second blocks. Post-hoc p<.05). Such a difference necessitated a normalization procedure to remove the influence of differences in luminance across video categories, as well as individual differences. We identified 2 normalization strategies. The gray screen that proceeded and followed each video had the same average luminance as that video. The raw average pupil diameter data from each video was therefore divided by the average pupil diameter during the gray screens flanking each video and multiplied by 100 to provide pupil diameters in terms of a percent of the gray screen value. An alternative approach was to use the same within-subject strategy as described for the gaze data above. The raw average pupil diameter for each video was divided by that animal's average pupil diameter across the entire testing session on that day and multiplied by 100 to provide pupil diameters in terms of a percent of the daily average. Analysis of the pupil diameter data was conducted using both normalization strategies and nearly identical results were found for both strategies. Therefore, for the sake of consistency with the gaze data, we will display results for the pupil diameter dataset using the within-subject normalization strategy. The results generated using the gray screen normalization strategy are included as As an index of cognitive processing load F = 14.74, p = .01, p2η = .747].Examples of 1 Nature, Naturalistic Social and Subject Directed Social video are shown online with the pattern of visual attention (or point-of-gaze) from 1 animal mapped onto the video with black cross-hairs  = 10.45, p<.01, p2η = .676; post-hoc: Naturalistic Social>Subject Directed Social p = .066, Naturalistic Social>Nature p = .015]. We also found that sub-categories within the Naturalistic Social videos differed in the total frequency of fixations . In particular, the animals fixated more frequently when watching videos showing conspecifics engaged in mounting behavior relative to those showing foraging [post-hoc: Mounting>Foraging p = .021]. No other sub-categories within the Naturalistic Social videos differed significantly.Based on the frequency of fixations, the monkeys' discriminated between the Nature, Naturalistic Social and Subject Directed Social video categories. As shown in F = 4.685, p = .037, p2η = .484; post-hoc: Subject Directed Social = Naturalistic Social = Nature, all p>.11]. However, discrete fixations were significantly longer when animals watched Subject Directed Social videos . More focused analysis of the 3 sub-categories within Subject Directed Social videos did not indicate any significant differences in average fixation duration based on type of content .In contrast to the total number of fixations, Subject Directed Social videos commanded the greatest total fixation duration , but theF = 9.108, p = .006, p2η = .646; post-hoc: Subject Directed Social>Nature p = .036, Naturalistic Social>Nature p = .065]. There was no significant difference in average dwell duration between the sub-categories of Subject Directed Social videos. However, within the Naturalistic Social category, aggression and mounting videos captured the animals' attention for longer periods of time than foraging videos . Finally, the measures of total fixation frequency, total fixation duration, average fixation duration and average dwell duration were all consistent throughout the entire video , indicating that these measures of visual attention were not consistently driven by 1 particular segment of the videos.In addition to measures of gaze frequency and duration, we also measured the average time that the animals' gaze remained or “dwelled” within the video frame area of interest without leaving . This meF = 101.53, p<.001, p2η = .953; post-hoc: Subject Directed Social and Nature>Naturalistic Social, both p<.001]. Pupil diameter was also largest during the first 10 seconds of a given video (Block 1 of 3), regardless of content , but remained static between Blocks 2 and 3. By contrast, when animals watched Nature videos, their pupil diameters decreased slightly across the 30-second video, but did not change significantly. We also examined the sub-categories within the Subject Directed Social and Nature videos to determine if 1 or more specific type of content could be driving the larger pupil diameters observed for the more general category. There were no significant differences in pupil diameter between the 3 sub-categories of Subject Directed Social videos (p<.05). Pupil diameter was intermediate and did not differ between videos showing birds, insects/invertebrates and landscapes/flowers, but each of these 3 sub-categories still produced pupil diameters greater than land mammals .Average pupil diameter during each video was also collected to provide a measure of sympathetic nervous system activity l videos . HoweverThe present study assessed how visual attention and autonomic nervous system arousal are differentially modulated by social and nonsocial information. This was assessed using noninvasive video eye-tracking and pupillometry to simultaneously measure differences in visual attention and sympathetic nervous system arousal when adult male rhesus monkeys watched high-quality videos. Overall, adult male rhesus monkeys fixated more frequently and for longer durations when watching videos with social content relative to nonsocial, nature documentary footage. Relative to nature documentary footage, videos showing macaque social signals directed towards the viewer commanded the longest average fixation duration, total fixation duration and average duration of continuous fixation or “dwell” within the video frame. Videos showing conspecifics engaged in naturalistic social interactions resulted in the highest total number of fixations, but videos in this category typically fell between those showing subject-directed social signals or nature documentary footage in fixation duration measures of attention .These results confirmed our expectations that were based on previous eye-tracking studies with humans, apes and monkeys indicating a strong preference for attending to social stimuli, especially faces As is common for most infrared eye-tracking systems currently available, we were also able to measure 1 index of autonomic nervous system arousal in our animals – pupil diameter. The diameter of the human and nonhuman primate pupil is controlled by muscles in the iris that contract and relax In contrast to the clear visual preference for videos with conspecific social content, we showed here that both subject-directed social signals and nature documentary footage, relative to those with naturalistic social interaction content, resulted in larger pupil diameters indicating elevated sympathetic nervous system arousal. The elevated autonomic response in the subject-directed videos was expected. Although the subject-directed social videos varied in the type of social signals displayed , all consisted of close-up footage of unfamiliar monkeys displaying direct eye contact toward the camera/viewer. In rhesus monkey society, direct eye-contact is a highly threatening gesture and readily elicits behavioral expressions of fear, passive avoidance and/or generalized tension The increased pupillary response to the Nature category of videos was not expected. One possible explanation for the elevated pupil diameters could be the novelty of the content in those videos. The monkeys used in this study were raised in semi-naturalistic outdoor enclosures. As a result, they had limited exposure to non-primate animals  during development. The rural landscape in which the housing enclosures are embedded is very consistent. Thus, the marine mammals or fish, as well as the oceanic, forest, desert and polar landscapes shown in the nature documentary footage were highly novel to our subjects. There was evidence of habituation in the temporal characteristics of pupil diameters (significantly lower pupil diameters after the first 10 seconds) for the Subject Directed and Naturalistic social videos, but not for the Nature videos  between the animals and the video camera. Such an extensive stimulus set does not currently exist in the fields of social neuroscience or primatology, and therefore may be of value to other investigators. For example, the normative point-of-gaze and pupil diameter data already generated in the current study could be used to select specific stimuli for future studies. These stimuli could also be used during functional neuroimaging or electrophysiological recording studies, alone or in conjunction with static images, to ask questions about how the primate brain represents social context or sequential information during social interactions. These video stimuli will be made available to other investigators and should be requested from the corresponding author.In summary, the study of social neuroscience, like other areas of neuroscience, can benefit greatly from comparative studies in humans and animal models. This is especially true if studies utilize stimuli that closely reflect naturalistic conditions and use similar methods (such as eye-tracking and pupillometry) across species. Understanding how different kinds of experimental manipulations  perturb specific aspects of social cognition  will greatly facilitate the development of new insights into the underlying neurobiology of human psychiatric illnesses.Table S1Pupil diameter analysis using the gray screen normalization method and comparing the three main video categories.(DOCX)Click here for additional data file.Table S2Pupil diameter analysis using the gray screen normalization method and comparing the Subject Directed Social video sub-categories.(DOCX)Click here for additional data file.Table S3Pupil diameter analysis using the gray screen normalization method and comparing the Nature video sub-categories.(DOCX)Click here for additional data file.Video S1A sample video from the Nature video category.(MOV)Click here for additional data file.Video S2A sample video from the Naturalistic Social video category.(MOV)Click here for additional data file.Video S3A sample video from the Subject Directed Social video category.(MOV)Click here for additional data file.Video S4A sample video from the Nature video category with black cross-hairs superimposed to indicate one animal's point-of-gaze.(MOV)Click here for additional data file.Video S5A sample video from the Naturalistic Social video category with black cross-hairs superimposed to indicate one animal's point-of-gaze.(MOV)Click here for additional data file.Video S6A sample video from the Subject Directed Social video category with black cross-hairs superimposed to indicate one animal's point-of-gaze.(MOV)Click here for additional data file.


Drosophila indirect flight muscles (IFM) rely on an enhanced stretch-activation response to generate high power output for flight. The IFM is neurally activated during the male courtship song, but its role, if any, in generating the small amplitude wing vibrations that produce the song is not known. Here, we examined the courtship song properties and mating behavior of three mutant strains of the myosin regulatory light chain (DMLC2) that are known to affect IFM contractile properties and impair flight: (i) Dmlc2Δ2–46 (Ext), an N-terminal extension truncation; (ii) Dmlc2S66A,S67A (Phos), a disruption of two MLC kinase phosphorylation sites; and (iii) Dmlc2Δ2–46;S66A,S67A , expressing both mutations. Our results show that the Dmlc2 gene is pleiotropic and that mutations that have a profound effect on flight mechanics  have minimal effects on courtship song. None of the mutations affect interpulse interval (IPI), a determinant of species-specific song, and intrapulse frequency (IPF) compared to Control (Dmlc2+ rescued null strain). However, abnormalities in the sine song (increased frequency) and the pulse song (increased cycles per pulse and pulse length) evident in Ext males are not apparent in Dual males suggesting that Ext and Phos interact differently in song and flight mechanics, given their known additive effect on the latter. All three mutant males produce a less vigorous pulse song and exhibit impaired mating behavior compared to Control males. As a result, females are less receptive to Ext, Phos, and Dual males when a Control male is present. These results open the possibility that DMLC2, and perhaps contractile protein genes in general, are partly under sexual selection. That mutations in DMLC2 manifest differently in song and flight suggest that this protein fulfills different roles in song and flight and that stretch activation plays a smaller role in song production than in flight.The  Drosophila, is generally considered one of the main driving forces in the evolution of insects due to the fact that it facilitates escape from predators, dispersal, colonization of new niche, and subsequent speciation Drosophila, males generate species-specific courtship songs of rhythmic pulses and sinusoidal hums generated by small amplitude wing vibrations Drosophila) required for powered flight Drosophila mating does not occur aerially and therefore the males do not need to overcome drag and lift forces during singing. Furthermore, males beat only one wing at a time during singing  ranging from 150–350 Hz Muscle is well known for its ability to generate force through a contractile mechanism that involves sliding of myosin-containing thick filaments pass actin-containing thin filaments. A fundamental outcome of the force is movement, principal among which is animal locomotion. The ability to fly, present in the majority of insect species including Drosophila myosin regulatory light chain (DMLC2) (see mlc2Δ2–46 or Ext) and alanine substitutions of two myosin light chain kinase phosphorylation sites  have no major effect on IFM ultrastructure mlc2Δ2–46 (Ext) reduces flight ability, wing beat frequency, and the frequency of maximum power output by moving the myosin heads farther away from actin target zones mlc2S66A,S67A (Phos) further reduces all three aforementioned parameters by increasing the angular mobility of myosin heads which effectively reduces the proportion of myosin heads that are properly oriented towards actin target zones for strong binding Drosophila transgenic line Dmlc2Δ2–46;S66A,S67A  In order to understand how muscle protein genes are utilized for the two distinct IFM-driven behaviors, and to gain insight into the contractile mechanism used for song generation, we tested the effect of mutations of the LC2) see , a highlThe above findings on the DMLC2 mutations indicate that the N-terminal extension and the phosphorylation of serines 66 and 67 serve to enhance power output and improve flight performance. In this study, we examined the effect of Ext, Phos, and Dual on courtship song properties, mating behavior, and reproductive success. We report that males from all three DMLC2 mutant strains are capable of generating a courtship song with sine and pulse elements. Deletion of the N-terminal extension affects several song parameters; however, most song parameters are normal in Dual. The results indicate that the two attributes of DMLC2, N-terminal extension and phosphorylation, interact very differently in the mechanisms responsible for flight and song.Miller et al. All the mutant males  are capable of generating pulse songs and sine songs as shown by representative oscillograms  and the song audio clips , and S4.Males of the single mutant strains produce sine songs with abnormally high SSF (215±5 Hz for Ext and 176±4 Hz for Phos) compared to Control males (131±1 Hz). In contrast, the SSF of Dual males is similar to that of Control males  indicating that the single mutations are mutually suppressing each other’s effects . There wDmlc2 mutant males . While neither single mutant affects PDC, Dual males show approximately 85% decrease in PDC compared to Control males . AMP-RT We tested males in single pair mating assays with wild type (OR) females and found that all the mutant males engage in courtship, albeit at much reduced levels compared to Control . The winTo understand if the mutations’ effects on song and courtship affect their reproductive success, we conducted pairwise mating competitions of each mutant strain with a Control male for an OR wild-type female. All the mutant males were outcompeted by Control males as determined by female preference index (FPI) . In the Dmlc2 mutations studied here have been extensively characterized for their effect on IFM and myofilament lattice structure, muscle fiber mechanical properties, and whole organismal flight performance Dmlc2 gene are under sexual selection.The Our finding that all three DMLC2 mutant strains are capable of producing pulse and sine songs  show tha2+ activation. This possibility is rendered more likely by the observation that neither Ext nor Phos affect calcium-activated tension in skinned IFM fibers Second, song production may rely largely or entirely on CaResults showing that none of the DMLC2 mutations have an effect on IPI, the interval between successive pulses  suggest Drosophila modulates IFM power output during flight through a calcium-dependent mechanism Recent studies have shown that The observed differences in pulse song among the mutant strains also point to a calcium-dependent mechanism. Unlike Phos and Dual, Ext shows longer PL compared to Control . Calciumin vivo lattice spacing with Dextran T-500. As inertial power is the main contributor to mechanical power at flight initiation The nature of the interaction between N-terminal mutation and the phosphorylation mutation in the Dual strain is intriguing. While the two mutations have been reported to combine additively to impair flight performance in Dual Despite the relatively mild effects on courtship song, the mutant males do not perform well in single pair mating assays with wild type OR females. Ext and Phos males, but not Dual males, showed significant reductions in CI while all three strains exhibited greatly reduced WEI compared to Control males . As the In the courtship competition assays, wild-type females overwhelmingly preferred Control males over males of any of the three mutant strains . Ext andWhile questions remain about which specific pulse song component is most influential in female choice, several studies have indicated that PDC is more important than IPF and IPI Drosophila courtship that have specific effects in other physiological processes Dmlc2 (and perhaps other muscle protein genes) are pleiotropic with respect to flight and song. As these two behaviors are subject to different selection pressures, a comparative analysis of mutation effects on flight and song can reveal how individual genes respond to competing pressures and evolve under different selection mechanisms.Here, we show for the first time that mutations in a gene encoding a muscle contractile protein (myosin regulatory light chain) have distinct effects on courtship song and mating behavior. This adds a new category of genes known to influence D. melanogaster stock is a laboratory strain of Oregon R (OR). The generation of the following transgenic strains used in this study has been previously described: (i) Control, strain expressing full length myosin regulatory light chain (Dmlc2+) mlc2Δ2–46) mlc2S66A,S67A) mlc2Δ2–46; S66A,S67A) The wild type http://stockcenter.ucsd.edu/info/food_cornmeal.php for ingredients and recipe). or directly digitized using Goldwave v5.58 software The recorded songs from the INSECTAVOX were extracted either with Audacity  female introduced into a plexiglass mating chamber (1 cm diameter × 4 mm height). The courtship activities were video recorded until successful copulation, or longer (30–50 minutes) in the absence of copulation, using a 65X SD camcorder (Samsung) mounted on a tripod. The assays were done under room light at 22°C temperature and 70% humidity. From the videos, CI and WEI were calculated for each male as described in 2 and one of them was marked on its thorax with a neon-orange paint using a fine point paintbrush. Two males (one marked and one unmarked) of different transgenic strains and one wild type OR female were introduced into a plexiglass mating chamber (1 cm diameter× 4 mm height) and courtship activities were video recorded for 30–50 minutes using a 65X SD camcorder (Samsung) mounted on a tripod (Vanguard). The assays were done under light at 22°C temperature and 70% humidity. The competition videos were observed and the strain of the male that succeeded in courting and copulating with the wild type female was noted. Female preference index (FPI) was calculated as the relative advantage of the mutant male over the Control male , with values considered significant at p<0.05. One-way ANOVA followed by a post-hoc test by Fischer’s LSD pairwise comparisons between any two groups was used to examine differences between the Ext, Phos, Dual and Control for all variables. For statistical analysis on courtship song data, the average value of each song parameter was calculated for each fly; hence the number of statistical samples is the number of flies.Table S1Male courtship song and behavioral parameters.(DOCX)Click here for additional data file.Audio S1Male courtship song (sine and pulse) sample of Control male in the presence of a wild type (Oregon R) female.(MP3)Click here for additional data file.Audio S2Male courtship song (sine and pulse) sample of Ext male in the presence of a wild type (Oregon R) female.(MP3)Click here for additional data file.Audio S3Male courtship song (sine and pulse) sample of Phos male in the presence of a wild type (Oregon R) female.(MP3)Click here for additional data file.Audio S4Male courtship song (sine and pulse) sample of Dual male in the presence of a wild type (Oregon R) female.(MP3)Click here for additional data file.Video S1Success of Control male over Ext male for wild type (Oregon R) female in courtship competition.(MP4)Click here for additional data file.Video S2Success of Control male over Phos male for wild type (Oregon R) female in courtship competition.(MP4)Click here for additional data file.Video S3Success of Control male over Dual male for wild type (Oregon R) female in courtship competition.(MP4)Click here for additional data file.Video S4Success of Phos male over Ext male for wild type (Oregon R) female in courtship competition.(MP4)Click here for additional data file.Video S5Success of Dual male over Ext male for wild type (Oregon R) female in courtship competition.(MP4)Click here for additional data file.


Cucumis sativus L.).High-resolution cytogenetic map can provide not only important biological information on genome organization but also solid foundation for genetic and genomic research. The progress in the molecular and cytogenetic studies has created the basis for developing the cytogenetic map in cucumber (in situ hybridization (FISH) analysis on cucumber pachytene chromosomes. Together with our previously constructed cytogenetic maps of three cucumber chromosomes , cucumber has a complete cytogenetic map with 76 anchoring points between the genetic, the cytogenetic and the draft genome assembly maps. To compare our pachytene FISH map directly to the genetic linkage and draft genome assembly maps, we used a standardized map unit—relative map position (RMP) to produce the comparative map alignments. The alignments allowed a global view of the relationship of genetic and physical distances along each cucumber chromosome, and accuracy and coverage of the draft genome assembly map.Here, the cytogenetic maps of four cucumber chromosomes  were constructed by fluorescence We demonstrated a good correlation between positions of the markers in the linkage and physical maps, and essentially complete coverage of chromosome arms by the draft genome assembly. Our study not only provides essential information for the improvement of sequence assembly but also offers molecular tools for cucumber genomics research, comparative genomics and evolutionary study. Cucumis sativus L., 2n = 2x = 14), which belongs to the family Cucurbitaceae, is an economically important crop as well as a model system to study biologically relevant characters such as sex determination[http://cucumber.genomics.org.cn/page/cucumber/index.jsp), other two cucumber lines (the North American pickling type inbred line ‘Gy14’ and the North-European Borszczagowski cultivar ‘B10’ ) were also sequenced[Cucumber . To intuitively display the relationship between the genetic and physical distances, the positions of FISH mapped loci were transformed into the product of fraction length and the total length (cM) of corresponding linkage group in some studies,22,27,28In the present work, we constructed the molecular cytogenetic maps of the remaining four cucumber chromosomes  by FISH analysis using 8–14 fosmid clones per chromosome. These fosmid clones distributed at regular intervals across the chromosome-level cucumber draft genome assembly maps and some clones carried major genetic markers. Together with our previously published data,28, cucuTo construct the molecular cytogenetic maps of the remaining four cucumber chromosomes, we selected a set of fosmid clones distributed at regular intervals across the chromosome-level cucumber draft genome assembly maps. We first determined the physical order of adjacent fosmid clones based on their positions in draft genome assembly map by dual-color FISH on somatic metaphase chromosomes Figures a1-a4. OnA total of 16 fosmid clones distributed at an average distance of 1.68 Mbp along the cumulative physical length (29149675 bp) of the chromosome 1 were selected for FISH mapping. Among these 16 clones, we confirmed that 13 clones (1–1 to 1–13) showed unique hybridization signals on mitotic metaphase chromosomes by FISH  were selected for FISH mapping. Two clones which showed repetitive FISH signals in mitotic metaphase chromosomes were discarded (data not shown), one clone Ch3-1) yielded strong signals on other chromosomes but not signals on chromosome 3 was also discarded  were selected for FISH mapping. Two clones which showed repetitive FISH signals in mitotic metaphase chromosomes were discarded (data not shown). The remaining 11 (4–1 to 4–11) with unique hybridization signals were chosen to make a multicolor FISH cocktail mix together with the probe Type III  were selected. 6 clones which showed repetitive FISH signals in mitotic metaphase chromosomes were discarded (data not shown), one clone (Ch5-1) showed strong FISH signals on other chromosome pair but not signals on chromosome 5 was discarded [We next compared the distributions of fosmid clones on the 9930 draft genome assembly map and our cytogenetic map Figure . The RMPType IV). Each ofWhile a total of 72.2-fold genome coverage was generated for the cucumber genome. However, the total length of the assembled cucumber genome was only 243.5 Mb, about 30% smaller than the 367 Mbp cucumber genome. Consistent with this proportion, several types of satellite sequences accounted for about 20-30% of the total nuclear DNA in cucumber based on the proportion of rDNA (3.3% of the genome), Type III (4.04%), Type I/II and Type IV (15.92%) on mitotic chromosomes. These rMis-assemblies are common when draft genome sequences have been generated by de novo assembly of sequences obtained with NGS technologies,33. SincThe present developed molecular cytogenetic map integrated with genetic linkage map and physical map not only verifies the quality of 9930 draft genome assembly and provides essential information for the improvement of cucumber genome assembly but also offers molecular tools for cucumber genomics research. The majority FISH-mapped fosmid clones carried major genetic markers thus our analysis revealed the genetic and physical relationships in specific chromosome regions. This can provide crucial physical information to positional cloning projects that might otherwise be fruitlessly aimed at a target gene on the basis of markers that are very tightly linked but physically distant. In addition, the integrated molecular cytogenetic map also forms a solid foundation for future FISH-based comparative genomics and evolutionary studies. The reference set of fosmid clones can serve as a universal set of cytogenetic markers to study synteny and chromosomal rearrangements between cucumber and other cucurbit genomes like our previous work.The cytogenetic map incorporating genetic, cytological and physical data can contribute significantly to the improvement of sequence assembly by confirming the physical positions of markers on the linkage groups, identifying mis-assembled clones and evaluating the size of the putative remaining gaps. Such map also offers molecular tools for cucumber genomics research, comparative genomics and evolutionary study. In the present work, we constructed the molecular cytogenetic maps of four cucumber chromosomes. Together with our previous results, cucumber has a complete molecular cytogenetic map. Furthermore, we used a standardized map unit—relative map position (RMP) to produce the comparative map alignments. The alignments showed that draft genome assembly map provided excellent coverage of the corresponding chromosomes. The reference set of fosmid clones can serve as a universal set of cytogenetic markers for comparative genomics study between cucumber and its close relatives.C. sativus ‘Chinese long’ inbred line 9930 was used for cytological studies. Root tips were harvested from germinated seeds, pretreated in 0.002 M 8-hydroxyquinoline at room temperature for 2 h to accumulate metaphase cells, and fixed in methanol:glacial acetic acid (3:1). Root tips were macerated in 2% cellulase Onozuka R-10  and 1% pectolyase Y-23 (ICN) at 37°C for 2 h and squashes were made in the same fixative. Young panicles were harvested and fixed in 3:1  Carnoy’s solution. The procedure for meiotic chromosome preparation was largely the same as that used for preparing mitotic chromosomes from root tips with the following modification: anthers were digested in the enzyme mixture for 4.5 h at 37°C. The digested anthers were macerated on glass slides in 50% acetic acid solution with fine-pointed forceps and then “flame-dried” over an alcohol flame.All fosmid clones were provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. The fosmid clones screened from the 9930 fosmid library distributed at regular intervals across the chromosome-level cucumber draft genome assembly maps and some clones carried major genetic markers. Fosmid DNA was isolated using QIAGEN plasmid midi kit and further purified by Plant DNeasy spin columns (QIAGEN). The centromere-specific DNA probe Type III repeat was usedhttp://cucumber.genomics.org.cn/). These RMP values were used to produce the comparative map alignments.We used relative map position (RMP) units for direct comparative analysis between the cytogenetic, the genetic linkage, and draft genome assembly maps of cucumber. The RMP values for the pachytene cytogenetic map were the percentage of the distance (in μm) from the FISH site to the end of the short arm relative to the total length of the chromosome (in μm). In order to establish the position of each clone along the chromosomes, hybridization signals on the same 5 pachytene chromosomes without morphology distortion showing all hybridization spots were measured. The RMP values for the SSR linkage map were theThe authors declare that they have no competing interests.YHH designed the study, performed the analysis and wrote the paper. SWH and ZYL participated in the design of the study, analyzed the data and critically revised the manuscript. JYS, ZHZ and XZ carried out the experiments and acquisition of original data. JYS analyzed the data and participated in writing. All authors read and approved the final manuscript.FISH-mapped fosmid clones and their corresponding RMPs in genetic, cytogenetic, and draft genome assembly maps.Click here for filePositions of three identified misassembled clones.Click here for fileFISH results of three identified misassembled clones. a The signals of Ch3-1 (red) and 3–2 (green) weren’t in the same chromosome pair. b The signals of Ch5-1 (red) and 5–10 (green) weren’t in the same chromosome pair. c Ch5-2 (red) was not in the long arm with 5–11 signals but in the short arm of chromosome 5.Click here for fileHeterochromatin distribution on cucumber pachytene chromosomes 1, 3–5.Click here for file


Spheroid formation is one property of stem cells—such as embryo-derived or neural stem cells—that has been used for the enrichment of cancer stem-like cells (CSLCs). However, it is unclear whether CSLC-derived spheroids are heterogeneous or whether they share common embryonic stemness properties. Understanding these features might lead to novel therapeutic approaches. Ovarian carcinoma is a deadly disease of women. We identified two types of spheroids (SR1 and SR2) from ovarian cancer cell lines and patients' specimens according to their morphology. Both types expressed stemness markers and could self-renew and initiate tumors when a low number of cells were used. Only SR1 could differentiate into multiple-lineage cell types under specific induction conditions. SR1 spheroids could differentiate to SR2 spheroids through epithelial–mesenchymal transition. Alkaline phosphatase (ALP) was highly expressed in SR1 spheroids, decreased in SR2 spheroids, and was absent in differentiated progenies in accordance with the loss of stemness properties. We verified that ALP can be a marker for ovarian CSLCs, and patients with greater ALP expression is related to advanced clinical stages and have a higher risk of recurrence and lower survival rate. The ALP inhibitor, levamisole, disrupted the self-renewal of ovarian CSLCs in vitro and tumor growth in vivo. In summary, this research provides a plastic ovarian cancer stem cell model and a new understanding of the cross-link between stem cells and cancers. This results show that ovarian CSLCs can be suppressed by levamisole. Our findings demonstrated that some ovarian CSLCs may restore ALP activity, and this suggests that inhibition of ALP activity may present a new opportunity for treatment of ovarian cancer. Indeed, spheroid formation is a unique common phenomenon in ascites fluid of patients with ovarian cancers. It is still unclear whether spheroids formed from ovarian cancer are a single kind of CSLC [Differentiated descendant tissue cells do not exhibit some capacities of stem cells, such as being viable in suspension culture, and spheroid formation . Stem ce of CSLC -8. To daOur group previously characterized tumor-initiating spheroids expressing the surface markers CD44 and CD117 (c-Kit) . Others In the current study, we hypothesized that ovarian CSLCs would prove to possess different stemness status. We observed two types of ovarian CSLCs. Both of them fulfilled the definition of a CSLC, but only one of them possessed translineage differentiation capability. We also found that ovarian cancer development involves an epithelial–mesenchymal transition (EMT) in ovarian CSLCs. The suppression of alkaline phosphatase (ALP) activity inhibited the self-renewal and tumorigenicity of ovarian CSLCs. These findings provide evidence of a stemness hierarchy in ovarian cancer development. We also suggest that ALP might be a therapeutic target for women with ovarian cancers.+CD133+ population increased significantly in SKOV3SR1 and A2780SR2 cells, and the CD44−CD133+ population also increased in A2780SR2 cells  cell lines using stem cell suspended-culture conditions , which ps Figure  and 1C. s Figure  and 1E. s Figure  and 1G dA diversity of morphology was observed in the suspension cultures of original CP70 cells . To assein vivo animal model of tumorigenesis. As few as 1000 CP70SR1SC cells could propagate a visible tumor nodule 6 weeks after implantation  and pathogenesis model [P<0.001) (p=.02) and lower overall survival (p=.004). Multivariate analysis revealed that ALP expression was also strongly associated with cancer stage and nuclear grade, indicating ALP was not an independent factor in the recurrence and survival in patients  , Table 2P<0.001)  and 6F , in which the mesenchymal type GSCs are more aggressive and tumorigenic than the proneural type . AlthougTranslineage differentiation is one capacity of normal stem cells, and it determines the hierarchical status of cells. The stemness definition of CSLCs remains controversial ; howeverAccording to a recent study, there is a cancer-prone stem cell niche at the junctional area of the ovarian surface epithelium, and one population of normal cells bearing stem-like properties exists in this specific site . This fiWe speculate that the presence of cancer tissues with high ALP expression might imply that ovarian cancer cells have gained some stemness characteristics and that this might be related to the high rates of recurrence and death of patients with such tumors. As for stemness, the regulation of ALP expression is associated with β-catenin signaling  and epigThe association between EMT and stemness remains controversial. The induction of EMT promoted the growth of cancers exhibiting stemness properties -37. HoweIn summary, this research supports a plastic ovarian cancer stem cell model and provides a new understanding of the cross-link between stem cells and cancers. ALP was confirmed as a potential therapeutic target for treating women with ovarian cancers. An immediate trial of a clinically proven ALP inhibitor may facilitate the translation of cancer stem cell research to patient therapy.http://www.atcc.org). The original human ovarian cancer cell line A2780 and its cognate cisplatin-resistant CP70 were obtained in 2007 from Dr. Tim Huang's lab . The cell lines used were tested by the Bioresource Collection and Research Center  for identity verification by DNA profiling of short tandem repeat sequences. DNA profiles were compared manually to the ATCC and European Collection of Cell Cultures database. Earlier passages of all cell lines were maintained in several cryovials in liquid nitrogen in our laboratory. All original cell lines were cultured in RPMI-1640 (Invitrogen) supplemented with nonessential amino acids (Invitrogen), sodium pyruvate (Invitrogen), and either 10%–20% standard fetal bovine serum  or different growth factors, and plated in standard cell culture dishes (Corning). To enrich spheroid cells carrying stemness properties, the original cancer cells from cell lines or primary culture were harvested and cultured in ultra-low attachment plates (Corning). Following the culture protocols for ESCs and neuronal stem cells, cells were cultured in Dulbecco's modified Eagle medium (DMEM) with a high or low glucose concentration, or DMEM/F12 medium containing 0%–5% FBS qualified for use with human ESCs  or KnockOut™ Serum Replacement (Invitrogen), supplied with or without basic fibroblast growth factor , and either human recombinant epidermal growth factor  or 2-mercaptoethanol (Millipore), depending on the protocol modified for different ovarian cancer cell lines or patients' samples. The cells were cultured in suspension, and starting from 14 days, the cultures were examined every day for sphere formation. Spheres were then dissociated and passaged at least eight times in 2 months to generate spheres, which are henceforth referred to as “SR cells”. Collection of each clinical specimen adhered to the protocol approved by the institutional review board.The original human ovarian cancer cell lines OVCAR-3 and SKOV3 were purchased from ATCC . The stem-like phenotypes of the parental CP70 and SR cells were assessed by detecting specific stem cell gene markers using flow cytometry (BD Biosciences) and immunocytochemistry. Cells were stained intracellularly with antibodies against human NANOG (GeneTex), OCT4 (Millipore), stagespecific embryonic antigen 4 , and SOX2 (Millipore), according to the manufacturer's instructions as described previously , 21. Thein vivo tumor xenograft studies of CSLCs, female nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice were obtained from the Laboratory Animal Center of the National Taiwan University College of Medicine . Six-week-old mice were used for the experiments unless otherwise indicated. The rules of the Animal Protection Act of Taiwan were strictly followed, and all animal procedures were approved by the Laboratory Animal Care and Use Committee of the National Defense Medical Center. Various numbers of SR cells and original cancer cells were injected subcutaneously or intraperitoneally (i.p.) into NOD/SCID mice. Tumor vessels in the tumor nodule in live mice were quantified after 14 days by measuring the tumor uptake of the blood pool probe AngioSense™ 750 [For nse™ 750 , 78 and For differentiation studies, SR cells were dissociated and cultured in standard medium containing 5% standard FBS in standard dishes (Corning) coated with FNC Coating Mix® (AthenaES). The morphology of spontaneously differentiated SR cells was observed, and the translineage-differentiating capacity was tested based on various protocols for inducing the differentiation of adipocytes, osteocytes, chondrocytes, and neural cells. SR cells were dissociated and allowed to form aggregates for 2 days, and neural differentiation was induced. The cells were transferred to poly-d-lysine-coated dishes and cultured in common medium containing retinoic acid for 4 days. Neural differentiation of SR cells was also induced using Neurobasal® (Invitrogen), DMEM/F12 (Invitrogen), or DMEM/F12 knockout (Invitrogen) medium supplied with B27 (Invitrogen), N2 (Invitrogen), human recombinant bFGF (PeproTech), and EGF (PeproTech). Neural cell differentiation was confirmed using immunostaining with antibodies against α-internexin (GeneTex) and βIII-tubulin (GeneTex), and pan-neuronal markers . Mesenchymal differentiation of SR cells was induced using a commercial StemPro® Differentiation Kit (Invitrogen). Adipocyte formation was confirmed using oil red O staining. Osteogenic differentiation was confirmed by detecting ALP activity using a BCIP/NBT tablet kit (Sigma) and intracytoplasmic calcium crystals were visualized using Alizarin red staining (Lifeline Cell Technology). Chondrogenic differentiation was confirmed with an Alcian blue staining kit (Lifeline Cell Technology).6 CP70 cells. Three days after the injection, the mice were divided into two groups that received either 50 mg/kg levamisole (Sigma) or vehicle orally every day for 21 days. To evaluate the levamisole treatment response of tumors, fluorodeoxyglucose (FDG)/micro-positron emission tomography (PET) scanning was performed 4 weeks after tumor inoculation. The mice anesthetized with isoflurane were injected intravenously with 250 µCi 18F-FDG and were imaged 30 minutes later using a BioPET system .All NOD/SCID mice were initially inoculated i.p. with 1 × 10Paraffin-embedded tumor tissues and tissue microarray slides were analyzed by the Department of Pathology, National Defense Medical Center, Taipei, Taiwan , 46. Sev2 test or Fisher's exact test. The disease-free interval and overall survival time were assessed by Cox regression analysis. Kaplan–Meier survival curves were compared using the log-rank and Breslow test. The significance level was defined as p<.05.The SPSS software package  was used for statistical analysis. Correlations between ALP expression and clinicopathological characteristics were identified by the χ