An investigation was conducted in West Central Florida, USA to evaluate the efficacy of either topically applied fluralaner or topically applied selamectin to control flea infestations, minimize dermatologic lesions and reduce pruritus in naturally flea infested cats over a 12-week period. When dogs were present in the households, they were treated with either oral fluralaner  or oral sarolaner .Thirty-one cats in 20 homes were treated once with fluralaner topical solution on day 0 and 18 dogs in these homes were administered a single fluralaner chewable. Twenty-nine cats in 18 homes were treated once monthly with a selamectin topical solution for 3 treatments and 13 dogs in these same homes were treated once monthly for 3 treatments with a sarolaner chewable. Fleas on cats were counted by flea combing, fleas on dogs were estimated using visual area counts and fleas in the indoor premises were assessed using intermittent-light flea traps. Blinded-assessments of feline dermatologic lesions were conducted monthly and pruritus severity was evaluated by pet owners.A single topical application of fluralaner reduced flea populations on cats by 96.6% within 7 days and by 100% at 12 weeks post-treatment. This efficacy was significantly greater than selamectin treatment where single topical application reduced flea populations on cats by 79.4% within 7 days of initial treatment and 3 consecutive monthly treatments reduced flea populations by 91.3% at the end of 12 weeks. At the end of the 12-week study, all fluralaner-treated cats were flea-free and this was significantly greater than the 38.5% of selamectin treated cats that were flea-free. At the end of the study, fleas were completely eradicated  in 95.0% of fluralaner treatment group homes, significantly greater than the 31.3% of selamectin/sarolaner treatment group homes with complete flea eradication. Owner reported cat pruritus was reduced similarly in both treatment groups. Significant improvements in dermatologic lesion scores were achieved by day 30 in fluralaner treated cats and by day 60 in selamectin treated cats.An in-home investigation in subtropical Florida found that 1 application of topical fluralaner eliminated flea infestations on cats and in homes significantly more effectively than 3 consecutive monthly doses of selamectin. Ctenocephalides felis felis [Dipylidium caninum, as well as pathogens including Rickettsia felis, Bartonella henselae and Mycoplasma haemofelis [Flea allergy is considered the most common cause of dermatitis in dogs, and flea infestations in cats are also extremely common causes of irritation and pruritus leading to erythema, excoriations, papules and alopecia –4. The fis felis , 2. The emofelis –3. RapidA new class of ectoparasiticides, the isoxazolines shows rapid residual speed of kill of fleas infesting dogs in both laboratory and field trials –9. RecenThe present study was conducted to evaluate the efficacy of topical fluralaner in naturally flea infested cats in subtropical Florida, USA. This area of Florida is known to carry a year round risk of intensive flea challenge. Selamectin , a flea control product with a monthly retreatment interval, was chosen as the positive reference control. The treatments were compared in their ability to eliminate existing flea infestations on cats, minimize pruritus and reduce dermatologic lesions and assess the impact on flea control within the home environment. Most homes in the area with cats also own dogs; therefore, co-habiting dogs were also treated for fleas. Dogs were treated with fluralaner in households with fluralaner treated cats. However, to match the monthly treatment recommendation of selamectin, dogs in these households were treated with the isoxazoline sarolaner . Sarolaner was selected for dogs based on the results of a prior flea efficacy trial conducted in the same area .Home owners with flea infested cats contacted the “Flea Team” through referrals from the Sunshine Animal Hospital, Tampa, FL, Animal Dermatology South, New Port Richey, FL, and advertisements on Facebook® and CRAIGSLIST®. Members of the team visited each residence, and 40 private residences were selected for inclusion in the study from May 17 through June 14, 2017. Selection criteria included: (i) ≥ five fleas in comb counts on at least one cat at the residence; (ii) ≥ five fleas trapped during a 16–24 h period in two intermittent-light flea traps; (iii) one to 10 healthy cats and dogs living at the private residence; (iv) cats spend ≥ 12 hours/day inside the residence; (v) homeowners agree not to use any other topical, oral or premise flea control products during the study; (vi) cats and dogs cannot be pregnant or nursing; (vii) all cats and dogs must be > 6 months of age and cats need to be at least 1.18 kg and dogs > 2 kg; and (viii) owners sign a consent form and fill out a questionnaire concerning pet habits, flea control history and personal observations concerning potential flea hosts around their residence.Flea infestation in indoor residence areas was assessed using two intermittent-light traps  , 15. TraPrior to initiating the study all personnel were trained in proper cat handling techniques using American Association of Feline Practitioners and American Animal Hospital Association recommended methods for proper and safe handling of cats. Fleas on cats were counted using a modified combing procedure due to time and safety constraints. Six regions were examined using 10 strokes of a standard flea comb per region: (i) back of the head behind the ears and extending along the dorsal midline to the tail; (ii) left and (iii) right side from axillary region to posterior aspect of the cat’s body, including hair on posterior of each leg; (iv) ventral aspect from chest to inguinal region; (v) ventral neck region from chin to chest; and (vi) top of the head between the ears. As fleas were collected they were placed in a plastic bag with collected hair. Fleas were counted and immediately placed back on the cat.Fleas on dogs were estimated using a previously described area count methodology . Fleas wAll on-animal and premises flea counts were conducted ± 1 day on days 0, 7, 14, 21, then once between days 28–30, 40–45, 56–60 and 82–86. Personnel conducting pet and premises flea counts were not blinded to treatment groups.Pet owners assessed the severity of pruritus of dogs(s) in each home during each scheduled visit using a previously validated and described non-numeric scale , 18. In In both assessments owners did not see, nor were they informed of the numerical score given to their dog(s) or cat(s). In households with more than one owner, the same owner was required to assess the pruritus level of the pet(s) throughout the study.Blinded clinical dermatologic observations were made on days 0, 30, 60, and 84 of the study (± 3 days) of all qualifying cats resident in the homes. The extent and severity of dermatologic lesions were assessed using the validated scale, Scoring Feline Allergic Dermatitis (SCORFAD) . For thiQualifying homes and all pets in that household were randomly allotted to 1 of 2 treatment groups on day 0. Household entry numbers (1–40) were assigned a random number by Excel  and blocked into groups of 2. The highest random number within each block was assigned to group 1 and the lowest to group 2.In treatment Group 1 cats were administered a topical fluralaner solution  at the recommended labeled dose (minimum 40 mg/kg). While this study was focused on cats, dogs resident in these households were administered an oral fluralaner chew  at the recommended labeled dose (minimum 25 mg/kg).In treatment Group 2 cats were administered a topical selamectin solution  at the recommended labeled dose (minimum 6 mg/kg). While this study was focused on cats, any dogs resident in these households were also administered an oral sarolaner chew  at the recommended labeled dose (minimum 2 mg/kg).All animals were weighed on a calibrated scale prior to treatments and products were administered according to product labeling by study personnel. Fluralaner was administered once on day 0. Selamectin and sarolaner were administered three times; once on study day 0; once between days 28–30 and finally between days 56–60. All dogs and cats living at a residence were administered group appropriate treatment and no alternative flea treatments were used during the study on pets or premises. During this study no corticosteroids, antihistamines, antibiotics or medicated shampoos were used to alleviate pruritus or skin lesions. No restrictions were placed on the animals regarding swimming, non-insecticidal baths or movement outdoors.This study was conducted without a placebo control group because the heavy and constant flea challenge experienced by cats and dogs in subtropical Florida would make inclusion of a non-treated group inhumane. Withholding flea adulticide treatment would be detrimental to the health and welfare of the pets and potentially also to humans in these households.e(x+1) transformation. The log-transformed flea counts on animals were analyzed by a mixed linear model with repeated measures including treatment, day, treatment*day as the fixed effects; and household, and animal as random effects. The log-transformed flea counts in traps were analyzed by a mixed linear model with repeated measures including treatment, day, and treatment*day as the fixed effects and household as random effect.The animal and trap flea count data were transformed prior to analysis using the Y= logA Kenward-Rogers adjustment was used to determine the denominator degree of freedom for hypothesis. Akaike’s information criterion (AIC) was used as the criterion to select the covariance structure for repeated measures. The dermatology, pruritus and SCORFAD scores were analyzed by the same mixed linear model with repeated measures as that for the flea counts on dogs. Percentages of animals without fleas were analyzed and compared using Fisher’s exact test. All comparisons were made between treatment groups on each data collection day and also between each collection day and the baseline (day 0) values within each treatment group.P < 0.05; 90% confidence intervals were constructed for the differences between treatment groups for the equivalence declaration. The primary software was SAS version 9.3 .A two-tailed t-test was used for the comparison and significance was declared when Percent control of flea counts were calculated using geometric means with Abbott’s formula:C is the geometric mean number of fleas on flea count day and MB is the geometric mean number of live fleas count on baseline.where MPercent reduction of clinical scores were calculated using arithmetic means with Abbott’s formula:C is the arithmetic mean of clinical scores on score collection day and MB is the arithmetic mean of clinical scores on baseline.where MInitially 40 private residences were enrolled in the study, although four residences in the selamectin-sarolaner treatment group did not complete the study. Two homes were dropped within the first 2 weeks because of severe cockroach infestations and data from these households were excluded. The owners in one residence moved because of the infestation severity and owners in the other residence had a professional pest management company spray their entire home with an insecticide/insect growth regulator combination. The third residence, with two enrolled cats, was lost after the 28–30-day assessment because the home was vacated after a small electrical fire. The fourth home, with one enrolled cat and one dog, was lost following the day 56–60-day appointment because the owners moved to a new home. Data from these two residences and their enrolled pets were included in analysis up to the point they were lost from the study.Additionally, there were three times during the study when data was not collected at a single counting period on an individual pet or an entire home because the animal was unavailable, or the owners were not at home that week. On day 7 data were collected from 19 of 20 homes in the fluralaner group and on day 14 data were collected from 17 of 18 homes in the selamectin/sarolaner group.In the 20 homes in the fluralaner treatment group, 31 cats  were officially enrolled on day 0. These cats were administered a mean topical dose of 58.9 mg/kg (range 40.7–86.3 mg/kg) fluralaner. There were also 18 dogs  officially enrolled on day 0 and they were administered a mean oral dose of 36.6 mg/kg (range 26.5–52.4 mg/kg) fluralaner. Additionally, there were 28 cats and 14 dogs that were treated but did not meet qualifying criteria. These animals had < 5 fleas; spent the majority of their time outside the residence; or could not be safely handled. These households had a total of 91 pets (59 cats and 32 dogs) treated with fluralaner.In the 18 homes that remained in the selamectin/sarolaner treatment group for at least 4 weeks, 29 cats  and 13 dogs  were enrolled. On day 0, cats were topically treated with a mean dose of 10.4 mg/kg (6.6–18.4 mg/kg) selamectin and dogs were orally administered a mean dose of 2.8 mg/kg (2.0–3.8 mg/kg) sarolaner. There were an additional 12 cats and 12 dogs in these residences that did not qualify for inclusion in the study for reasons previously described. These households had a total of 66 pets (41 cats and 25 dogs) administered group appropriate treatments.Pre-treatment geometric mean flea counts for cats in both groups Table  and dogsFollowing a single topical fluralaner dose, 80.6% 25/31) of treated cats had no fleas recovered in comb counts on days 28–30 and 100% (31/31) of cats were flea free at 12 weeks  in the fluralaner group traps and 28.0 (range 5–152) in the selamectin/sarolaner treatment group traps  scores of 5.87 (range 1.1–10) and 6.62 (range 0.3–10), respectively on day 0 Table . Owner aOn day 0 of the study, blinded pre-treatment feline dermatology lesion scores were similar in both treatment groups Table . DiffereC. f. felis were commonly observed by pet owners on their properties. Many pet owners reported opossums , raccoons  and/or feral cats  in their yards.Client interviews showed that reservoir hosts for No adverse events reported for any of the 100 cats in this study, regardless of treatment administered; however, two treated dogs had adverse events reported during the study. One fluralaner treated qualifying dog vomited one day before and one day after treatment administration. One sarolaner treated non-qualifying outdoor dog was found dead by its owner 7 weeks into the study (3 weeks after the second dose). The owner suspected that one of her other larger dogs killed this dog. The cause of death could not be determined as the dog’s remains were disposed of prior to reporting of this event to the study personnel. No other adverse events in treated dogs were reported.A single topical dose of fluralaner provided excellent flea control in cats, achieving > 96% reduction in flea counts, a significant decrease compared to baseline, within 7 days and 100% control at 12 weeks  from baseline was observed in both fluralaner and selamectin treatment groups, however, this was achieved sooner (30 days post-treatment) in fluralaner-treated cats compared to selamectin-treated cats (60 days) although treatment groups were not significantly different Table . Owner aThe flea control efficacies of the two oral isoxazoline formulations administered to dogs in this study were very high; were not significantly different throughout the study Table ; and werDogs administered oral sarolaner had significantly more fleas (geometric mean 37.9) than fluralaner treated dogs (geometric mean 26.9) prior to treatment (Table Intermittent-light flea traps used in this investigation provide an estimate of flea biomass, flea reproduction and emergence in homes . FollowiC. f. felis undergoes protogyny, where the first adults to emerge from a cohort of eggs are females, followed by both males and females and finally by an almost exclusively male emergent population [Previous field studies have shown that halting flea reproduction leads to a shift in the sex structure of the household emergent flea population. An initially predominately female flea population will shift toward a male dominated population , 27. Thipulation . In thispulation , 27. TheC. f. felis. These animals move readily throughout urban environments and contaminate protected outdoor premises such as crawlspaces, decks, and under bushes with flea eggs [A challenge for veterinarians and pet owners is continued flea re-infestation pressure on cats and dogs from flea-infested free roaming dogs and cats and certain urban wildlife. Urban wildlife in North America, including opossums and raccoons, can be infested with lea eggs , 2. AlthA single topical fluralaner application to cats living in an intense flea challenge environment was more effective than three consecutive monthly doses of selamectin for flea control on cats and for flea eradication in households. For dogs, both one dose of fluralaner and three doses of sarolaner delivered a very high level of flea efficacy with no significant flea control difference between the treatments. Successful household flea control requires effective treatment of both cats and dogs in the home.


Anoplophora glabripennis, the Asian long-horned beetle (ALB), is highly polyphagous and can colonize a wide range of broadleaved host trees causing significant economic damage. For this reason, it is considered a quarantine pest in Europe and North America. Although the global spread of ALB has been depicted recently, no comprehensive studies exist on the genetic pattern of populations’ establishment and dynamics at fine-scale (i.e. within invasive outbreaks), before eradication measures are applied. This information may, however, be particularly important for an efficient management and control of invasive pests. Here, we characterized population genetic diversity and patterns of spread of ALB within and among the four outbreaks detected in Switzerland between 2011 and 2015. For this, we genotyped 223 specimens at 15 nuclear microsatellite loci and conducted specific population-based analyses. Our study shows: (1) At least three independent introductions and a, human-mediated, secondary dispersal event leading to the four outbreaks in the country; (2) An overall low intra-population genetic diversity in the viable and several years active invasive populations; (3) A colonization of single trees by homogeneous ALB genotypes; And (4) an establishment of populations several generations prior to its official discovery.The xylophagous cerambycid  Insights into the species biology and genetic structure of newly established populations, as well as reconstruction of introduction events and invasion routes have the potential to define the factors underlying the invasion success and to outline the pattern of local pests’ outbreaks . This information can be particularly helpful for developing successful management strategies to prevent new invasions and control new outbreaks.The intensification of global trade and worldwide mobility has humanitarian and economic benefits, but it also involves potential challenges for native ecosystems due to the introduction of alien species. In recent years, an increasing number of invasive pests and pathogens have been reported in ecosystems worldwideColeoptera, Cerambycidae) Anoplophora glabripennis (Motschulsky) is a well-known invasive insect in Europe, in North America, and in artificial plantations in East Asia5. ALB can attack a wide range of deciduous trees and survive in cut wood. Trace-back investigations have shown that introductions into Europe and North America occurred via infested wooden packing material from East Asia6. In China, the beetle presumably co-evolved in its native range with relict plants like Eucommia ulmoides7 and, due to reforestation programs in the 1980s, spread as a pest through artificial forests  that were frequently established with tree species introduced from Europe . In invaded areas, the favourite host trees are species in the genera Aesculus, Acer, Betula, Fraxinus, Platanus, Salix, and Ulmus5. Female beetles can produce 30–60 (up to 200) eggs annually, which they oviposit in the cambial region of the host trees12. Although ALB adults, according to laboratory experiments14, have the potential to fly 14 km, in open environments they fly only 1–3 km during their life span, and when suitable host trees are available most ALB remain in proximity (hundreds of meters) to the tree from which they emerged15. Larvae feeding on the phloem and xylem tissues, before emerging as young adults from the trees (after 1–2 years), cause the most damage by disrupting the vascular system of the tree16.The xylophagous Asian long-horned beetle  in a private garden in the remote village of Brünisried, Canton Fribourg17. The source of the infestation could not be determined. During a second survey and sampling in 2013, the presence of exit holes suggested that at least one generation of ALB developed and emerged in Brünisried17. In 2012, a new ALB outbreak was found in the industrial area of the city of Winterthur, Canton Zurich9. The pest was detected on sycamore maples with new and old exit holes within three infestation spots situated about 250 m apart9. In 2014, a large outbreak was detected in Marly, an industrial city situated about 10 km away from the first detected population in Brünisried18. In Marly, exit holes were observed on several dozens of trees of Acer spp. and Aesculus hippocastanum, and ALB was found in all development stages . The infestation area in Marly included two spots (North West and South East), at about 1.5 km distance17. The fourth and last Swiss population was detected in Berikon (Canton Aargau) in 201519. A female beetle with fertilized eggs was initially found on a construction site. Later on, a sycamore maple with beetles, egg depositions and exit holes was detected 240 m away from the first finding. Because ALB is an A1 quarantine organism (EPPO 2017), all trees after sampling within outbreaks were eliminated by chipping and/or burning9. ALB was also found several times in wooden packaging material at the border of the country and in industrial sites within the country. The relatedness of these beetles to the four outbreaks remained unclear9 until the present study.Here, we aimed to investigate the invasion genetics of ALB in the four outbreaks detected in Switzerland between 2011 and 2015 , markers to explore the invasion genetics in the four ALB populations described above and in single ALB findings. The SSR markers previously developed specifically for ALB enabled to assess genetic diversity within and among invasive populations and offered a great potential to reconstruct history and the pathways of beetles’ spread using Approximate Bayesian computation analysisTherefore, with an emphasis on genetic population structure and history of local spread, we addressed the following questions: (1) How many introduction events in Switzerland led to the four outbreaks? (2) What is the genetic relatedness of the beetles in the outbreaks and single findings? (3) How genetically diverse and structured are the invasive populations?A. glabripennis. However, three different COXI haplotypes were observed: specimens from the geographically close outbreaks of Marly and Brünisried showed the COXI haplotype C, whereas specimens from Winterthur and Berikon the haplotype A .Locus-specific deviation from HWE and significant correlation between allele frequencies were not consistent among populations  was observed between all populations pairs, apart from Brünisried and Marly  was 1.5-fold higher than between the geographically more distant (~ 10 km apart) Marly #1 and Brünisried (FST_Ma1Br = 0.08).The population structure analyses described above suggests that populations in Marly and Brünisried were clearly interconnected and substantially differ from the other two Swiss populations  and the Structure results, six different scenarios of the demographic history were tested for the ABC analysis , failed to establish new populations.15. Results of our genetic population structure analysis confirm the field observations. In all populations, only one or maximum two genetic clusters were found on a single tree. Remarkably, riparian broadleaf stands that are potentially attractive for the polyphagous ALB exist in a proximity of 140 m to the first infested area (Marly #1). However, the second population (Marly #2) was established at a distance of about 1.3 km from the first, along a main road, which could be explained by local human-mediated transportation (e.g. hitch-hiking).The strong population differentiation coupled with deficit of heterozygotes suggests no gene flow between the geographically distant populations, which is expected considering the limited natural dispersal of ALB. As already observed in open environment, when suitable host trees are available most ALBs remain in proximity to the tree from which they emerged17, that would support the oldest origin of the population in Marly. The genotypes with admixed assignments indicate that beetles had probably several generations for breeding prior to sampling. The multiple genetic clusters in the oldest population may be also the result of introductions from a diverse population, presumably in South Korea as suggested in the study of Javal, et al.22. The presence in the stepwise population of Brünisried of a cluster that was not detected in the initial population of Marly, might be explained either by genetic drift that had occurred, or by the introduction of specimens from another, unidentified population. However, considering that i) Brünisried is a relatively small and remote village and ii) there is no evidence of such an unknown population in the surrounding region, we think that the last explanation is unlikely. As expected, the founder population (Marly #1) was most likely of limited effective size, owing to a transitory bottleneck. According to the supported ABC scenario and the estimated parameters, this first population experienced a lag invasion phase, i.e. a period of low growth and reduced effective size24. Intriguingly, this period was probably twice as long in the initial population as in the step-wise populations. It could be that during this period natural selection acted in favour of the phenotypes which performed best in the new environment. Thus, the beetles that established subsequent populations were already better adapted than those that initially arrived. Despite a possible uncertainty in the specific quantitative results of our ABC analysis, it shows that all three populations with common origin increased their effective sizes in about the same time period and, given also to the high fecundity of female beetle , had the potential to restore populations of up to a few thousands specimens. Although, field observations during sampling suggest that the populations in Marly and Bruenisried were less abundant in reality.Reconstruction of the invasion history of the interconnected populations of Marly and Brünisried with Approximate Bayesian Computation (ABC) suggests that initially up to two hundred ALB individuals arrived in the industrial area of Marly . ALB genotypes originating from a single tree were mostly genetically homogeneous and belonged to the same genetic cluster. However, our analyses also revealed an efficient secondary dispersal of ALB within Switzerland, most likely by human activity. Consequently, when a new outbreak of an invasive pest is detected, intensive monitoring should also target trees along the main transportation and communication routes  near the outbreak. Remarkably, the largest Swiss population (Winterthur) was genetically homogeneous but have been successfully active by several years before eradication. It seems that low genetic diversity does not compromise the ability of ALB to become established outside its native range.9.A total of 223 ALB specimens were sampled at different development stages and analysed in this study Table . Most of25. PCR products were purified with Illustra™ ExoProStar™ mix , following the manufacturer’s instructions. Sequencing reactions were performed at both strands separately, using the Big Dye Terminator sequencing kit , on an ABI 3130 Genetic Analyzer at Swiss Federal Research Institute WSL . The obtained sequences were edited manually using GeneStudio (TM) Professional Edition 2.1.2.3 and analysed with CLC Main Workbench 7. The sequences were then BLAST searched against the non-redundant nucleotide collections of the NCBI database (https://www.ncbi.nlm.nih.gov/). A COXI haplotype was assigned when match similarity of the BLAST search was greater than 98%.Until DNA extraction, the ALB specimens were preserved in absolute ethanol at −20 °C. DNA extraction was performed using the DNeasy Blood & Tissue Kit , following the manufacturer’s protocol. DNA was extracted from one leg (adults), the head  or entire eggs. The barcoding region of the mitochondrial gene cytochrome oxidase I (COXI) was amplified using the standard primer pair HCO2198 and LCO1490et al.26. Amplifications were multiplexed in six reactions as follows: (1) ALB38/ALB77, (2) ALB10/ALB53/ALB59, (3) ALB9/ALB14, (4) ALB15/ALB35/ALB44, (5) ALB40/ALB24, and (6) ALB43/ALB19/ALB30). Multiplexed PCR reactions were performed using the Multiplex PCR Kit (Qiagen), as described by Javal, et al.22. All PCR amplifications were performed on a Veriti 96-Well Fast Thermal Cycler (Applied Biosystems), and PCR products were subsequently denatured by formamide before fragment sizing on ABI PRISM 3500 . Allele sizes were scored with the GeneMapper v5 software (Applied Biosystems). Ambiguous loci and genotypes that could not be reliably scored were re-amplified and re-analysed.Samples were genotyped at 15 microsatellite loci developed by Carter, 27, the number of private alleles, the evenness of multilocus genotypes28, fixation indices, and the deviation from Hardy-Weinberg equilibrium29 were estimated using Arlequin 3.5.2.130 and the R-package poppr v 2.3.031. Pair-wise linkage disequilibrium (LD) between loci was tested with the log-likelihood ratio using a Markov chain algorithm (default parameters), as implemented in the web version of Genepop 4.232. The statistical significance of LD was inferred using 1000 permutations and a sequential Bonferroni correction with α = 0.05. Genetic differentiation among populations was assessed by calculating pairwise FST-values29 and corresponding P values (α = 0.05) with Arlequin 3.5.2.1.For population genetics analyses in the four outbreaks in Switzerland , each outbreak was considered as a population. In each ALB population, the number of alleles per SSR locus, the observed and expected heterozygosityadegenet33. First, multilocus genetic data were transformed into principal components (PCs) and the optimal number of PCs was determined with cross-validation33. Thereafter, ALB groups were predefined according to the origin of the specimens . Samples were then plotted along the first two discriminant functions of the analysed PCs.Genetic relatedness of the ALB specimens among populations as well as single findings was studied with a multivariate clustering method, i.e. discriminant analysis of principle components (DAPC), implemented in the R-package et al.34, by (1) considering the maximal mean and small standard deviation of the posterior probability of K among runs35, (2) applying ΔK methods36, using Structure Harvester37, and (3) analysing the alterations of individual assignment probabilities with increasing K . Average assignment probabilities of specimens to the genetic clusters were computed with Clumpp 1.1.238 using the greedy algorithm for K ≥ 10 and visualized using Distruct 1.139 and R graphic functions. Spatial distribution of the defined genetic clusters and their probabilistical assignments in populations were mapped with ArcGis 10.4.1.The population genetic structure of the four outbreaks was analysed with the Bayesian model-based cluster analysis, as implemented in STRUCTURE v 2.3.4. ALB specimens were probabilistically assigned to genetic clusters using allele frequencies at each locus. No prior geographic information was used (LOCPRIOR = 0 option), and the admixture ancestral model with correlated allele frequencies was applied. Analyses were run with 200,000 burn-in iterations, followed by the same number of iterations for Markov chain Monte Carlo (MCMC) in ten independent runs for each number of clusters (K) from 1 to 20. The most likely K was determined, as suggested in Janes, 40. For this analysis, the two infestation areas (North West and South East spots) in Marly were considered as different populations, that is Marly #1  and Marly #2 , geographic coordinates for the rest of 14 individuals in Marly were not recorded, thus they were excluded from the analysis. The demographic scenario that best explained the observed genetic diversity in outbreaks was inferred from two analysis steps (for details see Supplementary). First, the most likely sequences of demographic events and defined topology of ALB populations’ spread were tested. In this step, we used a broad frame of prior population parameters to compute simulated data-sets. Then, considering information from the field observations we restricted the first event of the ALB arriving to 10 generations ago and defined the time of each demographic event in a likelihood scenario. In both steps, the ABC analysis was conducted following Cornuet, et al.41 and included the following steps: (1) assume realistic competing scenarios considering structure, FST ratio between sampled populations, and field observations; (2) simulate 1 × 106 pseudo-observed datasets (PODs) for each scenario and compute correspondent summary statistics; (3) evaluate posterior probabilities of each scenario on 1% PODs with the closest summary statistic to the observed dataset and identify the best scenario in 95% confidence interval; (4) assess the confidence level of the chosen scenario as the proportion of times that this scenario was falsely rejected (type-I error) or accepted (type-II error); (5) evaluate the goodness-of-fit of the selected scenario to the data.The demographic history of the ALB spread in the two related outbreaks of Marly and Brünisried (see Results) was investigated using a coalescent approximate Bayesian computation approach implemented in DIYABC v.2.1.0The article does not contain any studies with human participants or vertebrate animals.Supplementary information


The nanoparticle material was formed as a scattering layer on a glass substrate by a coating method, which is generally used in the commercial display manufacturing process. Additionally, a planarization layer was coated on the scattering layer with the same method. The implemented nanoparticle material and planarization material endured, without deformation, the subsequent thermal annealing process, which was carried out at temperature ranged to 580 °C. We demonstrated a practical and highly efficient OLED device using the conventional display manufacturing process by implementing the YSZ nanoparticle. We obtained a 38% enhanced luminance of the OLED device and a decreased angular color change compared to a conventional OLED device. High refractive index nanoparticle material was applied as a scattering layer on the inner side of a glass substrate of a bottom emission organic light emitting diode (OLED) device to enhance light extraction and to improve angular color shift. TiO In flat-panel display devices, organic light emitting diodes (OLEDs) are widely used, from mobile phones to large size TVs because OLED has excellent electro-optical properties for display products compared to widely used liquid crystal displays (LCDs). Among the properties, the efficiency of OLED devices is an important factor for practical OLED display products. There are many studies on enhancing the efficiency of OLEDs in various technology fields, such as materials, device structures, fabrication processes, circuit design, and circuit driving. The efficiency of OLEDs has been enhanced by improving the characteristics of organic materials comprised in OLED devices and by optimizing the structures of OLED devices ,2,3,4. HAs shown in In this study, we applied high refractive index nanoparticle material as a scattering layer on the inner side of the glass substrate of the bottom emission OLED device to enhance light extraction and to improve angular color shift. The nanoparticle scattering layer is located near the emitting layer of the OLED device, compared to previous approaches mentioned above, so it rarely affects the image quality of the OLED display. We demonstrated a practical highly efficient OLED device using the conventional display manufacturing process by implementing the YSZ nanoparticle as the high refractive index scattering layer. We obtained a 38% enhancement of luminance of the OLED device and a white angular dependency (WAD) of 0.005 at 60 degrees from normal direction.2 and YSZ  were investigated as high refractive index nanoparticle materials for scattering layer. The refractive index of TiO2 nanoparticles is 2.5 and that of YSZ is 2.13, respectively. Both TiO2 and YSZ nanoparticles are suitable for high refractive index materials compared to that of glass substrate. The size of the TiO2 nanoparticle was 250–300 nm, while the size of the YSZ nanoparticle was 100–150 nm. The nanoparticles were dispersed in propylene glycol methyl ether acetate (PGMA) solvent for applying to the slit coating method, which is used in the conventional display manufacturing process for large areas. The nanoparticle suspension was coated on the glass substrate, forming the high refractive index scattering layer, followed by a thermal curing process at 300–500 °C in a furnace for 30 min. The curing temperature can be controlled according to the state of the coating layer, without any deformation. A SiO2 solution, which was normally used at spin-on glass (SOG), was used as a planarization material, which was coated on the nanoparticle scattering layer. The refractive index of the SiO2 planarization layer was 1.45, which was similar to that of the glass substrate. The planarization layer was formed on the scattering layer with the same method as the scattering layer. It was cured at 500 °C in a furnace for 30 min. The planarization layer had a flat surface covering the roughness of the scattering layer caused by the distribution of nanoparticles. Therefore, subsequent manufacturing process can be performed easily, like on the glass substrate surface. Next, the conventional low temperature poly silicon (LTPS) thin film transistor (TFT) manufacturing process was performed on the planarization layer. In the LTPS TFT manufacturing process, the thermal annealing process is required at the highest temperature, approaching 580 °C, which can cause deformation of the nanoparticle scattering layer and the planarization layer. The proposed materials should endure the high temperature during LTPS TFT manufacturing process without any deformation.TiOA conventional bottom emission white OLED display device was used to investigate the effect of the high refractive index nanoparticle scattering layer, as represented in Measurements of OLED properties were performed by recording current–voltage characteristics as well as electro-luminescence (EL) spectra. The current–voltage was acquired using a Keithley 236 voltage source unit, while the EL intensity and spectral characteristics of the devices were measured with a calibrated silicon photodiode , a photomultiplier tube, and a spectroradiometer .2 nanoparticles was fabricated using the conventional slit coating method. However, TiO2 nanoparticles are hard to disperse in PGMA solvent, therefore they were not dispersed uniformly. The coating thickness of the TiO2 nanoparticle scattering layer was 1.5 μm, then 1 μm thick SiO2 was coated as the planarization layer. 2 nanoparticle scattering layer, such as transmittance and haze. We observed 70% of total transmittance at 550 nm wavelength and 55–82% of haze. The surface of the TiO2 nanoparticle layer was rough, even though the planarization layer was applied on it. Considering its surface roughness, it would cause poor characteristics in the subsequent LTPS TFT process. Due to poor dispersivity of TiO2 nanoparticles in the PGMA solvent, the curing temperature was limited to 300 °C, which was lower than the annealing process temperature of 580 °C in the subsequent LTPS TFT process. Despite the high refractive index of TiO2 nanoparticles, the use of conditions such as particle size, sorts of solvent, and better dispersion of TiO2 nanoparticles is required.The scattering layer with embedded TiO2, comprising YSZ has a monoclinic crystal structure at room temperature. It changes to a tetragonal crystal structure and a cubic crystal structure as temperature increases. YSZ comprised of Yttria doped ZrO2 has a stable crystal structure even when temperature increases. YSZ has characteristics of chemical stability, thermal resistance, low thermal conductivity, and high strength. It has similar optical properties to ZrO2, including a high refractive index. The YSZ nanoparticle scattering layer was coated with various thicknesses of 1.5 μm, 2.5 μm, 4.0 μm, and 7.0 μm, respectively, and then was cured at 500 °C. Next, 1 μm of SiO2 was coated as the planarization layer. 2 solution permeated into the vacancies of the nanoparticle scattering layer. The large difference between refractive index of the YSZ nanoparticles (2.13) and the SiO2 material (1.45) within the interpenetrating areas enhanced the scattering effect. On the other hand, the scattering layer with embedded YSZ nanoparticles was successfully fabricated using the conventional slit coating method. ZrOA conventional bottom emission white OLED device for flat-panel display products was fabricated. The characteristics of the OLED devices with and without the YSZ scattering layer are illustrated as In the previous study, employing a scattering layer to OLED devices as a layer or film coated on outer surface of the glass substrate reported enhanced light efficiency of OLEDs . HoweverThe implemented YSZ nanoparticle material and planarization material endured, without deformation, the subsequent thermal annealing process at the temperature ranged to 580 °C. Highly efficient OLED devices can be manufactured using the conventional display manufacturing process by implementing YSZ nanoparticles. A 38% enhancement in luminance of the OLED device was achieved by the scattering layer with a thickness of 2.5 μm, compared to the device without the scattering layer. Moreover, the decreased angular color change was obtained by implementation of the nanoparticle scattering layer, where the white angular dependency (WAD) Δu’v’ was reduced to 0.005 at 60 degrees from normal direction. Thus, we believe that this study can provide a simple, practical, and low-cost method for improving the performance of OLED display products.


Cebus apella), a new-world nonhuman primate, maternal exposure to constant light during last third of gestation induces precocious maturation of the fetal adrenal and increased plasma cortisol in the newborn. Here, we further explored the effects of this challenge on the developmental programming of adrenal function in newborn and infant capuchin monkeys. We measured (i) plasma dehydroepiandrosterone sulphate (DHAS) and cortisol response to ACTH in infants with suppressed endogenous ACTH, (ii) plasma DHAS and cortisol response to ACTH in vitro, and (iii) adrenal weight and expression level of key factors in steroid synthesis (StAR and 3β-HSD). In one-month-old infants from mothers subjected to constant light, plasma levels of cortisol and cortisol response to ACTH were twofold higher, whereas plasma levels of DHAS and DHAS response to ACTH were markedly reduced, compared to control conditions. At 10 months of age, DHAS levels were still lower but closer to control animals, whereas cortisol response to ACTH was similar in both experimental groups. A compensatory response was detected at the adrenal level, consisting of a 30% increase in adrenal weight and about 50% reduction of both StAR and 3β-HSD mRNA and protein expression and the magnitude of DHAS and cortisol response to ACTH in vitro. Hence, at birth and at 10 months of age, there were differential effects in DHAS, cortisol production, and their response to ACTH. However, by 10 months of age, these subsided, leading to a normal cortisol response to ACTH. These compensatory mechanisms may help to overcome the adrenal alterations induced during pregnancy to restore normal cortisol concentrations in the growing infant.In the capuchin monkey ( In order to cope, the fetus modifies its developmental program to allow for adaptation to the ensuing new physiological contexts as a newborn, infant, and adult. Mounting experimental and epidemiological data has shown that detrimental conditions experiencedult life –5.As most species, human beings are subjected to environmental stressors, which continuously shape their evolutionary trajectory. Industrialized society is engaged in a myriad of activities that generate environmental pollution including particulate matter, high-level noise, and distorted lighting schedules . There is little doubt that the lack of adequate adaptive responses to these modern-life stressors is likely linked to chronic disease –9. HowevIn primates, maternal exposure to constant light  during the last third of gestation induced precocious maturation of the fetal primate adrenal  and resuBuilding on our previous findings , 21–23,  in utero [ in vivo plasma DHAS and cortisol levels immediately after birth and (ii) plasma DHAS and cortisol response to exogenous ACTH at one and ten months of age in infants pretreated with dexamethasone to suppress endogenous ACTH. To assess adrenal function at 10 months of age, we measured adrenal weight, mRNA, and protein expression levels of key factors in steroid synthesis (StAR and 3β-HSD) as well as DHAS and cortisol responses to ACTH and the circadian transducer melatonin in vitroEvidence suggesting that the onset of adult diseases, such as hypertension and metabolic disorders, may originate from alterations of the hypothalamus-pituitary-adrenal (HPA) axis settingin utero –27 in coCebus apella) offspring along 3 developmental stages: at birth (n=8), 1-month-old infants (n=8), and 10-month-old infants (n=8) at the Chilean Primate Center, Pontificia Universidad Católica de Chile, Santiago, Chile. For each comparison, 4 newborns were delivered by mothers which had been exposed to constant light  during the last third of gestation , whilst the other 4 were control newborns; i.e., whose mothers had undergone pregnancy under normal 14:10 light:dark conditions of the colony (lights on at 0700 h). In the facility, females and weaned newborns were maintained in individual cages, in a room with controlled temperature and humidity; with food administered twice a day and water available ad libitum [We studied capuchin monkey  were measured at 10-day intervals under i.m. Ketamine  as reported . After dhttps://www.asp.org/society/resolutions/).Animal handling and care were in compliance with the recommendations of the NIH Guide for Animal Experimentation Care. The study protocol was approved and supervised by the Commission on Bioethics and Biosafety of the Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile (CAB-Fondecyt-1050839). The research adhered to the American Society of Primatologists (ASP) Principles for the Ethical Treatment of Nonhuman Primates  and nadir (clock time 2000 h) of cortisol circadian rhythm reported previously by us in capuchin monkey [Stringent conditions were used in the design and performance of the protocols to minimize discomfort and health effects to the newborns. Venous blood samples were drawn by venipuncture using sterile 30G needles, under local (lidocaine gel) or general anesthesia . Single 0.2 ml blood samples were taken between 0900 and 1100 h at birth (3-6 hours after delivery) and at 2000 h at one and 10 months of age.n monkey .The day of birth, the mother was sedated with ketamine (10 mg/kg of body weight) and mother and newborn were removed from the cage. The newborn leg was rubbed with Lidocaine gel  and 0.2 ml of blood was collected from one saphena vein, without separating the newborn from the mother. A similar procedure was used at one month of age (lactating newborns) to draw a blood sample at 2000 h, followed by an i.m. dexamethasone injection , using dexamethasone doses previously tested by us . After tμg/kg; Cortrosyn, Organon Laboratories, Oss, Holland) was injected into one saphena vein. Blood samples were taken from the other saphena vein 15 and zero min before the administration of ACTH and at 30, 60, and 120 minutes after ACTH. Plasma was separated and stored at -20°C until assayed. During the experiment, newborn wellbeing was assessed by measuring oxygen saturation, heart rate , and respiratory frequency. Dexamethasone and ACTH doses were selected based on data from a previous study in adult capuchin monkeys [At 0700 h, newborns and infants  were sedated with ketamine  and atropine , brought to the surgical theatre at the animal facility and anesthetized with halothane (0.5-1% in 100% oxygen). At 0800 h, a single dose of 1-24 ACTH . Immediately after necropsy adrenal glands were obtained in sterile condition and cut in pieces. A piece was processed in TRIzol  for total RNA and protein isolation, whilst other piece was cryopreserved for histological studies. The remaining adrenal tissue was used fresh in explants culture as previously reported . Other o2, and 95% air with 2 ml DMEM-F12. After 6 h preincubation, the explants were incubated by 48 h in triplicate in basal condition , in presence of 100 nM ACTH (ACTH) and 100 nM ACTH plus 100 nM melatonin (ACTH + Mel). After 48 h, the adrenal explants were harvested and weighed and the supernatants were frozen at -20°C until DHAS and cortisol measurement. In each experiment, we checked cell viability by trypan blue exclusion after dispersion with collagenase. Cell viability was measured in one explant aliquot at the beginning of the experiment, and one explant aliquot of each treatment at the end of the 48-h incubation. Percentages of dead cells (n=5 LD and n=6 LL) were 8.1 ± 3.0% for LD and 7.9 ± 4.1% for LL at the beginning of the incubation. After 48-h incubation, the percentages of dead cells were 5.7 ± 2.3 for LD and 6.0 ± 1.9% for LL.Explants from each adrenal pair were prepared as described previously . Explantβ-HSD, and Mt1 melatonin receptor were determined by semiquantitative RT-PCR using 18S-rRNA as housekeeping gene by a methodology previously described [β-HSD were measured by an amplified slot-blot technique using β-ACTIN as housekeeping protein, as reported previously [After total RNA isolation using TRIzol reagent , the mRNA expression levels of StAR, 3escribed , 30. FolAdrenal gland frozen sections were cut using a cryostat  and stored at -20°C. The sections were stained with hematoxylin-eosin after drying at room temperature for 30 min, rinsing in phosphate-buffered saline and fixing for 2 min in 4% paraformaldehyde. Transmitted-light images were obtained using an Olympus microscope .Cortisol and DHAS were measured in plasma and culture supernatants using RIA assays previously validated for capuchin monkey .β-HSD and Mt1 mRNA and StAR and 3β-HSD protein in adrenal at 10 months of age were assessed by Student's t-test. The effects of ACTH on DHAS and cortisol concentration in vivo and in vitro within groups were assessed by ANOVA for repeated measures followed by the post hoc Newman-Keuls test. The effect of LL treatment on the response of DHAS and cortisol to ACTH in vivo was assessed by two-way ANOVA followed by the post hoc Bonferroni test. Statistical analyses were performed using GraphPad Prism software . Results were considered significant when P values were < 0.05.Data are expressed as mean ± SEM. The effects of maternal exposure to constant light during late pregnancy (LL group) on plasma DHAS and cortisol concentrations in the newborn at birth and infants at 1 and 10 months of age, as well as on the expression levels of StAR, 3Chronic maternal exposure to constant light during the last third of gestation induced pronounced and divergent changes in plasma DHAS and cortisol at birth, which tended to normalize by 10 months of age. At the adrenal level, we found a decreased steroidogenic capacity partially compensated by an increase in adrenal weight.μg/ml versus LL: 4.98 ± 1.17 μg/ml P=0.82 Student's t-test), and pregnancy length (157.2 ± 0.4 days versus LL: 157.8 ± 1.3 days P=0.35 Student's t-test).As reported previously  we did nImmediately after birth, newborns from mothers exposed to constant light during the last third of gestation had a markedly reduced concentration of DHAS and almost twice the concentration of cortisol than control newborns . At one As expected, dexamethasone treatment reduced DHAS and cortisol concentration the following day at clock time 0800 h to values about 1/10 lower than those at 2000 h the evening before ACTH treatment in the two groups of infants at one month of age . ACTH inConcentrations of DHAS and cortisol were measured at 0800 and 2000 h . At 0800Dexamethasone treatment effectively suppressed DHAS and cortisol to similar values in both groups of infants. ACTH induced increased DHAS and cortisol in LL and control infants, maximal responses for both steroids being observed at 120 min. As shown in , the incComparison of the ACTH response in control animals at one and 10 months of age shows that DHAS response decreases between these ages, whereas cortisol response increases. In contrast, these developmental changes in response to ACTH were not present in LL animals.Immediately after necropsy we analyzed the effect of maternal exposure to constant light on adrenal weight and adrenal morphology. We found that adrenal weight was increased in the infants from mothers exposed to constant light, which displayed a combined adrenal weight about 30% higher than control infants . When weβ-HSD, and Mt1 melatonin receptor mRNA levels , as reported previously [The differences in DHAS and cortisol levels at birth induced by maternal exposure to constant light most likely represent the decrease in fetal adrenal size due to a decrease in the fetal zone cells (DHAS producing cells) and an increase of 3eviously . in vivo. As observed at birth, plasma DHAS concentration remained lower than in control newborns. Nevertheless, in both groups ACTH increased DHAS in the same proportion with respect to basal values, supporting that the observed effect could be a consequence of a reduced number of cells producing DHAS. In line with this, the cortisol response to ACTH was enhanced in LL newborns. Altogether, these results are consistent with maintenance of the early maturation of the adrenal gland at one month of age, secondary to maternal exposure to constant light during gestation. The physiological role of increased plasma DHAS concentration present soon after birth is not clear. The possibility that DHAS may participate in adequate fetal adaptation for the transition from maternal to external environment must be considered. For instance, it has been demonstrated that DHAS is involved in preadipocyte maturation [At one month of age, when the initial effects of late gestation and delivery have been subsided, chronic exposure to constant light during the last third of gestation still had effects on adrenal function, detected on plasma DHAS levels measured at 2000 h and on the DHAS and cortisol response to ACTHturation , in the turation . In humaturation , supportturation . Howeverturation , 39. in vivo. However, at 2000 h plasma DHAS concentration in LL young animal was much lower than in control infants whilst cortisol levels were similar. The absence of clock time differences in DHAS but not cortisol production suggests a selective and long lasting effect of maternal exposure to constant light during gestation on DHAS production and/or on the clock contained in the capuchin monkey adrenal gland. Although cortisol and DHAS circadian production is controlled by several mechanisms, it is strongly regulated by the photoperiod in adult nonhuman primates [At 10 months of age, seemingly the scenario was different to that found at birth and one month of age. Plasma levels of cortisol and DHAS were similar in LL and control young animal at 0800 h and the ACTH responses of these steroids were also similar, suggesting that there is an “improvement” in adrenal functionprimates , 40, poiprimates  and in rprimates ). Therefprimates , 43. AccThere is scant information about the role of either the maternal photoperiod or the circadian system on the long-term newborn physiology. In this context, the interesting possibility that the alteration of the adrenal steroidogenic capacity may be secondary to the lack of maternal melatonin (induced by exposure to constant light) must be considered. In fact, we previously demonstrated that melatonin shifts the circadian expression of clock genes in the fetal and adult adrenal gland under culture conditions –46, and β-HSD mRNA, and protein levels, as well as the DHAS and cortisol responses to ACTH in vitro. Consistent with a compensatory response, we found that adrenal weight was higher in the LL infants than in control infants and that basal cortisol production per gland was similar to that of control infants even with a lower expression of StAR and 3β-HSD and a decreased cortisol production per mg of tissue. However, the increase in adrenal weight did not compensate the low basal DHAS production evoked by maternal exposure to constant light during gestation. These in vitro studies also indicated a decreased steroidogenic capacity to respond to ACTH. These results are in contrast with the DHAS and cortisol response to ACTH in vivo.Next, to assess whether the differences between plasma cortisol and DHAS discussed above reflect a selective compensation for cortisol at the adrenal level, we measured adrenal weight, expression of adrenal StAR and 3The observed normal plasma levels of DHAS and cortisol in response to ACTH may imply several compensatory mechanisms operating at both intra- and extra-adrenal level. A first possibility may be a low conversion of cortisol to cortisone; however, we did not find differences between the groups, whilst the plasma concentration of cortisone and the response to ACTH were similar between the groups (data not shown). Another aspect potentially modified by maternal exposure to constant light could be the level of catecholamines. In the rhesus monkey, it has been shown that exposure to constant light induced a decrease in maternal catecholamines . We did The present results clearly suggest that a compensatory mechanism may reside in the adrenal gland through the regulation of the enzymes involved in the synthesis of cortisol. However, factors such as altered clearance of steroid hormones or adaptive changes in innervation may also contribute to the observed effects. On the other hand, the present report demonstrates that the alteration of the photoperiod experienced by the mother during gestation induced several alterations in the adrenal function evidenced in the newborn. Recent data obtained in rats is in line with this finding, in which alternated photoperiod (shift photoperiods) during gestation induced a widespread of negative consequences in the adult offspring's health, including heart hypertrophy, decreased glucose tolerance, decreased spatial memory, and increased blood pressure and heart rate variability –21, openIn conclusion, our results support that maternal chronodisruption during late gestation has profound effects on primate adrenal gland maturation and it may set the DHAS and cortisol response to ACTH observed in capuchin infants. The long-term impact of these changes during postnatal and particularly adult life remains to be investigated.


Oryctolagus cuniculus) were randomly allocated into two groups. Following complete surgical transection, rabbits in group A (ECM applied) (n = 8) had their Achilles tendons reconstructed using both, nylon suture and extracellular matrix scaffold, whereas in group B (without ECM) the tendons were reconstructed using nylon suture only. After four weeks, the rabbits were euthanized and tendon samples harvested and stained with hematoxylin eosin, Mallory, and Gomory and subsequently histologically analyzed according to modified Bonnar scale. Group B had significantly stronger inflammatory response, including abundant cell infiltration and neovascularization. In group A collagen fibers were predominantly found, whereas in group B reticular fibers were more abundant. Extracellular matrix scaffold has been found to have the real potential for promoting tendon healing through accelerating collagen formation, which is crucial for restoring biomechanical properties of a tendon, decreasing peritendineous adhesion formation, and reducing inflammatory edema and subsequently pain.Extracellular matrix (ECM) has been intensively used in cardio surgery. The main goal of this research was to determine if Achilles tendon healing could be promoted by applying extracellular matrix scaffold . Sixteen (n = 16) New Zealand white mature rabbits ( The biggest, thickest and strongest tendon in human, as well as in animal body, is the Achilles tendon Injuries and subsequent inflammatory response in tendineous tissue are the common cause of pain and movement disorders. The adequate treatment usually includes a long term recovery period In the last decade a series of papers dealing with the improvement of tendon healing were published; the healing has been stimulated with ultrasound waves ®  is the extracellular matrix in a form of tissue scaffold that originates from porcine intestine and is widely used in cardiovascular surgery. However, biological and biomechanical properties of that material have not yet been sufficiently explored. The predominant component of CorMatrix® is collagen, which is frequently used in tissue engineering due to simplicity of use, remodelling properties, lack of immunogenicity, absorbability, and high potential to promote native tissue growth [® has been the focus of recent clinical studies when it was successfully used in reconstruction of the left coronary artery in a child ® was successfully used due to lack of calcification, ability to promote cellular and tissue infiltration, reduction in scar inflammatory response and hyperneovascularisation. Taking into account research data found in a number of recent studies that were predominantly published in the field of cardio surgery, the aim of the current study was to determine whether CorMatrix® could be having a positive effect on tendon healing.CorMatrixe growth , 13. Cor22.1The current experiment comply with the ARRIVE guidelines and it has been carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, or the National Institutes of Health guide for the care and use of Laboratory animals  and we confirm that such guidelines have been followed in the manuscript.2.2Ethical committee at Faculty of Veterinary Medicine, University of Zagreb, Croatia and ethical committee at Ministry of Agriculture, Croatia approved the current study , founding by Ministry of Science, Republic of Croatia.Oryctolagus cuniculus), body weight ranging from 4 to 5 kg were randomly allocated into two groups. Rabbits were sedated with 2-alpha agonist xylazine in combination with dissociative agent ketamine both applied and anaesthesia was maintained with 2% isoflurane applied through the face mask. Immediately after induction of anaesthesia, a single dose of 2% lidocaine was applied epidurally to obtain local analgesia. Surgical area was aseptically prepared. Surgery was performed without magnification tools. Achilles tendons were exposed, clamped with Pean haemostatic forceps and completely transected in their middle part. Tendon reconstruction was done with non-absorbable monofilament nylon  using Kessler suture pattern. In group A (n = 8) extracellular matrix scaffold (CorMatrix®) was attached to tendon with nylon  using simple interrupted suture pattern. In group B (n = 8) tendons were surgically reconstructed in the same manner, but without using the extracellular matrix scaffold (CorMatrix®). In both groups talocrural joint arthrodesis was performed using Kirschner wires and the joints were kept in physiological angle. In both groups paratendon was reconstructed with nylon suture  using simple running suture pattern, while skin closure was also performed with nylon suture  using simple interrupted suture pattern. In order to minimise the impact that variable surgical technique could be having on the results of this study, the same surgeons performed the surgery in both groups of the animals. Four weeks after surgery, the rabbits were euthanized and tendon samples harvested. Tendon samples were fixed in 4% buffered solution of formaldehyde, embedded in paraffin blocks and sliced in 6 μm thick slides. Histological samples were stained with haematoxylin eosin (HE), Mallory and Gomory. Unlabelled histological samples were presented to the pathologist for obtaining more accurate semi quantitative histological assessment according to modified Bonar scale. Bonar scale score was used over Movie scale for simplifying results and histology assessment of results. Bonar scale has 4 factors for evaluation and Movin eight. Both techniques are commonly used for histologically evaluation of tendon healing. Maffulli 2008 Sixteen (n = 16) New Zealand white mature rabbits  whereas in B group was 3 (2.7 ± 0.67). Significantly more inflammatory infiltrate was in B group (p = 0.00016). Neovascularisation in A group was assessed as 1 (1.42 ± 0.51) in B group as 2 and 3 (2.5 ± 0.53) also, neovascularisation was abundantly significant in B group. p value was 0.00043. Collagen fibers in A group (with ECM applied) were predominately 3 (2.92 ± 0.29) whereas in B group (without ECM) was 2 (1.8 ± 0.42) that is also considered significantly different (p = 0.00002); more collagen fibers were in A group. In contrast statistically significant higher value of reticulin fibers were in B group 3 (2.6 ± 0.52) whereas in A group was 1 (1.25 ± 0.45). P value was 0.00009. In all assessed parameters statistically significant difference between groups was noted. The results are summarised in 5 ± 0.45 ®) had a beneficial effect as it decreased neovascularization and increased inflammatory response.In our study reconstruction and tendon overgrowth were observed in the middle third of the tendon where the natural vascularization is weaker and overgrowth is slower. In the group A extracellular matrix (CorMatrix4®). In this study, we used the porcine processed extracellular matrix as a mechanical support to tendon repair, for regeneration of the target tissue without precursors for cell growth, fibrin, antibiotics, platelet-rich plasma and other substances that promote growth.The main objective of the study was to improve and accelerate Achilles tendon healing in a rabbit model by applying a porcine processed extracellular matrix (CorMatrix®), thereby extending healing time.Formed scar in the tested ligament did not have a fibrous orientation or form as in the intact ligament, indicating that the reparation and regeneration of ligaments is disputable Myostatin production has a favourable effect on physically active tendons, it stimulates proliferation and growth and the first stage of overgrowth ® acted as an accelerant for rapid collagen fibre formation, therefore reducing local inflammation. Ossification was not detected in rabbit tendons treated with extracellular matrix. This is an undesirable side effect in the chord during assisted overgrowth, as it implies a loss of elasticity of the tendon, which is prone to increased fragility under physiological strain.A previous study confirmed that morphogenetic protein-2 (BMP-2) in native cartilage enhanced callus formation and provided strength to the cartilage, thereby reducing the amount of reticulin fibres and enhancing collagen formation In vitro cell culture of the flexor tendon of rabbits, chickens, dogs and monkeys was examined, and the thickness of epitenon cell differentiation, cell migration and phagocytosis were evaluated. After 12 weeks, rabbit tendon showed almost complete overgrowth PRP is the best stimulator of stem cell migration i.e. collagen.Alsousou et al. ® influence in animals and humans tendon healing.Based on the knowledge of the effect of matrix metalloproteinases and the results, it can be concluded that its activity was higher in group A due to the greater quantity of collagen fibres In 55 patients with a painful Achilles tendon, the initial review of neovascularization was present only in 30 tendons and neovascularization spontaneously disappeared within 3 months. Inhomogeneity was found in 35 of 55 tendons ®).Hyperneovascularisation in the tendon healing is an indicator of the success of surgical treatment. No differences were found in collagen quality between the operated and non-operated group according to immunohystochemical analysis An earlier study stated that it could not be determined with certainty as to whether neovascularization is necessarily associated with oedema and pain, and that the final outcome cannot be determined based solely on the intensity of neovascularization in the area of the Achilles tendon i.e. reticulin fibres, were also found in the regeneration phase. In the early stage of overgrowth of tendons, those reticulin fibres were gradually and partially replaced by collagen fibres. Therefore, in group B, overgrowth of the tendon was significantly slower, as reticulin fibres were in a significantly higher percentage than in group A. It is assumed that the intensive inflammatory infiltrate and hyperneovascularization slowed the process of tendon overgrowth. A defect in the tendon was found in the middle third, as this is the site of the physiologically minimal vascularization of the tendon. Our research may exclude the beneficial effects of blood clot on tendon overgrowth.In the present study, neovascularization was observed in the context of the incidence of oedema and the inflammatory infiltrate. In group A, which recorded a significantly lower percentage of neovascularization, there was also less inflammatory infiltrate. It could be assumed that tendons in group A were less painful and that the regenerative properties at the end of the experiment in week 8 were significantly better, with histological examination showing a greater amount of collagen fibres. In group B, intensive neovascularization and inflammatory infiltrates were recorded. More immature fibres, ®, but without cell supplements. It is therefore easier to apply and the body is less compromised in terms of an immune response to agents. In all three cases, rapid formation of collagen was noticed, which is essential for tendon functioning.In an earlier study, mesenchymal cells impregnated with cytokines reduced inflammation in tendon healing and increased the concentration of procollagen type I , 11. Hum®) (group A) had less inflammatory infiltrate and blood vessels with more collagen fibres. The second group (B) without the use of CorMatrix® had more inflammatory infiltrate, blood vessels and immature collagen fibres.The study results confirmed the hypothesis that the group of tendons reconstructed with a Kessler suture and with the extracellular matrix set (CorMatrix5In the current research extracellular matrix accelerated collagen formation which is the essential compartment of tendon responsible for biomechanical properties. Furthermore reduced peritendinous adhesion and subsequently reduced edema and pain as a result of very easy application in injured tendon.Marija Lipar, Marko Samardžija: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Boris Zdilar, Mario Kreszinger, Marko Pećin: Conceived and designed the experiments; Performed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.Marijana Ćorić, Berislav Radišić: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Rado Žic: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.No additional information is available for this paper.


SWEET gene family even before the seedling reaches the light. Presumably, by participating in the control of major metabolic pathways, phyA sets the stage for photomorphogenesis for the dark grown seedling in anticipation of light.Phytochromes comprise a small family of photoreceptors with which plants gather environmental information that they use to make developmental decisions, from germination to photomorphogenesis to fruit development. Most phytochromes are activated by red light and de-activated by far-red light, but phytochrome A (phyA) is responsive to both and plays an important role during the well-studied transition of seedlings from dark to light growth. The role of phytochromes during skotomorphogenesis (dark development) prior to reaching light, however, has received considerably less attention although previous studies have suggested that phytochrome must play a role even in the dark. We profiled proteomic and transcriptomic seedling responses in tomato during the transition from dark to light growth and found that phyA participates in the regulation of carbon flux through major primary metabolic pathways, such as glycolysis, beta-oxidation, and the tricarboxylic acid (TCA) cycle. Additionally, phyA is involved in the attenuation of root growth soon after reaching light, possibly via control of sucrose allocation throughout the seedling by fine-tuning the expression levels of several sucrose transporters of the  Plants use light not only as a source of energy but also as an environmental cue that can trigger developmental changes. As sessile organisms, sensing the surrounding environment and coordinating their development in response is necessary for plant survival. Photoreceptors, such as phytochromes, cryptochromes, and phototropins, sense light conditions and direct cellular and developmental responses .Phytochromes, which specifically perceive the ratio of red light (R) to far red light (FR), play a well-studied role in photomorphogenesis by coordinating signal transduction pathways leading to changes in gene expression and eventually to de-etiolation . Key to PHYA-E), it has been suggested that light-activated phyB may play a role in the seed before germination, possibly preconditioning the developing seedling for photomorphogenesis while still growing in the dark  and phyA mutant and WT seedlings under the same experimental conditions as described for published work in Arabidopsis , and built co-expression networks. We then used gene ontology (GO) enrichment analysis on both the DE proteins and transcripts and the co-expression networks to better characterize the functions of proteins and transcripts regulated by phyA.To determine the molecular role of phyA during skotomorphogenesis and seedling transition to light and to compare the tomato photomorphogenic response with that of the well-studied model plant Arabidopsis , we profbidopsis . We mademin of R . Using cphyA mutant comparisons both in the dark and in R  significantly differentially expressed proteins (DEPs) . Of thesArabidopsis thaliana using microarrays . The shared genes that are induced by R include the phy responsive transcription factor HY5, circadian clock transcription factors RVE1, RVE7, REV8, and LHY1, HSP70, β-amylases, and several photosynthesis genes. The shared genes repressed by R include circadian clock genes RAV1, a transcription factor, which may be a negative growth regulator , “pyruvate metabolic process” (GO:0006090), and the “tricarboxylic acid (TCA) cycle” (GO:0006090). In contrast, GO analysis of the DEPs and DETs from comparisons between seedlings in the dark to R did not show enrichment of any of the same categories  of transcripts in phyA mutants, in fact showed lower abundance in phyA mutants than in WT regardless of light exposure (phyA mutants relative to WT but were relatively unaffected by R. For gene ID Solyc06g072580 (one of these eight transcripts annotated as belonging to the pyruvate dehydrogenase complex), the protein was also DE, but it was significantly downregulated in the mutant and again relatively unaffected by R. The consistent differential expression between WT and phyA mutants but not between dark and R , “GTPase activity” (GO:0003924), and “GTP binding” (GO:0005525)  . Like thparisons . Like thn the WT . Two genn the WT . This DEphyA mutant dark versus R-treated set. These values are consistent with what we would expect from random chance shown by permutation testing , suggesting no correlation between the proteome and transcriptome in the dark to R transition after 60 min of light. However, of 39 DEPs and 5651 DETs in the dark grown WT to phyA comparison, there were 23 overlapping gene IDs between the data sets, more than would be expected by chance (p = 0.0001). Similarly, in the WT to phyA in R comparison, of 62 DEPs and 5423 DETs, there were 29 overlapping gene IDs, again more than would be expected by chance (p = 0.01). Of these 21 and 29 genes, 15 genes were DEPs and DETs found in both the dark and R WT to phyA comparisons.While we found corresponding GO categories between DEPs and DETs, we found very little else coordinately regulated in the same direction (up versus down) between the transcript and protein data. First, we compared our proteomic and transcriptomic analyses to find gene IDs that were identified as DEPs and DETs in the same comparison. Of the 52 gene IDs that are common between the DEPs and DETs across the four comparisons, none were from the set comparing WT dark versus R-treated plants, and only two were from the phyA mutant comparisons, the overlapping DEPs and DETs did not behave similarly overall. Of the 20 gene IDs unique to one comparison, only nine were regulated in the same direction, for example higher expression in the mutant than WT, in the protein and transcript data while the remaining eleven were regulated oppositely to each other. Of the 15 overlapping gene pairs, 11 were oppositely regulated in both WT to mutant comparisons. These genes include many of the primary metabolism genes from phyA mutants, the regulation of the transcriptome and that of the proteome are not synchronized.Despite more DEPs and DETs encoded by the same gene than one would expect by chance in the WT to phyA mutants transcriptional upregulation may be induced, leading to the unsynchronized levels of proteins and transcripts observed here. Interestingly, phyA mutants have low expression of many translation-related transcripts , suggesting that steady state ATP levels are not affected by the differences in transcript or protein expression of the primary metabolic enzymes , meaning they are among the most similarly expressed genes and strongly contribute to the expression pattern that defines the network  suggesting negative regulation by phyA, but not significantly correlated to condition, suggesting little to no regulation by R . Purple  enzymes  fell int enzymes , S7. The network . When we network , such as network , which a network , S8, whi network . The purcategory , S8, thuphyA mutant was low in both conditions  and histone acetylation (GO 0016573) respectively and contained genes known to be involved in the phyA response such as HY5 , but had statistically significantly surpassed them after 15 days of growth in the dark, according to a t-test , and only used synchronized seeds for the phenotypic experiments described in Besides the safe green light during collection, ambient light exposure during sterilization was the only light to which these seeds were exposed as there is no light treatment required to induce uniform germination in tomato. To additionally ascertain that the use of safe green light did not inadvertently trigger photomorphogenesis, we also performed a biological assay in which we measured chlorophyll concentrations in cotyledons of seedlings grown in the various conditions and found the same very low levels of chlorophyll in all conditions (about two orders of magnitude less than light-grown tomato leaves)  and no dIn summary, the results suggest that phyA plays a subtle role in the dark hypocotyl growth of these seedlings at 15 days after imbibition that cannot be explained by ambient lab light exposure during sterilization or green light exposure during collection.phyA plants, but also implicated sucrose transport as a potential mechanism by which phyA regulates early seedling development. Chen and co-workers showed that AtSWEET11, AtSWEET12, and AtSWEET15, all sucrose transporters, are involved in provisioning seeds with sugar during fruit development . phyA mutants grown in R did not show a statistically significant increase in root length in response to sucrose, while WT in R did  expression and reported elevated levels of ABI3 in dark-grown Arabidopsis phyB seedlings. These authors hypothesized that high ABI3 expression in the Arabidopsis phyB mutant might be responsible for putting the seedling in a quiescent stage in the absence of photomorphogenesis-inducing light conditions. Interestingly, our data (ABI3 homolog (Solyc06g083590) in dark-grown tomato phyA mutants is also highly transcriptionally upregulated (∼15-fold) compared to WT. It is possible that the absence of phyA in tomato seedlings has a similar effect on inducing quiescence as the phyB mutation does in Arabidopsis. Since phyA, at least in Arabidopsis, is unlikely to be the direct signaling agent between seed and seedling, a more likely scenario is one in which light-activated phyA-mediated signaling occurred during seed development and is propagated to the germinating seed and seedling through an intermediary, such as ABI3. Active phyA signaling could be initiated by phyA in the fruit of the mother plant or by phyA in the developing seed, an activity that is then lost during maturation or dormancy. Our data support this scenario of signaling from developing seed to germinating seedling, but our results cannot determine whether the active phyA signal comes from the mother plant, the seed itself, or both.If phyA is indeed absent from the mature tomato seed, then, as proposed in the second possibility above, active phyA in the developing seed or in the fruit of the parent might trigger downstream cascades that would then be active in the germinating seed and seedling. our data  show thaLastly, the third possibility suggested above where either minute amounts of active Pfr are formed in the dark by stochastic conformation changes, or PIFs are activated by the normally inactive Pr form at very low rates, is also consistent with our observations.phyA seedlings. Mutants grown in the dark exhibited stark gene expression differences from WT  has been shown to be a central player in the transcriptional reprogramming in response to declining energy levels leading to the induction of catabolic processes and the reduction of some anabolic reactions  and homozygous phyA mutant (fri) in the MoneyMaker background , which have been described previously (-2 sec-1) before harvesting and flash freezing. Samples were stored at -80°C until RNA and protein extraction.eviously , were usTissue was grown in six biological replicates of which all six were used for proteomic analysis, and four of these same biological replicates were used for RNA-seq analysis.-1 urea, 2 mol -1 thiourea, 2% cholamidopropyl-dimethylammonio-propanesulfonic acid (CHAPS), 2% nonylphenoxylpolyethoxyl ethanol (NP)-40, 0.002% Bromophenol Blue, 0.5% IPG buffer and 100 mmol-1 dithierythritol] with vortexing and allowed to incubate at room temperature for 30 min. Samples were spun, the supernatants with rehydrated protein were collected, and the pellets were discarded. Protein concentrations were determined using a 2D Quant kit  according to manufacturer instructions.Proteins were extracted and rehydrated following a previously established protocol with slight modifications for plant tissue . SpecifiProtein samples were separated by 2D gel electrophoresis as previously described . BrieflyPHYA or red light treatment, a Student’s t-test was performed on normalized spot volumes (p < 0.01).Digitized images from biological replicates of each genotype/condition were fused into a composite image using Delta 2D  and spot boundaries were identified. The relative spot volume for each spot on a gel was normalized against total spot volume in the image. To determine which proteins changed in volume due to mutation in -1 trypsin  overnight at 37°C. Digested proteins were eluted and combined with matrix solution, then spotted onto an AnchorchipTM target plate  in duplicate. One replicate of the spotted proteins was washed with 0.1% trifluoroacetic acid (TFA) and recrystallized using an acetone/ethanol/0.1%TFA (6:3:1) mixture. Peptide mass fingerprints (PMFs) were obtained using a matrix assisted laser desorption ionization tandem time-of-flight (MALDI TOF-TOF) mass spectrometer . We used flexAnalysis  to detect peptide peaks (PMF threshold of 500 ppm for MS and LIFT threshold of 0.6 Da for MS/MS). Porcine trypsin (Sigma-Aldrich) was used for internal mass calibration. Proteins were identified using Mascot protein identification software  and combined PMFs and tandem mass spectra in a search against the NCBI Solanum protein database.All visible spots were excised from the gels using a gel corer. Gel plugs were distained, dehydrated, and digested with 11 ng μlhttp://www.ncbi.nlm.nih.gov/sra/SRP072067.Frozen tissue samples were ground into a fine powder in liquid nitrogen with a mortar and pestle. For each biological replicate, 100 mg of seedling tissue was pooled (∼5 seedlings) for each genotype/condition, and RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. Library construction and sequencing was conducted by BGI Americas  using paired end 100 bp reads on an Illumina Hi-Seq 2000 instrument. All data are available for public use at NCBI’s short read archive 2 using both Tophat2  regardless of mapping strategy by at least two of three DE methods and for which gene identifiers (Solyc numbers) existed in the Sol Genomics Networks database.RNA-seq reads were mapped to the SL2.4 version of the tomato genome with ITAG2.4 genome annotation from Sol Genomics Tophat2  and HISA Tophat2 . DETs we Tophat2 , edgeR ] and used as input for WGCNA  were used as the “gene universe.”Gene lists were tested for GO enrichment using topGO in R . P-valueed model . All catp-values were calculated by percentage of permutations at or greater than the observed overlap. This permutation testing was completed for each DE comparison randomly drawing the appropriate number of DEPs and DETs. The same approach was used to compare overlap between DETs found in A. thaliana , phyA mutants and MoneyMaker seedlings were sterilized in 50% bleach for 15 min rinsed, and placed on saturated filter paper in light-excluding plastic boxes in a dark incubator at 25°C. Sterilization and planting either happened in ambient lab light or in darkness with green safe light supplementation. Two days after sowing, seeds were checked for germination under green safe light. Only those seeds with slightly protruding radicles (∼ 2 mm long) were considered germinated and transferred to a new box for the experiment. Seedlings were grown in the dark for 15 days, with a subset of them collected at day 5 and day 15 under green safe light, and scanned on a flat-bed scanner. Root and shoot lengths were measured with ImageJ.In six separate biological replicate experiments  with or without 2% sucrose. At 5 days old, half of the seedlings were exposed to continuous R  for 24 h. Seedlings were collected and scanned at 6 days old. Roots and shoots were measured using ImageJ.phyA mutants and MoneyMaker seedlings were sterilized in 50% bleach for 15 min, rinsed, and placed on saturated filter paper in light-excluding plastic boxes all while working under green light. Boxes were then placed in a dark incubator at 25°C. After 5 days, dark grown seedlings were collected in pools of 5, weighed, and flash frozen under green light. The remaining seedlings were exposed to 1 h of red light  before collection, weighing, and flash freezing. Frozen tissue was ground in a mortar with a pestle and liquid N. Samples were boiled for 10 min in 2 mL of Tris-HCL Buffer . ATP standards were prepared in the same buffer (5X dilution series from 100 μM to 6.4 nM) with Tris-HCl used as blank. After boiling, samples were spun at 4°C for 10 min at 14,000 rpm in a table top centrifuge. Samples and ATP standard were loaded into a plate provided with the ATPlite 1step kit , substrate solution was added according to kit manufacturer’s instructions, and luminescence was measured by a SpectraMax M2 microplate/cuvette reader .-2 sec-1) before harvesting and flash freezing. Total RNA from dark grown and 60 min R grown tomato seedlings was extracted (3 μg for ambient light sterilized and 1 μg for dark sterilized) using a QIAGEN RNeasy kit as per the manufacturer’s instructions. Reverse transcription was performed using an iScript cDNA Synthesis kit (Bio-Rad) and a thermocycler program of 25°C for 5 min, 46°C for 20 min and 95°C for 1 min. QPCR was performed on a Bio-Rad Mastercycler C1000. The reactions were carried out using iTAQ Universal SYBR Green Supermix (Bio-Rad) and incubated at 95°C for 3 min, 40 cycles of 95°C for 10 s, and 60°C for 30 s followed by 95°C for 10 s. SAND (Solyc03g115810) and RPL2 (Solyc10g006580) genes were used for normalization. PCR specificity was checked by melting curve analysis, and data were analyzed using the 2-ΔΔCt method , rinsed, and then sown on saturated, sterile filter paper in light-excluding plastic boxes inside a dark growth chamber at 25°C. Five-day old seedlings of similar height were harvested in green safe light, and flash-frozen in liquid nitrogen. Half of the seedlings were exposed to R treatment for 60 min (660 nm LED custom display, 10 μmol mt method . StatistTranscriptomic, proteomic, and co-expression network analysis suggest that phytochrome A plays a role in primary metabolism in dark grown seedlings. Physiological experiments further show that phyA, via the SWEET family of sucrose transporters, fine-tunes root and shoot elongation in the transition from dark to light growth.AM conceived the original research experiments. KC, SB, DA, and AM performed the experiments and analyzed the data. LT supervised the proteomics experiments. KC and AM wrote the article with contributions of all the authors.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.