Bubo bubo) in the Iberian Peninsula. During the last century, the species was the object of extermination programs, suffering direct persecution by hunters until the 70’s. Moreover, during the last decades the eagle owl was severely impacted by increased mortality due to electrocution and the decline of its main prey species, the European rabbit (Oryctolagus cuniculus). In recent times, the decrease of direct persecution and the implementation of some conservation schemes have allowed the species’ demographic recovery. Yet, it remains unknown to which extent the past population decline and the later expansion have influenced the current species’ pattern of genetic diversity. We used eight microsatellite markers to genotype 235 eagle owls from ten Spanish subpopulations and analyse the presence of genetic signatures attributable to the recent population fluctuations experienced by the species. We found moderate levels of differentiation among the studied subpopulations and Bayesian analyses revealed the existence of three genetic clusters that grouped subpopulations from central, south-western and south-eastern Spain. The observed genetic structure could have resulted from recent human-induced population fragmentation, a patchy distribution of prey populations and/or the philopatric behaviour and habitat selection of the species. We detected an old population bottleneck, which occurred approximately 10,000 years ago, and significant signatures of recent demographic expansions. However, we did not find genetic signatures for a recent bottleneck, which may indicate that population declines were not severe enough to leave detectable signals on the species genetic makeup or that such signals have been eroded by the rapid demographic recovery experienced by the species in recent years.The study of the demographic history of species can help to understand the negative impact of recent population declines in organisms of conservation concern. Here, we use neutral molecular markers to explore the genetic consequences of the recent population decline and posterior recovery of the Eurasian eagle owl ( Animal populations may be reduced and isolated by both natural and anthropogenic factors. Habitat fragmentation by means of the transformation of natural and semi-natural habitat may lead to a patchy distribution of populations and communities and it is considered as a major paradigm in conservation biology . FragmenBubo bubo) in Europe experienced such dynamics in their recent history. This owl is the largest strigiform in the world and it is widely distributed in the Palearctic    and the K method , as implarvester . The secDistruct .GenAlEx to calculate pairwise FST and F’ST values between subpopulations. We used Mantel tests to analyse the relationship between the linearized estimates of pairwise FST and F’ST and the logarithm of geographical distances between pairs of populations [Ne) of groups using the LD method implemented in Neestimator 1.3 [Ne estimates assuming monogamy, excluding all allele frequencies lower than 0.05, and selecting a parametric procedure to construct 95% confidence intervals.In order to further explore the spatial pattern of genetic differentiation, we used ulations . Moreoveator 1.3 . We calcBottleneck 1.2.02 [ps), 22% multi-step mutations (pg) and a variance of 3.1 among multiple steps, as recommended for microsatellite data [The genetic signatures of the recent population bottlenecks and expansions of the Eurasian eagle owl in the Iberian Peninsula were explored using the software k 1.2.02 . We testk 1.2.02 . We assuite data . The sigk) over the range of allele sizes (r) since it is expected that during a bottleneck the random loss of alleles will result in a reduced M-ratio (M = k/r). To determine the significance of the reduction of M, the observed value is compared to a distribution of values obtained from simulated populations considering a hypothetical pre-bottleneck Ne, the mutation rates of loci (μ), the proportion of multi-step mutations (pg) and the mean size of multi-repeat mutations (Δg). Critical_M software was used to calculate critical values (Mc) set at the lower 5% tail of the distribution below which it can be assumed that the population has experienced a significant reduction in size [Neμ), considering a mutation rate (μ) of 5x10-4  and dif species .Msvar 1.3 [N1) started to decrease (or increase) linearly or exponentially some time ago (T) towards the current population size (N0). Based on this assumption and using the MCMC approach the method not only allows the estimation of N0 and N1, but also T. Mutation rates (θ = 2N0μ) were assumed under the SMM model and, as for the rest of the parameters, we considered wide uninformative priors under a lognormal distribution. The generation time of Eurasian eagle owl was set at 4 years, the average age of the reproductive population . Because we tested both recent or ancient expansion and bottleneck processes, we ran the program five independent times under the exponential growth model, using different hyperprior means for T and N0, to represent stable, decreasing or increasing demographic histories at different times , the median (50%) and upper quantile (90%) of the posterior distributions.Finally, we used svar 1.3 , 49 to int times . The totMicro-Checker indicated that null alleles may be present by detecting a significant heterozygote deficiency at five out of the eight employed markers, possibly as a consequence of cryptic population structure   . HoweverStructure analyses without considering prior population information indicated that the number of genetic clusters best fitting the data set was K = 1 (i.e. LnPr(X|K) reached a maximum value for K = 1; K = 3 was the most supported cluster solution , but in this case the distant Sierra de Huelva and Madrid SE showed the highest differentiation (0.387). Moreover, both statistics showed a significant correlation with the geographical distance between sites .For the eight populations with samples sizes higher than six individuals, all pairwise FCT = 0.02; FSC = 0.05; FIS = 0.17; FIT = 0.22; all p = 0.001). Considering these groups in a one-way ANOVA, we did not find significant differences in descriptive statistics of genetic diversity among them .Despite the low levels of genetic differentiation found among sampling sites, AMOVA analyses statistically supported the reported substructure and maximized the differentiation among the inferred clusters when Cádiz was assigned to the south-western cluster, revealing that 1.84% of the global variation is found among clusters, 4.39% among sampling sites within these three groups, 16.04% among individuals within sites and the remaining 77.73% corresponds to individual variation  F1,5 = 0.6; p = 0.58; (NE) F1,5 = 0.11; p = 0.9; (HO) F1,5 = 0.44; p = 0.67; (uHE) F1,5 = 1.15; p = 0.39; (FIS) F1,5 = 0.36; p = 0.72N, number of genotyped individuals; Bottleneck tests yielded similar results when loci that showed a significant homozygote excess were excluded from the analyses . The effective population N1 decreased over 10 times from a larger ancestral effective size estimated around 25,119 individuals , to the current effective size N0 of 3,162 . The obtained posterior densities of N0, N1 and T slightly varied over the different runs and the different priors used, depicting in all cases a population decline of one order of magnitude in the effective population size dated thousands of years ago  of 5x10-4; M, calculated as M = k/r, where k is the number of alleles and r is the range of allele sizes; MC, critical value set at the lower 5% tail of the distribution below which it can be assumed that the population has experienced a significant reduction in size.We did not find significant evidences of recent bottlenecks at any of the inferred groups using Bayesian clustering methods and AMOVAs analyses Aquila adalberti) [Aquila fasciata) [In this study we found some genetic signatures which could be in part attributable to the recent population dynamics experienced by eagle owls in the Iberian Peninsula, but also probably related with the species’ past demographic history and traits. Concretely, we found a weak but consistent pattern of genetic structure revealed by both Bayesian clustering and AMOVAs analyses, and a significant correlation between genetic and geographical distances. In the Iberian Peninsula, spatial genetic patterns like this have been reported for other long-lived raptor species that also experienced recent population declines as the Spanish imperial eagle  , 51 and asciata) . Such spasciata) , 54. Howasciata) . Insteadasciata) , 57.Bottleneck analyses detected population expansions in some of the inferred groups, a pattern that was even consistent when excluding loci that deviate from HWE. These findings were also supported by the moderated levels of genetic diversity and the large effective population sizes estimated for the studied populations. However, it remains unclear whether subsequent demographic changes can erase the genetic signatures of recent demographic bottlenecks, which is likely to depend among other things on the population growth rate and the impact of immigration and admixture with individuals from other populations during the expansion phase [Bottleneck analyses. SMM showed a higher power, despite for most microsatellite loci TPM is thought to be more adequate [Demographic analyses failed to detect genetic signatures of recent bottlenecks in the inferred groups, which may indicate that past population declines were not severe enough to leave detectable signals on the species genetic makeup. Thus, a possibility is that the decline of eagle owl populations in the recent past has not reduced effective population sizes below the threshold that is necessary to produce genetic signals associated with such demographic events. Alternatively, the recovery and range expansion of the species over the last 40 years could have eroded some of the genetic signatures associated with local population declines. Unlike most large raptors such as eagles or vultures, the eagle owl has high reproductive rates and this may have favored a rapid demographic recovery . Furtheron phase , 66. Stuadequate .Msvar analyses found signatures of an ancient population collapse that started after the end of the last glacial period. Both this and previous studies have generally found that the Storz and Beaumont method [Bottleneck analyses [Ailuropoda melanoleuca) [Martes pennanti) [Ulmus laevis) [At the whole population level, t method  shows a analyses . Accordinoleuca) , fishersennanti)  or Europ laevis)  and Iber laevis) . Althoug laevis) . Overall laevis) , 73, 74.2). In a wider geographical context, one of the most important anthropogenic negative impacts on population connectivity are probably electrocutions. Electrocution became the major source of non-natural mortality of eagle owls after the legal protection of raptors in Spain [Although eagle owls can potentially disperse large distances, our results agree with recovery data of banded individuals and evidence of limited dispersal among subpopulations , supportin Spain , 76, 77.in Spain . In densin Spain . This efin Spain , 79.Besides, some natural factors might have also increased population fragmentation. Eagle owl traits such as philopatry, the tendency to breed close to birthplaces , 81, or HE = 0.61; [Bubo blakistoni) on Hokkaido Island in Japan, a population with a significant decline of allelic richness over the last three decades [Overall, our analyses indicate that population declines were not severe enough to leave detectable genetic signals or that such signals have been eroded by the rapid demographic recovery experienced by the species in recent years. The combination of limited dispersal and high turnover and reproductive rates might have contributed to both the observed patterns of spatial genetic structure and the short-term erosion of the genetic signatures of recent demographic bottlenecks in Iberian eagle owls. In fact, we found moderate levels of genetic diversity and no differences in descriptive statistics of genetic diversity between the inferred clusters. Somewhat lesser levels of genetic variation were reported for the Scandinavian population of eagle owls, which passed a bottleneck below a few hundreds of individuals ; and a  decades . In our  decades .S1 FigK))  and the magnitude of ΔK as a function of K  over 11 runs.(a) Without considering prior population information and (b) using population identity as prior information (“Locprior” option). Plots show the mean (± SD) log probability of the data (ln Pr(X|(DOCX)Click here for additional data file.S1 Tablea, annealing temperature; NA, number of alleles; uHE, unbiased expected heterozygosity; HO, observed heterozygosity. *Significant heterozygote deficiency after Bonferroni correction (p < 0.05).For labelled primers the dye is reported within brackets. T(DOCX)Click here for additional data file.S2 TableIn bold those values changing from the previous simulation.(DOCX)Click here for additional data file.S3 TableFST values  and F’ST values .(DOCX)Click here for additional data file.S4 TableT) represents the date of the change in population size from N0 to N1.N0 and N1 are the current and ancestral effective population sizes respectively. Time ((DOCX)Click here for additional data file.


Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa permits real time monitoring of the pathogens. The murine model was employed to evaluate the performance of four different photosensitizers as mediators in Photodynamic Therapy. While all four photosensitizers, Rose Bengal, porphyrin TMPyP, New Methylene Blue, and TLD1411 demonstrated good to excellent antimicrobial efficacy in planktonic solutions at 1 to 50 μM concentrations, whereas in in vivo the growth delay was limited with 24–48 h delay in pathogen expansion for MRSA, and we noticed longer growth suppression of P. aeruginosa with TLD1411 mediated Photodynamic Therapy. The murine model will enable developing new strategies for enhancement of APDT for chronic wound infections.It is generally acknowledged that the age of antibiotics could come to an end, due to their widespread, and inappropriate use. Particularly for chronic wounds alternatives are being thought. Antimicrobial Photodynamic Therapy (APDT) is a potential candidate, and while approved for some indications, such as periodontitis, chronic sinusitis and other niche indications, its use in chronic wounds is not established. To further facilitate the development of APDT in chronic wounds we present an easy to use animal model exhibiting the key hallmarks of chronic wounds, based on full-thickness skin wounds paired with an optically transparent cover. The moisture-retaining wound exhibited rapid expansion of pathogen colonies up to 8 days while not jeopardizing the host survival. Use of two bioluminescent pathogens; methicillin resistant  Chronic wounds, generally comprising diabetic foot ulcers, pressure ulcers, venous leg ulcers, burn wounds, and wounds “older than 3 months of age”  are a woAny break in the barrier function of the skin and other epithelial layers, predisposes an individual to infections . One of Pseudomonas aeruginosa caused infections, whereas it appears to be less or ineffective against other bacterial strains  provides appe- aling properties. In APDT, a chemical compound (photo- sensitizer) absorbs light photons with sufficient quantum energy resulting in its electronic excited state, from which energy can be transferred to biomolecules or to molecular oxygen. The latter leads to the generation of Reactive Oxygen Species (ROS) or singlet oxygen formation, which can cause cell damage and microorganism’s death as recently reported by in vitro studies, transition to in vivo studies showed variable results, with some pre-clinical models showing good efficacy, whereas the majority of the studies demonstrated a poor translation of the APDT efficacy in vivo.While efficacy of APDT has been demonstrated in various planktonic P. aeruginosa (106) or Vibrio vulnificus (106), allowing observation of wound healing and infection development within individual animals. As wounds remain uncovered, mice had to be kept separate to prevent opportunistic contaminations between animals. 2) was cut down till the subcutaneous areolar tissue using scissors. In the second model a 25 mm2 square shaped wound was created by scarification using a 27G needle. Both skin preparations were inoculated with 108 CFUml-1 MRSA cells on the wounds. To assess the effectiveness of the therapy mice were sacrificed upon treatment and the number of surviving bacteria was quantified following incubation on agar plates.Typically, to quantify a therapy’s efficacy, based on the eradicated bacterial number, one group of hosts need to be sacrificed immediately after treatment and a second group need to be kept up to 50 h to determine the microbiota survival fraction. Blood samples can also be taken to analyze possible bacteremia development. in vivo experimental design stage.The ideal pre-clinical model of hard-to-heal wounds should be based on an immunocompromised host, thus permitting systematic infection. However, that model requires weeks to be established and carries significant risk of sepsis and survival failure. Also the use of immunocompromised animals can dramatically increase the cost of the research. Minimal animal number, wound drying, dressing changes causing wound oxygenation variations, unnecessary animal pain, or large surface area damage are some of the important factors to be considered during the TM based topical infection model for antimicrobial PDT as local antimicrobial therapy, and demonstrate its utility by testing Rose Bengal (RB), porphyrin TMPyP New Methylene Blue (NMB), or TLD1411 mediated APDT.Here we demonstrate a TegadermBy the use of strong skin adhering, vapor and light transmitting dressings , a moistin vivo as time delay until pathogen regrowth is noted.To demonstrate the utility of the murine model, we validated the efficacy of 4 different photosensitizers RB, porphyrin TMPyP, NMB, and TLD1411 as APDT mediators in planktonic solution, demonstrated the need for a covered wound for maintaining an ongoing infection and finally demonstrated the ability to quantify APDT outcome in this murine model, All procedures were approved by the Animal Care Committee (ACC) of University Health Network, Toronto, ON, Canada (protocol number AUP 3303.2). FVB/N mice of both sexes, originally obtained from Xenogen Corporation – Alameda, CA but tested negative for the luciferase gene were used. Animals were housed in 12-h day/night cycles, at 20°C room temperature, granulated food, and water were provided ad libitum.n = 3) to establish the duration of normal wound healing. Groups (ii) to (iv) represent infected wounds without APDT comprising: (ii) Tegaderm dressing, (iii) no dressing, and (iv) removal of dressing at the end of day 2 from established infection to evaluate the need for the transparent dressing to maintain the infection control throughout the observation period. For these groups n = 3 animals were used for each of the two bacterial strains employed here. The APDT treatment groups v) comprised n ≥ 3 × 2 bacterial strains for each of the four photosensitizers were tested. Group (vi) evaluated APDT efficacy versus potential LLLT effects employing a light only group for both bacterial strains.In total 51 mice were required for this study and randomly assigned to one of 6 groups: (i) non-infected mice  was held by tweezers and top layer of the skin is cut off to cause 5 to 6 mm diameter wound.7 CFU bacteria using a pipette tip. For mice in groups ii to vi TegadermTM was applied immediately as per group assignment and kept for the assigned time.Immediately after cutting the skin the wound area was inoculated with10 μl of PBS containing 10TM (Transparent Film Roll 3MTM) were cut and applied to the wound area. According to commercial specification, the dressing is breathable permitting oxygen in and moisture vapor out thus allowing the skin to function normally. TegadermTM is transparent and no light attenuation needs to be considered  strain (XEN)  and Pseudomonas aeruginosa (PAK) were used. PAK is a wild type, commonly studied P. aeruginosa strain containing and expressing a full complement of virulence factors. The strain has luxAB inserted into its chromosome thereby exhibiting bioluminescent properties . The light source was used previously for in vivo phototherapy studies and did not cause thermal effects  was employed and the output was focused on the wound area. The light source of non-coherent light, delivering an irradiance of 50 mWcm effects . The ligin vitro and in vivo: RB, NMB, TMPyP, and TLC1411. All photosensitizers were excited at 525 nm to minimize APDT efficacy differences due to variations in the wavelength dependent penetration depth into the moist infected wounds. Comparing the efficacy between the photosensitizers is thus only a function between their respective molar extinction coefficients at 525 nm.Four Photosensitizers were evaluated for their APDT efficacy max = 548 nm and the molar extinction coefficient at 525 nm is 3653 molL-1cm-1. The singlet oxygen quantum yield was reported as 0.74  (Sigma–Aldrich) is a sodium salt used in medicine as an eye drops to stain damaged conjunctival and corneal cells . Maximum as 0.74 .max = 590 nm, a molar extinction coefficient at 525 nm of 471 molL-1cm-1 and a singlet oxygen quantum yield of 0.66 ± 0.04 in air equilibrated acetonitrile  porp- hyrin tetra(p-toluenesulfonate)] (Sigma–Aldrich) is a positively charged, two-photon dye, that can bind to DNA in cell cultures and affect the cell viability. Its one photon peak absorption wavelength λ of 0.74 .-1cm-1. The singlet oxygen quantum yield was reported as >0.9 in complete media (TLD1411 is a Ru(II) coordination complex of the form  phenanthroline)]2+, originally synthesized by Dr. McFarland at Arcadia University and provided by Sigma Aldrich Fine Chemicals . Its use as an APDT agent has been previously described . Its abste media .Figure 1 shows the wavelength resolved molar extinction coefficient of these four photosensitizers.All photosensitizers were freshly prepared prior to the procedure as 1 mM stock solution in water, and diluted to the proper test concentration before being added to the bacterial culture.7 CFUml-1. The photosensitizer solution was added at predetermined concentrations ranging from 1 μM to 150 μM equivalent to a final concentration of 2⋅108 to 3⋅1010 PS molecules per bacterium. 100 μl of the PS solution was pipetted into wells followed immediately by photoactivation using the light source described above. Non-irradiated sample served as negative controls. Upon completion of light administration, samples were serial diluted and inoculated on fresh LB agar plates. A positive result required more than 3 magnitudes of log10 per CFUml-1 reduction in these planktonic conditions. PDT irradiation used a 96-diode laser array delivering 100 Jcm-2 total radiant exposure.All procedures were performed using sterile 96-well plates. Bacteria were diluted in fresh Luria–Bertani (LB) broth to a final planktonic concentration of 3⋅109 photosensitizer molecules per bacterium. Photoactivation was initiated after 30 min of wait period.All procedures of wound establishment and bacterial inoculation were executed as described above. 30 min post bacterial inoculation 30 μl of the 500 μM photosensitizer solutions was injected under the dressing by Hamilton syringe and allowed to spread over the wound for an estimated final concentration of ~2⋅10-1cm-2sr-1] and subjective evaluation of the wound’s appearance. The bioluminescence (BLI) radiance was determined by selecting equal size of the region-of-interest (ROI) directly over the wound and between the two shoulders blade to determine the cameras dark counts.Images were collected using the IVIS Spectrum imaging system . For imaging, mice were anesthetized using isoflurane, as described above. Mice were imaged at specific time points: immediately after bacteria inoculation, prior to and after PS injection, prior to and after light administration, then every 10 min post APDT therapy up to 2–3 h, followed then daily up to 5th day or the endpoint determined in the protocols. APDT efficacy for bacterial inactivation was measured by loss of bioluminescent signal, defined as average radiance [hυsTM covered wounds an increase in bioluminescent signal was observed after inoculation with either PAK or XEN strain, starting from day 0 up to 3rd (PAK) or 4th (XEN) day of experiment. Then, a plateau phase was reached lasting up to 8th day after wound inoculation with bacteria . On the contrary, in wounds left open from the beginning as well as those where the TegadermTM was removed after 48 h, a reversal of the initial increase in BLI radiance was observed. The wound’s appearance was dry after 2 days, and a pink appearance was retained.In TegadermTM dressed wound changed from the clear raw appearance to yellowish dense mucus on day 2 and 3, whereas they appeared yellow for the remainder of the observed duration. Often the wound expanded and the infection bulged the dressing outward as shown in Figure 3, for PAK inoculated wounds.The appearance of the TegadermFigures 4 and 5, for PAK and XEN results, respectively. The susceptibilities of bacteria to APDT mediated by one of the four sensitizers, was tested at increasing concentration of photosensitizers at three radiant-exposures ranging from 50 to 150 Jcm-2.There was no significant dark toxicity of the photosensitizers toward the investigated bacteria within the concentration range studied (up to 500 μM), except for TLD1411 at >10 μM for XEN. Whereas, upon illumination all studied photosensitizers showed activity against PAK, and TLD1411 also against XEN bacteria; see -2 radiant exposure. Increasing light dose up to 150 Jcm-2 resulted in a decreasing RB concentration required to elicit a response. 50 μM of RB and 150 Jcm-2 decreased below the level of detection CFU ml-1 units up to 6 log10 . APDT response reciprocity between radiant exposure and RB concentration breaks down for concentration above 50 μM presumably due to self-shielding within the 1 cm path length in the planktonic solutions used in these experiments, see below.Rose Bengal showed positive effect toward PAK with 75 μM concentration and 50 Jcm10 of survival cells when 100 Jcm-2 were applied, whereas 50 Jcm-2 failed to elicit a beneficial APDT response, both presumably due to self-shielding. At higher doses the photosensitizer showed no significant impact on efficacy. Unlike Rose Bengal, NMB had no dose-dependent efficacy, thus no significant difference in PAK inactivation from 10 to 150 μM when exposed the solution to 150 Jcm-2 .New Methylene Blue showed least antimicrobial activity against PAK. Only 10 μM concentration showed reduction of 3 log-2 were administered. Increasing the light exposure led to a higher inactivation effect at lower PS doses and even 10 μM concentration exhibit significant CFU ml-1 reduction. The combination of 75 μM concentration and 150 Jcm-2 of green light resulted in 6 log10 eradication of PAK . As for NMB 50 Jcm-2 did not initiate sufficient PAK inactivation again due to self-shielding within the 1 cm path length cuvette. Self-shielding also caused loss of reciprocity between light and PS doses.Porphyrin TMPyP reached a positive anti-bacterial effect against PAK when 150 or 100 Jcm-2 against PAK, so more than 6 log10 was achieved at the lowest concentration of 1 μM  effectively reaching sterilization levels.TLD1411 was tested only for 100 Jcm10 inactivation was achieved with the lowest concentration of 1 μM . However, while no significant dark toxicity against PAK was observed, TLD1411 showed significant dark toxicity, ~3 log10, at 10 μM against XEN as noted already above.Only TLD 1411 was tested for its activity against XEN in these studies, and similar to PAK, 6 logTable 1 shows the critical concentration required to achieve 3 log10 and 6 log10 reduction in bacterial cell count for the highest radiant exposure tested at 525 nm.in vitro planktonic cultures, the in vivo response was much more moderate, with none of the photosensitizers achieving sterilization of the wound equivalent to a 6 log10 reduction. In fact, not even clearing at 3 log10 was attainable based on BLI radiance measurements, see Figure 6.Unlike for t = 0 just prior to PS administration, permitting comparison of the APDT effects.As there was a variation in the group average BLI radiance for PAK studies, the BLI signal was normalized at Untreated wounds with expanding bacterial counts reached bioluminescence signal saturation at the first day. The two principal reasons for this saturation effect are increased infection thickness reabsorbing the bioluminescence photons and increased light scattering from the wound further confining the BLI radiance to the infection.Rose Bengal mediated APDT resulted in the least BLI radiance reduction, ~ one order of magnitude, or ~ 3.2 pathogen division times, presenting limited benefit to wound management. NMB had the highest BLI radiance reduction following APDT (~ two orders of magnitude); however, the BLI radiance regain was faster than in untreated wound suggesting that the radiance loss was due to biochemical luminescence quenching or bleaching rather than bacterial inactivation. TMPyP had the 2nd best APDT induced BLI radiance reduction, whereas post APDT signal gains were comparable to untreated wounds suggesting true PAK inactivation or destruction by APDT. TLD1411 showed signs of dark toxicity prior to PDT with accelerated BLI radiance loss during PDT. BLI radiance recovery over the first 2 h was slower and accelerated over the next 22 h, reaching BLI radiance increase rate comparable to untreated control wounds.Figure 6.Nevertheless, it should be noted that APDT mediated by either photosensitizer resulted in a lower BLI radiance intensity than non-treated control at 120 min, demonstrating the ability to delay PAK infection progression by APDT of induced wounds, see Figure 7, whereby untreated wounds reached BLI signal saturation at 2 days post infection. The TLD1411 concentration per XEN bacterium is estimated at ~2⋅107 and photo-irradiation showed a continuous decrease in BLI radiance up to 90 min post PDT (120 min time point). The regain of BLI radiance was not significant until day 4, followed by a signal increase rate comparable to the untreated control.The temporal evaluation of the XEN associated BLI in untreated controls and TLD1411 mediated APDT is shown in 8 and 106 hυs-1cm-2sr-1 of average normalized radiance for PAK and XEN, respectively, and remained constant for the remainder of the observation period (data not shown). Hence, LLLT does not appear to be a confounder to PAK or XEN survival at 525 nm.Group vi, included for evaluation of APDT efficacy versus potential LLLT stimulatory effects due to light irradiation with both bacterial strains were identical to group ii . Bioluminescence radiance signal increased for 2 days, reaching 1010 CFUml-1S. aureus cells. Biopsies were collected at specific time points to monitor wound healing and host cell growth. Mice were kept in individual cages to prevent cross contamination, affecting overall well-being of the animal by causing stress. 8 CFU of P. aeruginosa. The model mimics large, festering wounds and can cause high pain and stress levels. Application of an occlusive dressing to avoid cross contamination is favorable in this model. Similar wounds were established by 2) were immediately infected using 108P. aeruginosa cells.While various infected wound models have been published, there is still a need for an applicable model mimicking the clinical features of the disease, with short implementation time and efficient translation of the study results into clinical practice. TM covered wounds, thus it presents a favorable environment for testing APDT efficacy in vivo. The wound cover allows for oxygen and water vapor across the barrier and does not impede light penetration either for BLI, APDT, or LLLT  over the 6 mm diameter wound, which would result in an ~1 mm layer large compared to the diffusion distance of the ROS. As the PS acts also as a 525 nm light filter prior to the pathogens on the tissue surface, the resulting optical densities are RB = 1.83, NMB = 0.235, TMPyP = 0.653, and TLD1411 = 0.4155, respectively. In particular, for RB the high concentration employed in conjunction with the high molar extinction coefficient eventually caused a reduction of the radiant exposure at the microbes to 1.48 Jcm-2. Similarly, the worst-case radiant exposures for the other photosensitizers would be NMB = 58.2 Jcm-2, TMPyP = 22.2 Jcm-2, and TLD1411 = 38.4 Jcm-2, respectively. However, the solution was rapidly absorbed into the tissue and the filter effect by the photosensitizer had a lesser impact on the sensitizer specific APDT efficacy differences.Nevertheless, the added attenuation of PS in Conversely, trying to achieve a comparable OD in the tissue to eliminate effect of self-shielding as a confounding factor. There is a need for either adjustment of the photosensitizer’s concentration affecting the ratio of photosensitizing molecules per bacterium or selecting particular wavelength for each photosensitizer so that the molar extinction coefficients are the same, which in turn would impact the photon distribution in the tissue. Hence, our approach of using constant wavelength and radiant exposures in the sensitizer was compared.2 cm-1 and the penetration depth would be limited to < 100 μm  achieved at least 3 log10 of inactivation for 150 Jcm-2. The efficacy limits for RB and TMPyP are probably an artifact due to their high molar extinction coefficient and the high concentration used, which prevented effective illumination of the planktonic solution over the 1 cm sample depth. Radiant exposure with a much lower absorption coefficient of NMB compared to RB, its limited efficacy cannot be attributed to self-shielding. Additionally, as the photosensitizers have comparable singlet oxygen quantum yield one needs to conclude that NMB does not associate well with PAK. TLD1411 appears to present higher efficacy compared to other photosensitizers with a concentration less than 10 μM being sufficient for transient PAK sterilization at 100 Jcm-2. This may be explained by the ability of these ruthenium complexes to lead to DNA cleavage  employed for the different study groups. While this sample size is too small to allow statistical analysis of the results between photosensitizers, it is large enough to demonstrate an APDT effect and the report time line for BLI signal delay and time to signal saturation.One caveat to these experiments is the small number  methods are currently used for direct identification of selected Staphylococcus, Enterococcus, Klebsiella, and Candida species in less than 2 h (While only up to a 1.5 loghin 48 h . Peptidethan 2 h . While aStaphylococcus aureus (MRSA) and P. aeruginosa was similar to chronic wounds, without spontaneous clearing due to wound drying. The progress of the infection can be monitored real-time for bioluminescent bacteria, thus permitting the rapid evaluation of APDT agents and treatment protocols in vivo under physiologically relevant conditions for bacterial growth.Our murine model of consistent local wound infection with in vivo evaluation of antimicrobial effect of photo- sensitizers responded less effectively compared to what were seen in vitro, an observation also reported by others; however, the potentially significant clinical reduction of the bacterial load remains apparently feasible, causing delay in full activation of inflammatory response and hence reducing the risk of developing sepsis.The The presented murine model is a suitable platform to evaluate the feasibility and safety of repeated cycles of PDT, based on clearance time of photosensitizer from host mammalian cells.in vitro experiments with the use of RB, TMPyP, and NMB against P. aeruginosa, as well as all in vivo procedures. He wrote the first draft of the manuscript. KK assisted and supported the in vivo experiments. YA performed the in vitro experiments. JN: participated in the conception and design of the work; advised on the experimental design and writing of the manuscript. MG participated in the conception and design of the work; drafted the manuscript and revised it critically for important intellectual content. KB participated in designing of the experiments and helped drafting manuscript. LL designed the experiments together with Drs. MG, JN and KB, supervised GF, KK, and YA on a daily basis and is the senior responsible author for the manuscript together with GF. He also acts as the corresponding author on this manuscript.GF has performed The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chlamydomonas reinhardtii under varied CO2 inputs. The metabolic network model of Chlamydomonas was updated based on the genome annotation data and sensitivity analysis revealed CO2 sensitive reactions. Biological experiments were performed with cells cultivated at 0.04% (air), 2.5, 5, 8, and 10% CO2 concentration under controlled conditions and cell growth profiles and biomass content were measured. Pigments, lipids, proteins, and starch were further quantified for the reference low (0.04%) and high (10%) CO2 conditions. The expression level of candidate genes of sensitive reactions was measured and validated by quantitative real time PCR. The sensitive analysis revealed mitochondrial compartment as the major affected by changes on the CO2 concentrations and glycolysis/gluconeogenesis, glyoxylate, and dicarboxylate metabolism among the affected metabolic pathways. Genes coding for glycerate kinase (GLYK), glycine cleavage system, H-protein (GCSH), NAD-dependent malate dehydrogenase (MDH3), low-CO2 inducible protein A (LCIA), carbonic anhydrase 5 (CAH5), E1 component, alpha subunit (PDC3), dual function alcohol dehydrogenase/acetaldehyde dehydrogenase (ADH1), and phosphoglucomutase (GPM2), were defined, among other genes, as sensitive nodes in the metabolic network simulations. These genes were experimentally responsive to the changes in the carbon fluxes in the system. We performed metabolomics analysis using mass spectrometry validating the modulation of carbon dioxide responsive pathways and metabolites. The changes on CO2 levels mostly affected the metabolism of amino acids found in the photorespiration pathway. Our updated metabolic network was compared to previous model and it showed more consistent results once considering the experimental data. Possible roles of the sensitive pathways in the biomass metabolism are discussed.The development of microalgae sustainable applications needs better understanding of microalgae biology. Moreover, how cells coordinate their metabolism toward biomass accumulation is not fully understood. In this present study, flux balance analysis (FBA) was performed to identify sensitive metabolic pathways of  BiomassChlamydomonas reinhardtii has been extensively considered as a model organism to the study of different cellular mechanisms under distinct environmental conditions . This approach takes in consideration a linear programming strategy to solve and describe the fluxes in a systems at steady state   enzyme by increasing the cellular inorganic carbon (Ci) pools favoring the carboxylase activity of RubisCO; thus, carbon fixation through photosynthesis. Moreover, some Ci transporters located in the plasma membranes and chloroplast have been suggested as main proteins on the carbon uptake process  were retrieved from NCBI database  and the CO2 fluxes modified to evaluate the effects of different conditions of CO2 supply at the steady state of the cell metabolism and other parameters were established as previously described  is the objective function (biomass).Where 2 level response was performed through the identification of reactions affected under different CO2 fluxes through FBA simulations. FBA was resolved at five different CO2 conditions  and the resulting magnitudes defined the CO2-sensitive reactions by the flux variation coefficient ρ  in a R'ALF Plus solo 6.7 L bioreactor  in constant and continuous illumination with cool-white LED (average 400 μE·m−2·s−1). The cultures remained in a batch mode with starting culture volume of 4 L, in open system with automatic controlled CO2 gas flow in air, no pH control and continuous stir at 60 rpm. Cell inoculation was carried out with a 10 mL pre-inoculum taken from a sample at steady state. Cells were harvested by centrifugation at the late exponential phase of cell growth (O.D.750nm ≈ 0.9) for biomass characterization.The Cell growth monitoring was performed daily with measurements of O.D. at 750 nm and cell counting using Neubauer chamber. Growth rates were calculated as previously described  or two biological replicates , each one with three technical replicates, were considered for statistical analysis.Cell pellets from 50-mL cell culture aliquots at the late exponential phase were washed three times with 2 mL of de-mineralized water to remove inorganic salts and the final cell suspension was transferred to previously weigh empty Petri dishes. Plates containing the biomass were kept overnight at 90°C, and the dry weight was measured. Three (0.04 and 10% CO2 (10%) were harvested in 50-mL aliquots  and centrifuged at 3000 × g in a swing rotor bench centrifuge for 5 min at 4°C. Cell pellets were kept at −80°C until further processing. Frozen cell pellets were macerated until conversion into a light green powder. Protein extraction was performed using a buffer containing 100 mM Tris pH 7.5, 4 mM EDTA, 5 mM 2-mercaptoethanol, 10% glycerol, and 0.05% Triton X-100. Protein content was determined by Bradford assay  and high CO2 changes [low CO2  and high CO2 (10% CO2)] were selected to further gene expression analysis through the relative quantification by qRT-PCR. These candidate genes were previously annotated to the following routes and mechanisms: CCM, Calvin cycle, Glycolysis/Gluconeogenesis metabolism, and Glyoxylate/dicarboxylated.Based on our results on the sensitivity analysis using FBA approach on the model described by Chang et al.  and litewww.quantprime.de) following the criteria as follows: Tm = 60 ± 1°C, length 18–25 bases, preferentially on exon-exon junctions. When possible, primers were designed to have a GC content of 45–55%, generating a single PCR product sizing between 60 and 150 bp. Primers were synthesized by Macrogen .Cells for RNA extraction were harvested in 2-mL aliquots by centrifugation for 2 min, 3000 × g, at 4°C, and the cell pellets were immediately frozen and kept at −80°C until further use. Total RNA extraction was performed using the RNeasy Plant Mini Kit  as previously described  as primer for the synthesis of the first complementary DNA strand. Two genes [Actin (ACT) and Ubiquitin protein ligase (UBQ)] were selected as reference genes. All the cDNA samples were amplified in 96-well plates in an Applied BioSystems ABI7500 FAST system. The qPCR reaction was carried out in 10 μL containing 1 μM primers and SYBR Green qPCR Master Mix (Roche). The primer sequences used in this study are presented in Supplemental Table 2) were performed.Three micrograms of total RNA were used for cDNA synthesis employing the SuperScript III First Strand System  according to the manufacturer's instructions, using oligo-(dT750 ≈ 0.9) at low (0.04%) and high CO2 concentration (10%). Metabolites were extracted with methanol-chloroform-water HPLC grade (2.5:1:1.4) mixture and three biological replicates for low CO2 (0.04%) and two biological replicates for high CO2 (10%) were considered. Samples were immediately frozen in liquid nitrogen and lyophilized for storage. Extraction and derivatization of metabolites were performed as outlined previously , coupled to an Agilent 7890 A gas chromatograph—Leco Pegasus HT time-of-flight mass spectrometer . Identical chromatogram acquisition parameters were used as those previously described  or PMN (http://pmn.plantcyc.org/CHLAMY/) that report proteins and curated metabolic data, showed that only a small group of enzymes of Chlamydomonas has been discovered and functionally characterized. Considering this, our homology analysis was performed comparing all annotated proteins in KEGG database to identify the EC numbers and the associated reactions. In order to simplify the reconstruction, only the reactions with reported reactants in Chang et al.  were associated. After removing the reactions that already exist in the work of Chang et al. , 1803 neChlamydomonas. These results indicated that manual curation is still necessary and mandatory to guarantee feasible models.We found that some reactions and metabolites have generic names in KEGG database, especially those associated to lipid and starch metabolism. This inconsistent information provides new reactions and metabolites that cannot be interconnected affecting viable results from the FBA analysis. Gaps within the different pathways were observed when looking at the rows of the stoichiometric matrix and manual interconnection had to be assumed to consume products based on metabolic maps. Furthermore, unlikely reactions were found associated to homology parameters, reactions belonging to penicillin, and cephalosporin biosynthesis were rejected because no information was confronted with 2 input fluxes. However, in our complemented reconstruction, more, and novel sensitive reactions appeared related to the amino acid metabolism of alanine, threonine, tryptophan, and propionate metabolism  , and transport of compounds such as ethanol, ammonia, and O2. The mitochondria is important to the maintenance of intracellular redox gradients, impacting the rates of photorespiration, and efficiency of photosynthesis , reaching the highest content when evaluated at 10% ; this information corroborate previous results  and high (10%) CO2 conditions, showed a positive increase at high CO2 concentration. However, no significant changes were found for starch . Moreover, we observed that cell culture at 5% CO2 produced similar amounts of total biomass than cells cultured at 10% CO2, suggesting the existence of a saturation trend in the biomass production once cells are cultivated in CO2 concentration higher than 5%.Cell growth was measured daily Figure  and a si% Figure . Therefo2 conditions  showed similar profiles but cellular density was higher and statistically different from those cell cultures under low CO2 (0.04%). Thus, the exposure to high CO2 lead to an increased number of cells per culture volume and enhanced cellular capability to accumulate biomass, likely affecting mitosis-related cell cycle and energy metabolism.Furthermore, the cell growth curves observed on high CO2 sensitive metabolic reactions included gene expression analysis of selected genes and a metabolomics approach focused in the identification and relative quantification of the main metabolites of primary metabolism.The experimental validation of our results on the identification of CO2 related mechanisms may be determinant to biomass accumulation and CAHs play an important role on cellular carbon uptake . Similar results were shown by previous studies on the Carbon Concentrating Mechanism , glycine cleavage system, H-protein (GCSH), NAD-dependent malate dehydrogenase (MDH3), low-CO2 inducible protein A (LCIA), low-CO2-inducible protein 23 (LCI23), mitochondrial pyruvate dehydrogenase complex, E1 component, alpha subunit (PDC3), fructose-1,6-bisphosphate aldolase (FBA2), glyceraldehyde-3-phosphate dehydrogenase (GAP1), dual function alcohol dehydrogenase/acetaldehyde dehydrogenase (ADH1), and phosphoglucomutase (GPM2), were defined as sensitive nodes in the metabolic network simulations and found to be responsive to the changes in the carbon fluxes in the system.In Figure As discussed previously, the sensitivity analysis may generate different outputs depending on the completeness of the metabolic network used as input for simulation. Genes coding for GLN1, CIS1, ATP2, PRK1, and FBP1, which were found to be sensitive in Chang et al. , showed 2 concentrations. The metabolomics analysis performed using GC-TOF-MS permitted the quantification of 67 metabolites in the two compared CO2 concentrations  and high (10%) CO2 sensitive reactions of 13 out of the 67 metabolites identified. Our model was able to predict the behavior of these 13 metabolites at high and low CO2 concentrations regarding the amount and presence within the cells, being consistent with experimental results at the two growth conditions, except for the measurements of glycine, isocitrate, and sucrose, which were not good represented by the FBA.We further explored the CO2) and 50% of these did not show any alteration through FBA. From this perspective, it is shown that the model assumptions in general terms seem to be consistent with real behavior of maximizing biomass. However, others mechanisms may be activated resulting in saturation trends for CO2 processing and constraints.Other 50 metabolites quantified did not show significant differences between the two growth conditions  in order to satisfy the model constraints. The magnitude of the fluxes are >0 and are contributing to maximize the biomass function. Special attention to those magnitudes were taken into account for glycine transport from chloroplast to mitochondria, serine transport from mitochondria to chloroplast, hydroxypyruvate production, glycerate, and 3-phospoglycerate production in the chloroplast. All these processes may be related to photorespiration and showed positive fluxes in both Chang et al. and our complemented metabolic network models. Although genomic-scale restriction was not considered in the FBA, it is shown that these routes contribute to maximize biomass production.FBA analysis indicated that at low and high CO2 concentrations in the biomass composition of C. reinhardtii cells. Moreover, we identified candidate genes sensitive to the variations on the CO2 concentrations through the use of FBA in an extended metabolic network model presented here. Further experimental validation through gene expression analysis and metabolomics was performed. Our experimental results suggested that cells at high CO2 have increased capability toward biomass production. However, it also indicated that cells cultivated at CO2 concentrations higher than 5% achieved a saturation trend on total biomass accumulation. Although our FBA model was unable to describe this saturation trend, the magnitudes of the growth rate values were consistent between the different CO2 concentrations compared. This may be explained by the fact that the model was resolved using linear optimization and a change in input values, such as any external metabolite, represents a proportional increase in the objective function.In the present work, we compared the effects of varying CO2 concentrations. For the new sensitive routes and genes identified by our FBA, it was noted that many reactions were associated with energy metabolism. These sensitive routes have a number of genes which expression may be affected. Therefore, these genes may be interesting candidates on further biotechnological applications focused in the enhancement of biomass production. Reactions associated to the transport of amino acids, pyruvate, and carboxylate species into the mitochondria and chloroplast were shown to be the most sensitive ones. Moreover, mitochondria resulted as the most sensitive compartment as most reactions detected in our sensitive analysis may occur inside this organelle. Mitochondria presents a fundamental role in growth and biomass production, through its role on energy metabolism.Our results on the FBA sensitivity analysis of the two metabolic reconstructions showed a significant increase of flux in metabolic routes occurring in the chloroplast and mitochondria transport systems, including TCA cycle, glycolysis/gluconeogenesis, and amino acids biosynthesis in cells under high COSensitivity analysis also revealed a high dependence on the metabolic network quality and completeness on the identification of key routes. These routes can vary in differing models making it difficult to achieve consistent results; however, consensus in some main nodes were found and validated by qRT-PCR. Thus, our results revealed novel biochemical routes and candidate genes that may be relate to biomass production, through the modulation of the rate of biosynthetic processes.2 (10%) concentrations suggest that high CO2 concentration in microalgae cell culture may trigger mechanisms that are able to control the carbon fixation by the alternative synthesis of compounds that may have an inhibitory effect on the photosynthesis, or may enhance energy losses through photorespiration. These processes may have a role in the control of the cell biomass content and microalgae cell population, even under non-limiting availability of CO2. Further experiments are now necessary to provide more evidences of these effects.The changes observed in the metabolite profiles of cells at low (0.04%) and high CO2 concentrations. It was observed that many CCM-related genes are overexpressed at low CO2, indicating, as expected, that cells change their metabolism to produce enzymes involved in enhanced carbon uptake and carbonic acid conversion, instead of redirecting energy to build biomass precursors. A previous transcriptomics analysis compared Chlamydomonas cells at high (5%), low (0.05%), and very low (0.02%) CO2 concentrations  was found highly expressed at low CO2 concentration  itself does not depend on the CO2 conditions, we further compared which metabolites were modulated in the two CO2 concentrations (0.04 and 10%). Our metabolomics analysis indicated that the concentration of metabolites possibly related to photorespiration or other alternative route is modulated in response to high CO2 concentration.Besides the identification and validation of the expression of the main CA's under low and high CO2 concentration, indicating possible enhanced needs for light absorption and carbon fixation. However, starch content showed no significant changes which imply that the continuous light conditions may lead cells to control their carbohydrate stocks, probably due to the absence of a dark period and reduced need for starch accumulation or through the activation of alternative processes of carbon usage.Our results on the experimental biomass characterization showed that the pigment content per cell is the most sensitive component and its amount is almost duplicated at high CO2. The control of biomass accumulation may be closely connected to the regulation of biochemical pathways occurring in the mitochondria and the use of energy sources toward the accumulation of proteins and pigments.Altogether, these findings suggest that biomass accumulation does not enhances indefinitely with the enhanced availability of COFW, AG: conceived and designed the work; FW, DP, MM: performed experiments; FW and DP: drafted the manuscript; CC: performed metabolomics analysis; FW, DR, DP, AG: performed data analysis. All authors have read and approved the final version of the manuscript.This project was funded by Universidad de los Andes and FAPESP.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Conclusion: Guidelines to best practice management of 22q11DS based on a literature review and consensus have been developed by a national group of professionals with consideration of the limitations of available medical and educational resources.The commonest autosomal deletion, 22q11.2 deletion syndrome (22q11DS) is a multisystem disorder varying greatly in severity and age of identification between affected individuals. Holistic care is best served by a multidisciplinary team, with an anticipatory approach. Priorities tend to change with age, from feeding difficulties, infections and surgery of congenital abnormalities particularly of the heart and velopharynx in infancy and early childhood to longer-term communication, learning, behavioural and mental health difficulties best served by evaluation at intervals to consider and initiate management. Regular monitoring of growth, endocrine status, haematological and immune function to enable early intervention helps in maintaining health.  The effective and efficient use of resources in conditions with multiple disabilities is one of today’s major challenges. A United Kingdom (UK) government-led initiative, the National Service Framework for Children , aims foThe 22q11DS population prevalence is reported to be one in 4,000 to one in 6,000 , 50, altThe 22q11.2 deletion is a 1.5- to 3-megabase deletion on the long (q) arm of chromosome 22. The deletion contains TBX1, the major candidate gene, and other genes, controlling the third and fourth pharyngeal arches, brain and skeletal development. Haploinsufficiency results in the principal syndrome phenotype. No correlation between the size or site of the deletion with phenotype has yet been found , 39. TheThe major conditions occurring in approximately 70 % or more are congenital heart disease, immune deficiency, palate defects affecting feeding and speech, and learning difficulties . Those fGuidelines for the management of 22q11DS evolved from a consensus document initiated by the parent-led charity Max Appeal , to promThe SIGN  systematAge appropriate investigations and assessments were evaluated from the literature, the prevalence of 22q11DS associated disorders, and the need for information to guide management and advice giving , 50, 61.Fetal anomaly screening may result in identifying that the foetus is affected, and in some instances one of the parents, more likely the mother, is also affected. Careful multidisciplinary assessment of the pregnancy is required {C}.The individual with 22q11DS is likely to present changing clinical and psychosocial priorities from birth to maturity  and is rarely lifelong. Point prevalence of hypocalcaemia outside the neonatal period was 30 % of 27 subjects of all ages , with a Management: monitor by checking the (ionised) calcium level 3 monthly in infancy and annually thereafter {D}. Low calcium and raised phosphate should initiate a check for an inappropriately low parathyroid hormone and normal vitamin D level.Calcium supplements and various formulations of vitamin D and analogues are generally effective treatments {C} but careful monitoring is required in patients with associated structural renal anomalies such as unilateral renal agenesis or dysplastic kidney. Daily Vitamin D is advised for all ages {GP}. The dose should be the recommended daily allowance, or as therapeutically indicated {B}.(a) Immune disorders affect the majority of patients, are commonly mild and manifest mainly as reduced T cell numbers and function. The thymus may be absent. Reduced levels of IgA and IgM in older children occur more frequently than the general population. The T-cell deficiency undergoes spontaneous improvement in most individuals, usually by 2 years of age.Almost all patients have a greater than average tendency to infections, particularly of the respiratory tract, even when laboratory tests of immune function appear normal . PneumonMonitor: baseline immunology testing  Autoimmune phenomena. Auto-immune conditions are found in up to a third, and symptomatic in a fifth as juvenile chronic arthritis (JIA), autoimmune thrombocytopenia, haemolytic anaemia, Raynaud’s phenomena, and autoimmune thyroid disease , 16, 27.Monitor: clinical review and annual full blood count {D}.(a) Feeding problems. Feeding problems in the first year are common , affectiGastro-oesophageal reflux is common and dysphagia (10 %) may cause recurrent silent aspiration and pneumonia.Management includes thickeners, anti-reflux and propulsive medication {C}, and nasogastric feeding. Gastrostomy is indicated if the airway is significantly compromised, as demonstrated on X-ray barium contrast swallow or associated with recurrent aspiration pneumonia {C}.(b) Growth disorders. Weight faltering frequently occurs {2+}. Although 40 % fall below the 3rd centile in height and weight in the first year , 50, thiManagement: see Table Renal abnormalities are found in 7–36 %, comprising absent, dysplastic or multicystic kidneys, obstructive abnormalities, vesicoureteric reflux, and nephrocalcinosis secondary to calcium and vitamin D supplementation , 32, 50.Management: ultrasound examination generally determines the pathway for management {B}.(a) Hypotonia and ligamentous laxity are very common, as is a disinclination to walk even short distances, with frequent complaint of leg pains which are usually symmetric; asymmetry suggests other pathologies {3}. The pain may cause waking at night, contributing to daytime inattention. Unsteady gait, incoordination and clumsy hand skills are common. Patella dislocation and rheumatoid arthritis are increased  comparedManagement: assess gait, limb pain, joint swelling, early morning stiffness, or delayed motor development with these comorbidities in mind. Specialist referral to a paediatric rheumatologist and specialist physiotherapist may be indicated {GP}.(b) Cervical spine examination. Radiological abnormalities of the cervical spine region are very common . As in DManagement: seek advice expeditiously in the presence of any physical or neurological symptoms or signs of cord compression. Anaesthetists should be alerted to the possibility of cervical spine injury whilst manipulating the head and neck in the unconscious 22q11DS patient. No limitations on sports are imposed, subject to suitable supervision and support {GP}.(c) Scoliosis. The prevalence of scoliosis in 22q11DS is 9–24 % , 46, 61,Management: clinical examination of the spine at diagnosis and ages 10 to12 years is recommended {D}.Articulation and communication problems occur in two-thirds, characterised by hypernasal articulation due to VPD , cleft pDeafness is due to otitis media and secretory otitis media in 75 % {3}; up to 15 % , 50 haveManagement: universal hearing testing occurs at birth in many economically developed countries, and is repeated at 4 to 5 years {B}. Additional examinations are as indicated clinically, in the presence of delayed speech and language development {C}.Intelligence scores are shifted lower. The mean IQ is in the low 70s; 30 % lie in the low normal IQ range between 80 and 100 , 56, 68.Management: it is exceptional not to receive some learning support or a special needs teacher for reading comprehension and mathematics , 46. KeyIn common with other neurogenetic syndromes, emotional and behavioural issues and psychiatric disorders occur more frequently than in the general childhood population, affecting 20–50 % of 22q11DS individuals at any age. Often co-occurring, problems can include attention deficit hyperactivity disorder (ADHD), obsessive compulsive disorder, depression, anxiety disorders and autistic spectrum disorder (ASD) {2+}  to {3} . ChildreDuring adolescence and adulthood, risk of significant and persistent mental health difficulties is increased relative to comparable populations with mild to moderate intellectual disability, and mood disorders become increasingly common {3} , 17. TheMany young adults experience social isolation and employment difficulties and continue to be prone to the emergence of 22q11DS-related conditions.Management: At all ages ready access to a psychologist or psychiatrist is advantageous {GP}. The outcome of the assessment determines the intervention Table . When soIn drawing up guidelines, it was deemed appropriate to apply nationally accepted levels of clinical risk which are evidence based. These can be adapted to suit local circumstances and legal jurisdictions, for example where defensive medicine plays a significant role in clinical practice . ExampleChild advocacy is valuable in promoting understanding of the changing educational profile from among the 15 million children in the EU with special educational needs , 64. In The 22q11DS UK Guidelines group, by setting what it perceives as minimum recommendations and not optimal ones as advocated in other recent guidelines , aims toAdvocacy Table  is highlA consensus comprehensive care plan is presented. An acceptable standard of care for individuals with 22q11DS requires careful coordination and a multidisciplinary team approach, with access to services throughout the life cycle.


Yet it is vulnerable at many points and is in constant danger of subversion by cheaters that seem particularly adept at finding such points.”James F. Crow [-  F. Crow PLOS Genetics by de Villena and colleagues provides compelling evidence for a massive copy number expansion that is causally linked to female meiotic drive in mice [Mendelian transmission is established during meiosis, the cell division that generates haploid gametes  from diploid germ cells. Meiosis does not, however, have to be fair. Selfish genetic elements, or meiotic drivers, have evolved to cheat this process in order to be packaged into functional gametes more often than the expected 50% probability. By biasing allele transmission in their favor, meiotic drive alleles can short-circuit natural selection, causing their spread even if they are harmful to organismal fitness. Indeed, meiotic drive alleles are thought to be directly or indirectly associated with infertility in diverse eukaryotes, from fungi to flies to humans . Drive o in mice .Drosophila melanogaster [Didion et al. began their study by investigating the genetic basis of transmission ratio distortion (TRD) of a region of mouse chromosome 2. This TRD had been previously observed in a number of crosses, including, most recently, in the recombinant inbred Collaborative Cross (CC) lines designed for genetic analyses . TRD wasnogaster  and the nogaster .This finding left open two possibilities, the first being that the bias originates from a selective postfertilization defect. Indeed, Didion et al. find that the amount of TRD does negatively correlate with litter size, suggesting that lethality of oocytes or embryos inheriting the non-WSB/EiJ allele could contribute to the TRD. However, the observed decreases in litter sizes are insufficient to explain the magnitude of TRD observed. In addition, despite the decreased litter sizes, the absolute number of progeny inheriting the WSB/EiJ allele in crosses exhibiting TRD was significantly higher than the number inheriting the allele in crosses without TRD. Thus, although embryonic lethality occurs in crosses showing TRD, Didion et al. propose that “true meiotic drive” must occur to fully explain the TRD in favor of the WSB/EiJ allele. Such drive occurs because, in female meiosis, only one of four meiotic products is selected in the oocyte .R2d2 (Responder to drive on chr. 2). To map R2d2, Didion et al. again exploited the impressive genetic power of the CC and DO lines. By genotyping over 400 mice, they identified recombination events within the candidate region on chromosome 2. All eight recombinant chromosomes that showed TRD shared a 9.3 Mb region containing a 127 kb unit of DNA (R2d2) that is repeated ~36 times in WSB/EiJ  ancestral location spread over a 158 kb region (R2d1) ~6 Mb distal from the R2d2 locus  that bind R2d2 and endow it with microtubule attachment or motor function that results in neocentromere activity [R2d unit, Cwc2, might itself bind the R2d2 cluster and contribute to TRD.Not all dams heterozygous for activity . These factivity . One intR2d2, as well as their evolutionary origins, will broaden our general understanding of fertility and chromosome segregation. Such work also reinforces previous findings about the insidious and relentless nature of meiotic competition between chromosomes [In sum, the paper by Didion et al. represents a tour de force in characterizing a complex multilocus TRD system in a genetically tractable mammalian model system. Uncovering the molecular mechanisms underlying the transmission of exceptional meiotic drive alleles like omosomes , 2, 10.


Mamestra brassicae (Noctuidae) caterpillars to low intensity green, white, red or no artificial light at night and determined their growth rate, maximum caterpillar mass, age at pupation, pupal mass and pupation duration. We found sex-specific effects of artificial light on caterpillar life-history, with male caterpillars subjected to green and white light reaching a lower maximum mass, pupating earlier and obtaining a lower pupal mass than male caterpillars under red light or in darkness. These effects can have major implications for fitness, but were absent in female caterpillars. Moreover, by the time that the first adult moth from the dark control treatment emerged from its pupa (after 110 days), about 85% of the moths that were under green light and 83% of the moths that were under white light had already emerged. These differences in pupation duration occurred in both sexes and were highly significant, and likely result from diapause inhibition by artificial night lighting. We conclude that low levels of nocturnal illumination can disrupt life-histories in moths and inhibit the initiation of pupal diapause. This may result in reduced fitness and increased mortality. The application of red light, instead of white or green light, might be an appropriate measure to mitigate negative artificial light effects on moth life history.Rapidly increasing levels of light pollution subject nocturnal organisms to major alterations of their habitat, the ecological consequences of which are largely unknown. Moths are well-known to be attracted to light at night, but effects of light on other aspects of moth ecology, such as larval development and life-history, remain unknown. Such effects may have important consequences for fitness and thus for moth population sizes. To study the effects of artificial night lighting on development and life-history of moths, we experimentally subjected  Over the past decades, increasing levels of artificial night lighting have led to large-scale alterations of night-scapes worldwide  (Noctuidae). In Western Europe, this species has two generations per year of which the first generation pupates in summer and the second generation overwinters as a diapausing pupa  are most sensitive to short-wavelength radiation (Ichikawa and Hideki Mamestra brassicae (Linnaeus 1785) as a model species. This species is common in (sub-)urban areas in Western Europe, but in the Netherlands, its populations are declining . The spectral compositions of the red and green lamps  for 14 and 10 nights in block 3 and 6, respectively) was 16 ± 2°C. Average night temperature was 15 ± 1°C and was not affected by light treatment .In a greenhouse, we created 40 open-top compartments of 36 × 40 × 36 cm (L × W × H). These compartments were divided over 10 blocks of four compartments. The four compartments in each block were randomly assigned to one of the four artificial light treatments  artificial light at night), resulting in mps Fig. , we appl2, Harrod Horticultural, Lowestoft) after 23 days for better ventilation. Each caterpillar was provided with artificial diet ad libitum (see A random subset of 240 twelve-day old (second instar) caterpillars was selected. Each caterpillar was individually housed in a 12.5 cm high, 9.5 cm diameter white plastic pot, which was closed off with transparent plastic foil to prevent escape. Pots were randomly divided over the different compartments . Plastic foil was replaced by white insect mesh  until maximum mass were calculated as [ln(maximum mass) – ln(start mass)]/time , pupal mass and the duration of the pupal stage (henceforth: pupation duration) were averaged for males and females within the same compartment to avoid pseudo replication. All resulting variables fulfilled assumptions of normality and homogeneity of variances. We used a general linear model with treatment (fixed), sex (fixed), block (random) and a sex*treatment interaction as factors. In case of a significant sex*treatment interaction, analyses were performed for the two sexes separately using a general linear model with treatment (fixed) and block (random) as factors. All analyses were performed in SPSS Statistics version 20 .F3,35 = 1.489, P = 0.235). We determined sex in 37 of these 40 caterpillars, and we used data on their RGR and age at pupation for analyses. However, because pupation failure generally was accompanied by physical damage of pupae and loss of body fluids, we did not use data for analyses on pupal mass. Because we were unable to sex the other three caterpillars in which pupal molt fatally failed, data obtained from these caterpillars were excluded from both pupal mass and age at pupation analyses. Of the remaining 190 individuals, seven did not emerge from their pupae  and thus were excluded from analyses on pupation duration. In total, we obtained pupation durations of 183 individuals.Because of technical failure of one red lamp on day 17 of the experiment, data from the six caterpillars in this compartment were excluded from the analyses. In other compartments, four caterpillars died in the beginning of the experiment and data obtained from these caterpillars were also excluded from all analyses. Of the remaining 230 caterpillars, 40 caterpillars (17%) fatally failed during pupal molt. This effect was independent of light treatment . When analysing sexes separately, light treatment had an effect on maximum caterpillar mass in males . In sex-specific analyses, we found that male caterpillars that were subjected to white and green light pupated earlier than caterpillars in darkness . Caterpillars under red light were intermediate , with reductions of pupal mass of caterpillars subjected to white light compared to caterpillars that developed under red light or in darkness. Pupal mass of males in green light was significantly lower than that of males in red light, but not different from pupal mass of males in darkness or under white light . Moths that were under green and white light as caterpillar emerged significantly earlier (after 73 (± 4) and 73 (± 5) days on average (± SE), respectively) than moths that had experienced a dark treatment as caterpillars (139 (±1.8) days, on average). Moths that had been under red light as caterpillar, had intermediate pupal development times (118 (±2) days, on average) (Fig. Pupation duration differed greatly between light treatments (ter 73 ±  and 73 (ter 73 ±  and 73 (To date, studies on the effects of artificial night lighting on moths have almost exclusively focussed on attraction of moths to sources of artificial light (Van Langevelde et al. M. brassicae pupae are unlikely to survive the winter. Although, moths that were under red light also emerged on average earlier than moths in darkness, the pattern in emergence resembled that of moths in darkness (Fig. The initiation of pupal diapause in moths is strongly dependent on temperature and light conditions during the larval stage Adkisson . We founess Fig. .Spectral alterations have been suggested to provide a tool for mitigation of negative effects of artificial light on ecosystems (Van Langevelde et al. We conclude that low levels of artificial light at night affect key life history traits in moths and interfere with light as a cue for seasonal timing in moths, thereby disrupting the initiation of diapause. These effects can have important negative fitness consequences, which add up to other adverse effects of artificial light on moths such as attraction (Van Langevelde et al.


Vertebral fractures are a common burden amongst elderly and late middle aged people. Vertebral cross-sectional area (CSA) is a major determinant of vertebral strength and thus associated with vertebral fracture risk. Previous studies suggest that physical activity affects vertebral CSA. We aimed to investigate the relationship between leisure-time physical activity (LTPA) from adolescence to middle age and vertebral dimensions in adulthood. We utilized the Northern Finland Birth Cohort 1966, of which 1188 subjects had records of LTPA at 14, 31 and 46 years, and had undergone lumbar magnetic resonance imaging (MRI) at the mean age of 47 years. Using MRI data, we measured eight dimensions of the L4 vertebra. Socioeconomic status, smoking habits, height and weight were also recorded at 14, 31 and 46 years. We obtained lifetime LTPA (14–46 years of age) trajectories using latent class analysis, which resulted in three categories  in both genders. Linear regression analysis was used to analyze the association between LTPA and vertebral CSA with adjustments for vertebral height, BMI, socioeconomic status and smoking. High lifetime LTPA was associated with larger vertebral CSA in women but not men. Further research is needed to investigate the factors behind the observed gender-related differences. Osteoporosis is a bone deficiency disease with a high global prevalence among aging population and the elderly. It often leads to non-traumatic vertebral fractures with consequent morbidity and has negative effects on the quality of life1Vertebral bone strength is determined by architectural, material and geometrical properties56789168912Physical activity is known to influence bone through dynamic biomechanical loading, especially when performed frequently on a regular basis1415171819Bones respond to loading in a site-specific manner18According to literature, leisure-time physical activity (LTPA) has for long been proposed to associate with increased strength of the lumbar spine, as well as higher vertebral BMD23252713233The relationship between lifetime LTPA and vertebral bone size is not thoroughly explored. We have previously discovered a secular decrease in vertebral size from the Medieval times36In this study we attempted to clarify the role of lifetime LTPA (14–46 years of age) on vertebral dimensions and thus also on biomechanical strength of vertebrae. We utilized the large population-based Northern Finland Birth Cohort 1966 (NFBC1966) to study the association between LTPA from 14 to 46 years of age and vertebral size at 47 years of age. We hypothesized that high LTPA was associated with increased vertebral CSA.A total number of 524 males (44.1%) and 664 females (55.9%) were included in the analyses . The meaIn the latent class analysis (LCA), three lifetime LTPA clusters were identified as the most appropriate solution. The clusters were named as “inactive”, “moderately active” and “active”; the distributions of LTPA at 14, 31 and 46 years are shown for each cluster in 2 among males and 10.57 (SD 1.28) cm2 among females in our sample . The measurements of L3 and L4 showed a high correlation  in the CSA of these vertebrae and the level of intra-rater reliability was high (intraclass correlation = 0.963). The values of relative measurement error (%) distributed normally around the mean of 0.0 with a standard deviation of 4.9 (n = 400 repeated measurements).The mean vertebral CSA was 13.25 (SD 1.67) cm2 values, the unadjusted models, utilizing only the LTPA variables without adjustments, explained <1% of the variability in the CSA of the subjects. The adjusted models, in turn, explained 5.2–6.6% of the variety.When investigating the association between LTPA and vertebral CSA using linear regression, we found that unadjusted and adjusted analyses provided similar results  and 4. H2 (3.2%) larger vertebral CSA, compared to those who belonged to the “inactive” cluster (p = 0.012). No statistically significant differences were detected in the CSA between women classified as “inactive” and “moderately active”. Among males, no statistically significant differences were detected.In the analysis of the association between lifetime LTPA and vertebral CSA , those w2 (4.5%) larger vertebral CSA than the reference group (p = 0.003). We also detected that those men who were active 2 times per week at 14 years, had smaller CSA compared to the reference group (p = 0.027). No other statistically significant findings were obtained.In the additional analyses regarding LTPA at different time points , we deteIn this population-based birth cohort study, our main finding was that a high level of lifetime LTPA from 14 to 46 years of age was associated with larger vertebral CSA in middle-aged women. Our additional analyses showed that the participation frequency of ≥4 times/week at age 31 was associated with larger vertebral CSA in women at 47 years. However, the detected CSA differences were of minor magnitude, and physically active and inactive men had similar vertebral dimensions.In our previous study, physical activity and participation in different sports were not associated with changes in vertebral size at the age of 21 years336Several studies have analyzed the dimensions of other lumbar segments, e.g. L3, instead of L439There are many potential explanations for the observed gender differences. Given that women have smaller vertebrae, both absolute and relative to body size, there might be a higher ability or a greater demand for the vertebrae to enlarge in size. Furthermore, the men in our study sample were somewhat more physically active than women, which may prevent us from detecting a similar effect of LTPA on vertebral size. The ambiguous, inexplicable association between LTPA at a frequency of 2 times/week at age 14 and decreased vertebral CSA in men was not in line with our other results.Another potential hypothesis is that the female gender may have vertebral compensatory mechanisms, such as periosteal appositionWe are aware that the effect of physical activity on vertebrae may be expressed in other ways than changes in vertebral dimensions which were investigated in this study. The alternative mechanisms might be e.g. cortical shell thickening and/or changes in vertebral BMD. However, our previous study did not find any correlation between LTPA and trabecular bone density parameters in vertebraeOur study suggests that lifetime LTPA is positively associated with vertebral size, to a small extent, among women but not men. Further research is needed to confirm our findings and shed light on the factors behind the observed gender-related differences.st and December 31st 1966 in Northern Finland  were included in the prospective NFBC1966 study. Since their mothers’ recruitment during their first visit to the maternity health centers, data have been collected on the subjects’ health, lifestyle and social status. The study was conducted according the Declaration of Helsinki and approved by the Ethical Committee of the Northern Ostrobothnia Hospital District in Oulu, Finland. Cohort members and in youth also their parents provided written informed consent for the study. All personal identity information was encrypted and replaced with identification codes, providing full anonymity for the whole study population. We confirm that the study was carried out in accordance with the approved guidelines.Individuals whose expected date of birth fell between January 1Postal questionnaires enquiring about the participants’ health status and lifestyle were conducted in 1980, 1997–1998, and 2012–2014, when the participants were aged 14, 31 and 46 years, respectively . The queAt the age of 46 years, subjects who were living at known addresses in Finland  were invited to attend clinical examinations . A totalAfter the MR imaging, 352 subjects (23%) were excluded from the study due to 1) inability to measure vertebral dimensions , 2) bone-affecting medication , or 3) missing LTPA or covariate data (n = 179). Therefore the final eligible population consisted of 1188 participants.Physical activity was self-reported at 14, 31 and 46 years of age2). Socioeconomic status was evaluated based on the number of years the subject had attended school for . This was determined by asking: “What is your basic education?” 1) Less than 9 years of ground school, 2) ground school, or 3) matriculation examination. Smoking history and current smoking status were inquired with two questions: 1) “Have you ever smoked cigarettes (yes/no)?” and 2) “Are you currently smoking (yes/no)?” According to these questions, three categories were formed: 1) non-smoker, 2) former smoker, and 3) current smoker.At the age of 46, subjects underwent clinical examinations, where their height and weight were systematically measured by a trained study nurse. Body mass index (BMI) was then calculated for each subject  between 2012 and 2014 when the participants were averagely 47 years old. The imaging sequences followed routine lumbar spine protocol including T2-weighted fast-recovery fast spin-echo (frFSE) images in sagittal  and transverse planes .We measured 8 dimensions from the corpus of the L4 vertebra  to calcuCSA values were calculated by using the acknowledged formula CSA = π * a * b, where a = vertebral width/2 and b = vertebral depth/2All MRI measurements were performed by the same researcher, using the NeaView Radiology software  version 2.31, which is collectively in use on clinical workstations in Oulu University Hospital. The measurements were made prior to gathering or analyzing any other data on the subjects. To explore the correlation between dimensions of different vertebrae, we also measured the same dimensions from the L3 vertebra from a subsample (n = 110).In order to investigate intra-rater reliability, 50 MR images  were randomly selected for a second measuring three weeks after the first one, conducted by the original measurer. Based on the original and repeated measurements, intraclass correlation coefficient was calculated. Additionally, measurement errors were calculated. As the height, width and depth dimension values were of different magnitudes, we considered relative error (%) more informative as opposed to investigating absolute error.Latent Class Analysis (LCA) was used to obtain clusters, i.e. groups in which the subjects had a similar profile of LTPA during their lifetime (14–46 years of age). In LCA, the number of clusters is increased until the most appropriate model is found45We used linear regression analysis to reveal the association between LTPA and vertebral size. Vertebral size, i.e. the CSA of L4, was regarded as the dependent variable in all analyses, whereas LTPA variables acted as the explanatory variables. As both CSA47349First, we attempted to test our main hypothesis of high lifetime LTPA associating with increased vertebral dimensions. In order to investigate this, we analyzed the association between lifetime LTPA, represented by the LTPA cluster variable, and vertebral CSA. The “inactive” cluster was chosen as the reference category, and the other two clusters were compared to it. Two analyses were conducted (for both genders separately), one with and one without adjustments for vertebral height, BMI, education years and smoking.We also conducted additional analyses to assess the most important time point in terms of LTPA and increased vertebral dimensions. In order to investigate this, two linear regression analyses were conducted for both genders with the following variables included: 1) LTPA at 14, 31 and 46 years, 2) LTPA at 14, 31 and 46 years, vertebral height, BMI, education years and smoking. The LTPA variables of all three time points were included in every analysis and they thus acted as covariates for each other and their confounding effect was minimized. In the LTPA variables, the category with lowest LTPA frequency was chosen as the reference category, and the other 4 categories were compared to it.Statistical analyses were conducted using the SPSS software  version 22, 64-bit edition.How to cite this article: Oura, P. et al. Effects of Leisure-Time Physical Activity on Vertebral Dimensions in the Northern Finland Birth Cohort 1966. Sci. Rep.6, 27844; doi: 10.1038/srep27844 (2016).


Purpose: Cumulus cells have a critical role in normal oocyte development and fertilization. Prunus cerasus is an anthocyanin rich berry and performs strong antioxidant activity. The present study set to determine if Prunus cerasus can affect expression of HAS2  and progesterone receptor in Cumulus cells and its consequences outcome of the in vitro fertilization.Methods: 60 female and 15 male adult mice were used for mating and IVF (in vitro fertilization). Prunus cerasus extraction was added to the diet of female mice for 30 days. Ovulation induction and oocytes collection were done as routine. The cumulus cells were dissected apart, and the expression of progesterone receptor and HAS2 was detected using RT-PCR . Fertilization rate was evaluated by IVF. All data were analyzed using t-test.Results: Data was showed that expression of progesterone receptor and HAS2 in cumulus cells of mice that received prunus cerasus increased. Moreover, oocyte fertilization rate also increased significantly.Conclusion: Prunus cerasus as an antioxidant natural can become an important medication for improving oocyte quality and opening new opportunities for infertility treatment. It is concluded that Prunus cerasus consumption could improve fertility rate by increasing progesterone receptor and HAS2 activity in cumulus cells. Of these, 40%-55% are due to female factors, 20%-30% are due to male factors, and 15%-17% are unexplained infertility.1 Over the years, ART (assisted reproductive technology) has been a method for infertility treatment with different causes.6 In order to increase the success rate of fertilization, several herbal and chemical supplemental factors have been used.7 Because of less invasiveness and costliness nature of traditional medicine, recent research has focused on herbal use.9 Anthocyanin as secondary metabolite group of flavonoids has attracted much attention.10 Strong antioxidant activity was the best known property of flavonoids.11 Antioxidant activity of anthocyanin is associated with a variety of health benefits including inflammation, cancer, atherosclerosis, and diabetes.12 Recent studies have shown that reproductive processes may be influenced by hypothalamic-pituitary-gonadal axis via scavenging free radicals.15 However, interest in antioxidant has recently been intensified because of their possible effect on egg quality, fertilization, and pregnancy rates.17 Antioxidant therapy has been attended to alleviate infertility, and dietary antioxidant has been beneficial for female reproductive disorder.18 Fruits including Sour cherry (Prunus cerasus), containing anthocyanin and strong antioxidant activity, have attracted much attention.19 Prunus cerasus anthocyanins have a vast range of biochemical and pharmacological effect, and have been recommended as nutritional supplement.20 Granulosa cells consist of mural and cumulus cells in preovulatory follicles. Mural cells are in the follicle wall and cumulus cell in vicinal of oocyte.21 In the midcycle, increased level of LH result in ovulation by inducing resumption of meiotic maturation, transformation of granulosa cells, and expansion of cumulus-oocyte complex. The latter, is associated with accumulation of hyaluronic acid-rich extra cellular matrix in cumulus cells.22 During the process of cumulus expansion, the most important genes involved in the formation of matrix hyaluronic are hyaluronic synthase2 (HAS2).23 HAS2 mRNA is necessary for cumulus expansion and signaling during ovulatory response.24 Cumulus expansion facilitates the fertilization, and the failure of this process can lead to fertilization problem and is associated with low potential for implantation and decline in the quality of oocyte.26 Paracrine factor under the influence of progesterone is required for preimplantation embryonic development, but direct or indirect effect of progesterone is not clear.27 Previous studies in our laboratory showed that the presence of Progesterone receptor is essential for female fertility and ovulation control.29 In this study, Prunus cerasus effect on progesterone receptor expression, HAS2 in cumulus cells and the rate of in vitro fertilization are evaluated.Infertility is defined as inability to obtain fertility after one year of unprotected regular intercourse.Sour cherry were collected from the market and authenticated by a botanist . The extract, according to World Health Organization (WHO) protocol was prepared. 500g of Sour cherry was shed-dried powdered and added to 5 Li of 70°C ethanol (v/v) and left to macerate for 20 hours at room temperature. The basin was rotated slowly during this time. After filtration, ethanol at low pressure at 30 °C. The samples were stored frozen at –20 °C until used.A total of 60 female BALB/c mice (4-5weeks) and 15 adult male BALB/c mice were obtained from the animal house of Tabriz University of Medical Sciences (TUMS). For a period of 2 weeks prior to the experiment, the mice were kept in animal room for adapting. 1.5 kg of mice food was added to 1000ml of Prunus cerasus extract and mixed together completely. The mixed diet let to dry in 2 days. Female mice were randomly divided into two equal groups. In the control group, each animal received diet without Prunus cerasus. In the experimental group, each animal received 25g diet with Prunus cerasus for 4 weeks. After 4 weeks for induction of ovulation 10 IU HMG/PMSG (pregnant mare serum gonadotropin) and after 48 hours, 10 IU hCG (human chorionic gonadotropin) was injected intraperitoneally.One day post hCG injection, the mice were sacrificed and ovaries were removed from the female mice. Mice ovaries (n=100) were obtained and placed in sterile PBS and transported to the Tissue Engineering Laboratory in two groups. The stromal tissue surrounding the ovaries was removed, and oocyte were collected under flashing method and cultured in dishes with Universal IVF culture medium  under sterile mineral oil.The cumulus cells surrounding the oocyte (COCS) were separated from oocyte using hyaluronidase enzyme. Hyaluronidase enzyme causes the connection of oocyte and cumulus cells to be released and facilitates separation. Cumulus cells were transferred to another dish containing medium. PBS was added for washing and centrifuged 2 times for 5 min. After centrifuge, the pellet of cumulus cells was centrifuged for 1 h at -20 °C and then stored at -80 °C until RNA isolation.Male mice were killed by cervical dislocation, and the sperm were collected from the cauda epididymis by incubating the pieces of epididymis in 37°C Co2 incubator for 20 min. The sperm sample was added to the collected oocyte in the control group and experimental case group. The fertility success rate was assessed based on the formation of embryo.RNA expression was indicated using real time RT-PCR assay to measure HAS2 and PGR mRNA. The primers, used in PCR are presented in All statistical analysis was done using SPSS software version 22 and independent sample T-test. P values≤0.05 were considered statistically significant.Expression of HAS2 and PGR mRNA in cumulus cells, isolated from mice oocyte, was assessed by quantitative real time PCR. There was a significant increase in HAS2 mRNA in the experimental group compared to the control . HoweverIVF was performed in both control and experimental groups, and 100 oocytes were used per group. Counting the number of embryos revealed that in the control group which was not received Prunus cerasus, 77% of oocytes were developed into embryos, while in the experimental group receiving Prunus cerasus for 4 weeks, 89% of oocytes were developed into embryos (P≤0.0001). Developed embryos in the experimental group are shown in 10 Significant increase in the expression of HAS2 and PGR gene in the case group compared to the control group could be due to the loss of oxidative stress produced by antioxidant property of Prunus cerasus, which can also be justified for increasing fertility rate. Follicular fluid contains a lot of antioxidants that protect oocyte against free radicals. The disorder in pro oxidant/antioxidant system in follicular fluid can damage oocyte′s DNA, and cytoskeleton of membrane can lead to impaired fertility.31 Use of antioxidant rich food prevents progression of diseases related to oxidative stress.32 Free radicals that are indication of oxidative stress in many diseases are highly reactive, toxic, and short-lived molecules that hurt DNA, protein, lipid, and carbohydrates within the tissue.32 Oxidative stress specially affects microenvironment of oocyte, which has a detrimental influence on follicular development, ovulation, oocyte quality, implantation, and early embryonic development.33In the study of Damar and Eksi and other studies, anthocyanin and antioxidant capacity of Prunus cerasus is evidenced.35 Showell MG et al.’s study on the effects of antioxidants on fertility was contradictory, needing further investigation.32 Unlike the present study, Jozwick et al. did not report any relationship between free radical marker and pregnancy rate.36 The results of the present study is in agreement with Polak et al. who state that the concentration of antioxidant in peritoneal fluid of patients with unexplained infertility was significantly lower than the its concentration in fertile patients.37 Study of Agawarl et al is in line with the results of current study demonstrating that pregnancy rates in patients undergoing IVF, using antioxidant-supplemented media, were higher than standard media without antioxidants.32 Since studies have shown that antioxidants affect oocyte quality, high degree of expansion of cumulus cells is directly related to oocyte quality and expression of HAS2 and PGR gene is necessary for cumulus expansion. According to the results it could be postulated, it is antioxidant capacity of prunus cerasus that resulted in increased gene expression and fertilization rate.The results revealed that the addition of Prunus cerasus to laboratory diet resulted in increase of expression of HAS2 and PGR gene in cumulus cells and fertility success rate. Since Prunus cerasus contain significant levels of anthocyanins, strong antioxidant activity can influence fertility.It is concluded that Prunus cerasus consumption improves fertility rate by increasing progesterone receptor and HAS2 activity in cumulus cells..This article is resulted from a research proposal leading to the thesis of Fatemeh Namvar, MSc student of anatomical sciences and was approved by research deputy of Tabriz University of Medical Sciences, Tabriz, IranNot applicable.The authors declare no conflict of interest