Pinus. ponderosa (P. Lawson and C. Lawson) is a commercial tree and one of the most important forest species in North America. Ponderosa pine suffers hardship when going through vegetative propagation and, in some cases, 15–30 years are needed to achieve full reproductive capacity. Based on previous works on P. ponderosa regeneration through in vitro organogenesis and trying to improve the published protocols, our objective was to analyze the influence of different types of explants, basal culture media, cytokinins, auxins, and light treatments on the success of shoot multiplication and rooting phases. Whole zygotic embryos and 44 µΜ 6-benzyladenine showed the best results in terms of explants survival. For shoot organogenesis, whole zygotic embryos and half LP  macronutrients were selected. A significant positive interaction between whole zygotic embryos and half LP macronutrients was found for the percentage of explants forming shoots. Regarding the light treatments applied, a significantly higher percentage of shoots elongated enough to be rooted was detected in shoots growing under blue LED at a light intensity of 61.09 µmol m−2 s−1. However, the acclimatization percentage was higher in shoots previously cultivated under fluorescent light at a light intensity of 61.71 µmol m−2 s−1. Anatomical studies using light microscopy and scanning electron microscopy showed the light treatments promoted differences in anatomical aspects in in vitro shoots; needles of plantlets exposed to red and blue LEDs revealed less stomata compared with needles from plantlets exposed to fluorescent light. Pinus spp.)  stuk (1984)  analyzedk (1984)  investigk (1984) , and thek (1984) . Finallyk (1984) . Pinus. Moreover, as a result of priming cells during the somatic embryogenesis process in conifers, different metabolic, epigenetic, and proteomic profiles related to abiotic stress tolerance have been described recently [P. ponderosa by testing the chemical composition of the culture media or physical factors, such as type of lights (fluorescent versus LEDs) and their effect on morphogenesis, tissue micromorphology, and the overall success of the process.In our lab, several studies have been developed to optimize somatic embryogenesis  and orgarecently ,22,23. HP. ponderosa in order to develop an efficient protocol for in vitro propagation. To carry out this objective, we focused on improving (1) the multiplication phase using different types of explants, cytokinins, culture media, and light treatments, and (2) the rooting stage using different auxins and light treatments. Considering the abovementioned information, the main objective of our study was to optimize the organogenesis process of Contamination rates were registered after sterilization protocols A (21%) and B (16%). The sterilization protocol did not significantly affect the survival percentage. Based on this result, protocol B was selected as the preferred sterilization method in Experiment 2.When the type of initial explant was analyzed, a significantly higher survival percentage was observed when using whole zygotic embryos (89%) instead of isolated cotyledons (77%).Explants’ survival was also significantly affected by the 6-benzyladenine (BA) concentration used ; explantThe interaction between BA concentration and culture medium, and between explant type and sterilization protocol did not reveal statistically significant differences .When considering all the variables analyzed  and the interaction between them, after four weeks of induction medium (IM), statistically significant differences were only found for the percentage of explants forming shoots (EFS) depending on the culture medium, the explant type and the interaction between them . Whole zThe highest number of shoots (3.31 ± 2.8) were found in whole zygotic embryos cultured in HLP supplemented with 22 μM BA. p-value was bordering on significance. The survival percentage was 93% in whole zygotic embryos cultured with meta-Topolin (m-T) in the culture medium and growing under blue LEDs. On the other hand, 100% survival was found in whole zygotic embryos growing under the remaining treatments tested.In this experiment, the contamination rate registered was 1%. As shown in After five weeks of culture in the induction medium (IM), no significant differences were found for the percentage of EFS according to the cytokinin type, the light treatment, or the interaction between them . The EFSRegarding the number of shoots per explant (NS/E), no significant differences were observed for the variables studied (cytokinin type and light treatment), or the interaction between them . The NS/On the other hand, the cytokinin type and the interaction between cytokinin type and light treatment did not have a significant effect on the PSR . No statistically significant differences were observed in shoots exposed to different light treatments for root induction percentage (RI), number of roots per explant (NR/E), or the length of the longest root (LLR) . The RI The highest response for root induction was obtained in shoots exposed to white LEDs and the lowest response was recorded in shoots under blue LEDs . When shAcclimatized shoots were successfully obtained from shoots exposed to FL (50%), shoots under white LED (20%), and shoots exposed to blue LEDs (14%); it was not possible to acclimatize shoots grown under red LED.P. ponderosa needles from plantlets cultured with different cytokinin types and light treatment were analyzed. Regardless of the cytokinin type employed, needles from plantlets did not show microscopic alterations in the internal structure and organization.The effect of the chemical composition of the culture media and the physical factors on tissue micromorphology from needles of in vitro plantlets was studied, and then it was compared to the micromorphology from needles of ex vitro plants. In this way, adaxial and cross sections of Adaxial needle surfaces from in vitro plantlets indicated the presence of stomata arranged in parallel and uniseriate bands, while the margin areas were serrated with spinose teeth a. The longitudinal section of needles showed the parenchyma mesophyll cells and the large intercellular spaces between them. No structural changes in the mesophyll cell of needles from plantlets exposed to light treatments were found b. FurtheHistological analysis of needles from in vitro plantlets showed that the epidermis presented a single-cell layer and numerous tannin-rich cells were spread among the mesophyll, where large intercellular spaces were common d. No cleAs a general trend, needles of plantlets exposed to blue and red LEDs showed less stomata compared with the stomata of needles from plantlets grown under FL a,c,h. InThe morphology of the stomata of needles from shoots grown under white LEDs e was simThe internal structure and organization of needles from plantlets was similar for all light treatments b,d,g,i.SEM analysis showed similar anatomical aspects when needles from field-growing plants were compared with needles of in vitro plantlets a. The chThe middle zone showed the organization of the vascular bundle and surrounding tissues; xylem, phloem, endodermis, and transfusion tissue were recognizable a.The resin duct was generally localized adjacent to the epidermis and partially immersed in the mesophyll cells b. The prP. ponderosa material in vitro, contamination rates ranging from 1% to 16% were obtained using hydrogen peroxide (protocol B). In P. radiata [Pinus species as well. The most important initial step in the micropropagation process is an adequate sterilization protocol that minimizes tissue damage to achieve the greatest number of aseptic explants ready to be used in the next phases of micropropagation [The establishment of an aseptic in vitro culture is one of the most important factors of plant tissue culture and the effective elimination of contamination contributes to the better development of the explants ,27. When radiata ,28, hydrpagation .P. halepensis Mill. when using whole embryos or cotyledons as initial explants, over 90% of the embryos gave rise to adventitious buds, whereas isolated cotyledons slowly developed adventitious buds and these buds failed to elongate [P. pinaster and mentioned that this would increase the genetic diversity as well as considerably decrease the manual labor cost.As has been recently reviewed, factors such as explant source and plant growth regulators influence the in vitro regeneration of plants via organogenesis in different species ,31,32. Ielongate . These relongate  selectedP. ponderosa. Furthermore, several studies in P. halepensis, P. pinaster, P. ayacahuite, and P. ponderosa reported that salt concentration in the culture medium had an influence on morphogenesis [In our study, the capacity of explants to form shoots was significantly higher when whole zygotic embryos were cultured in HLP. Following the trend observed in previous studies ,8,14, siogenesis ,35,36.In woody species, it has been recognized that cytokinins are necessary to promote in vitro multiple shoot formation ,37. AmonPinus where the culture medium was supplemented with BA (from 1 to 50 µM) to obtain bud induction [In our experiments, BA at the concentrations tested  showed efficient organogenic response for NS, and, although not significant, a slightly higher response in whole zygotic embryos cultured in HLP supplemented with 22 µM BA was observed. A similar tendency was observed in several micropropagation protocols in nduction ,40,41,42Aloe polyphylla, the use of m-T resulted in a high multiplication rate [Spathiphyllum floribundum. In the same way, in P. sylvestris and P. pinaster, a higher rate of organogenic response was obtained by using m-T instead of BA [P. radiata and Sequoia sempervirens [When EFS and NSE were analyzed, no significant differences were found for the effect of BA or m-T, but a slightly higher response in explants cultured with BA was observed. BA in in vitro culture can cause hyperhydricity, shoot-tip necrosis, and histogenic instability . This waion rate . Likewision rate  found bead of BA ,46. Howeervirens ,32,41. SGerbera jamesonii in vitro shoots growing under red LEDs displayed the greatest elongation rates [Passiflora edulis reported an increase in chlorophyll content and plantlet quality when plants were grown under red LEDs. Additionally, the highest stem length was observed in in vitro potato plantlets under red LEDs [The influence of light in regulating growth, development, and adventitious root formation has been confirmed in different studies ,47. LED on rates . Chen eton rates  in Passired LEDs . Curculigo orchioides and Zingiber officinale where blue LEDs promoted in vitro shoot formation [Solanum tuberosum when cultured under blue LEDs. When studying the effect of the light treatment on PSR, shoots growing under blue LEDs showed the highest response. These results agree with those observed in ormation ,53. Furtormation  observedZ. officinale and Phalaenopsis pulcherrima indicated that LED treatments promoted in vitro root induction and development [Z. officinale and Citronella mucronata a higher rooting response was found when cultured under red or the combination of red and blue LEDs in a ratio of 1:2 [Studies performed in elopment ,54. In oo of 1:2 ,55.Cucumis metuliferus, explants showed high root induction rates when exposed to white LEDs and in a mixed circuit of blue and red [Cedrela odorata in vitro cultures grown under white LEDs presented a higher number of roots formed [In  and red . Furthers formed . In thiss formed  explaineP. radiata, in vitro shoots growing under FL showed high NR/E [Handroanthus ochraceus where plants showed higher root length values when exposed to low FL irradiances [P. radiata and Vitis ficifolia growing under red LEDs [Contrary to our results, in igh NR/E . Regardiadiances . In contred LEDs ,57.P. ponderosa in vitro shoots. The morpho-anatomic characteristics of needles from in vitro and ex vitro plants were similar and are in accordance with the study described for P. halepensis by [The light treatments tested promoted differences in anatomical aspects of the stomata in the needles of ensis by .P. halepensis and P. nigra [P. nigra by Mitic et al. [P. halepensis, [P. canariensis presented a volcanic shape [The teeth structures on the needle surface were morphologically similar to those found in primary needles of P. nigra ,59. In tP. nigra  describeepensis,  large anic shape .P. halepensis seedlings, primary needles were characterized by the absence of a hypodermal cell layer and the epidermis being in direct contact with the mesophyll tissue, but these differences could be attributed to the fact that these analyses were performed in seedlings 22–24 weeks after emergence [SEM analysis revealed the presence of hypodermis in needles from field-growing plants. Contrary to our results, in mergence . Musa spp.; the abaxial epidermis was thinner than the adaxial epidermis [Chrysanthemun leaves, the thickness of the adaxial epidermal cells increased under white LED; blue LED favored the anatomical development of the palisade parenchyma layer; and red LED reduced this parenchyma [Solanum tuberosum plantlets. Epidermises with greater thickness were induced and palisade parenchyma and spongy parenchyma were arranged neatly [P. ponderosa. LED sources improve the leaf anatomy of in vitro plantlets in pidermis . In Chryrenchyma . Red LEDd neatly . HoweverPfaffia glomerata in vitro plantlets, increased in the size of vascular bundles and vessel elements under combinations of red and blue LED. In the same way, the central vascular bundle of plants from Epidendrum fulgens grown with natural ventilation under combinations of white/high blue, deep red/white/medium blue, and deep red/white/far red/medium blue LED had a higher content of sclerenchyma [Z. officinale. Summarizing, the effect of different LEDs sources at different concentrations in morpho-anatomic characteristics seems to depend on the species cultured at in vitro conditions.Silva et al. , in Pfafrenchyma . Furtherrenchyma  found thP. ponderosa and their ex vitro acclimatization. Furthermore, our results demonstrated that different LEDs can be strategically used to improve micropropagation efficiency and reduce the costs of in vitro ponderosa pine plant production.All these aspects could, to some extent, explain the results obtained in our study, where the basal media, cytokinins, the explant type, and the light quality influenced the organogenic process and root formation of P. ponderosa (P. Lawson and C. Lawson) seeds were obtained from 14-year-old trees grown in a clonal seed orchard of the Rotonda, Trevelin Experimental Field,  (batch number: 25 U7519 JP) and they were also purchased from the Instituto Nacional de Tecnología Agropecuaria . −1 sodium hypochlorite) plus one drop of Tween 20® for 20 min, followed by three rinses in sterile distilled water for 5 min each; (2) 10% H2O2 (v/v) (30% active H2O2) plus one drop of Tween 20® for 20 min, followed by three rinses in sterile distilled water for 5 min each. The sterilization protocols were performed under sterile conditions in a laminar flow unit. Seeds were stored in moistened sterile filter paper at 4 °C in darkness. After four days, seed coats and megagametophytes were removed aseptically and two explant types (a) cotyledons (Seeds were rinsed under running water for 5 min and sterilized following two different protocols: (1) commercial bleach 5% (active chlorine 37 gLtyledons a or wholtyledons b were usw/v) sucrose and solidified with 8 gL−1 Difco Agar granulated. Moreover, three concentrations of BA  were evaluated. The pH of all media was adjusted to 5.8 before autoclaving . Cotyledons were excised from embryos and placed horizontally onto the induction medium; whole zygotic embryos were cultured in an inverted position with the cotyledons immersed in the induction medium. After sterilization, explants (cotyledons or whole zygotic embryos) were cultivated on Petri dishes (90 × 15 mm) containing 20 mL of bud induction medium (IM) a,b. Two −1 activated charcoal, 3% (w/v) sucrose, and was solidified with 8.5 gL−1 Difco Agar granulated. After 30 days in culture (when elongating needle fascicles were evident), explants were subcultured into baby food jars with Magenta TM b-cap lids containing 25 mL of LP or HLP elongation medium. The shoots were subcultured every six weeks into the same medium. When shoots were 10–15 mm long, they were separated and cultivated individually in a fresh medium. As soon as bud induction was observed (after four weeks), the explants were transferred to Petri dishes containing 20 mL of elongation medium (EM). EM consisted of hormone-free LP or HLP supplemented with 2 gL−2 s−1 light intensity provided by cool white fluorescent tubes .All the cultures were laid on the growth chamber at a temperature of 21 ± 1 °C, at a 16 h photoperiod with 120 µmol m−2 s−1; (B) red Light (peak wavelength 630 nm), 61.09 µmol m−2 s−1; and (C) white light (color temperature 4000 K), 61.09 µmol m−2 s−1, all of them provided by adjustable LEDs (RB4K Grow LEDs). As a control, whole zygotic embryos were cultured under cool white fluorescent light (FL)  at 61.71 µmol m−2 s−1 light intensity. The cultures were laid on the growth chamber at a temperature of 21 ± 1 °C for five weeks at a 16 h photoperiod.Based on the results of Experiment 1, sterilization protocol B and whole zygotic embryos were used as initial explant and HLP was selected to carry out this experiment. Two types of cytokinins were tested: BA and m-T at the same concentration (13.1 µΜ). Three different light treatments were also tested for the conditions named above: (A) blue light (peak wavelength 470 nm), 61.09 µmol mAs soon as bud induction was observed (after four weeks) c, the exw/v) sucrose and solidified with 8.5 gL−1 Plant Agar®. The different light conditions were the same as described above. After four weeks of culture in medium A or two weeks in medium B and C, explants were cultured in baby food jars with Magenta TM b-cap lids containing 25 mL of root expression medium (REM), which consisted of hormone-free HLP supplemented with 2 gL−1 activated charcoal, 3% (w/v) sucrose, and 8.5 gL−1 Plant Agar® for six weeks. Growth chamber temperature and photoperiod were the same as those described above.After the elongation phase, shoots of at least 20–25 mm long were used for root induction. The explants were cultivated into baby food jars with Magenta TM b-cap lids containing 25 mL of root induction medium, which consisted of HLP supplemented with three different auxins: (A) 5 µMNAA; (B) 10 µM NAA; and (C) a mixture of 5 µM NAA and 5 µM IBA; all media were supplemented with 3% (v/v) and acclimatized in the greenhouse under controlled conditions at 21 ± 1 °C with progressively decreasing humidity for one month from 95% to 80%  containing perlite:peat  moistened with liquid HLP. After six weeks in REM, explants with visible roots e were tr% to 80% f. Prior v/v) (1 h each).For structural analysis after eighteen weeks in the elongation medium, 16 needles were used following the procedure described in Giacomolli et al. . Two neev/v) for 120 min; ethanol 90% (v/v) for 90 min (2×); ethanol 95% (v/v) for 90 min; ethanol 100% (v/v) for 90 min (×2); ethanol: Clear Rite™ (1:1 v/v) and pure Clear Rite™ for 90 min (2×). Then, samples were embedded in paraffin wax at 65 °C for 120 min (2×) before the inclusion in paraffin molds. After paraffin inclusion , sections of 8–10 µm were obtained in a rotary microtome  and transferred to microscope slides previously prepared with albumin glycerol and kept at 30 °C for 12 h. Next, the deparaffinization was performed with Clear Rite™ for 20 min, 100% ethanol for 4 min, and washing under running water. The samples were stained with an Astra Blue (0.15%) and Safranin (0.04%) water solution for 10 min; and finally, they were observed in an optical microscope  and photos were taken with a Leica camera (Leica Application Suite version 4.13).Subsequently, the samples were embedded in paraffin by means of dehydration with ethanol and Clear Rite™ and the consecutive immersion in liquid paraffin at 65 °C, as described in Rossi et al. . The dehv/v) for 15 min each. For scanning electron microscopy (SEM), the analyses were performed following the procedure described in Giacomolli et al. . Two neeAll the samples were cut into small pieces and prepared following the procedure described in Marques et al. . Then, tThree Petri dishes and eight to ten explants per Petri dish (cotyledons or whole zygotic embryos) per sterilization protocol, were cultured in each culture medium and BA concentration. Contamination, survival, and explants forming shoots (EFS) percentages for each condition tested were measured after two months of culture. When the axillary shoots were isolated and cultured individually in an elongation medium, the EFS and the mean number of shoots were calculated with respect to the non-contaminated explants. A logistic regression model was used to analyze the effect of the sterilization protocol, explant type, culture medium, and BA concentration on survival and EFS (%). When necessary, Tukey’s post hoc test (α = 0.05) was used for multiple comparisons.Five Petri dishes and eight whole zygotic embryos per Petri dish, cytokinin type, and light treatment were cultured in HLP. When the axillary shoots were isolated and cultured individually in the elongation medium, the EFS, the NS/E, and percentage of (PSR) out of the total number of shoots produced per explant were calculated. A logistic regression model was used to analyze the effect of the cytokinin type and light treatment on survival and EFS. When necessary, Tukey’s post hoc test (α = 0.05) was used for multiple comparisons.The confirmation of the homogeneity of variances and normality of the data on the NS/E and PSR were performed, and PSR was A completely randomized design was carried out using six to twenty plantlets per cytokinin type and light treatment per each auxin treatment. The RI percentage (RI), the mean NR/E, and the LLR (cm) were recorded after six weeks of culture in a root elongation medium.® .To assess the effect of the cytokinin and light treatment on the RI, a logistic regression was performed. Data for NR/E and LLR were analyzed by ANOVA. When necessary, multiple comparisons were made using Tukey’s post hoc test (α = 0.05). The acclimatization percentage was calculated after four weeks under ex vitro conditions. The data were analyzed using R Core Team softwareP. ponderosa through organogenesis using whole zygotic embryos as explants was achieved. The best shoot induction percentage was obtained when whole zygotic embryos were cultured in half LP macronutrients. Following the novel use of LED lights for organogenesis, a higher number of rootable shoots were obtained when blue light was used. However, our results suggest that white LED could be more beneficial for rooting stages. Finally, in the greenhouse, the shoots cultured under fluorescent light showed the highest acclimatization success (50%). This may indicate that the use of different kinds of lights could be necessary during the process in order to optimize the organogenic process in P. ponderosa. Concerning the anatomy studies, this was the first microscopy assessment of in vitro plantlets exposed to LEDs treatments. The needles from in vitro and ex vitro plants showed similar morpho-anatomic characteristics. The regeneration of It would be interesting for future experiments to test other combinations and concentrations of auxins for rooting induction, and other LEDs, such as a combination of red and blue LEDs, to improve the rooting and acclimatization of shoots.


We found that the presence of more than 25% of mast cells in the bone marrow, the presence of concomitant marrow dysplasia, and the presence of disease-related complications affect survival (p < 0.001). In addition to the WHO classification, the IPSM scoring system is indicative of the prognosis in this rare disease.Mastocytosis is a very rare disorder and is divided into three prognostically distinct variants by World Health Organization: Cutaneous mastocytosis (CM), systemic mastocytosis (SM), and mast cell sarcoma or localized mast cell (MC) tumors. The wide range of complaints may cause patients to consult various clinics, with resulting mis- or underdiagnosis. Therefore, cooperation between different subspecialties is of paramount importance. In this article, we have compiled 104 adult mastocytosis cases diagnosed and followed in our Hematology and other clinics. 86 (82.7%) of 104 patients had systemic mastocytosis. Osteoporosis, disease-related complications, and secondary malignancies are important topics in this group. We know that indolent form has great survival. But smoldering or aggressive mastocytosis has a poor prognosis. CM and indolent SM have a significantly better prognosis compared to aggressive SM ( Mastocytosis is a rare disease with diverse presentation and variable prognosis, characterized by clonal mast cell proliferation. It is considered to be an orphan disease, and there is the limited number of epidemiological studies. In a Danish study, the overall incidence of systemic mastocytosis (SM) was 0.9 per 100,000 per year, and more than 80% of these patients were diagnosed with indolent systemic mastocytosis (ISM) . In a stIn patients with mastocytosis, various symptoms and signs may be observed due to mediators released from mast cells and organ dysfunction due to mast cell infiltration. These mediators can cause dermatological , gastrointestinal , respiratory and cardiovascular system , musculoskeletal , neurological and psychiatric complaints . The diagnosis can be missed due to this diversity. Regardless of the subtype, patients may complain of mediator symptoms and life-threatening allergies.According to the World Health Organization (WHO) 2016 update, mastocytosis is divided into three major groups: Cutaneous mastocytosis (CM), systemic mastocytosis (SM), and mast cell sarcoma or localized mast cell (MC) tumors. Diagnostic criteria are defined in Table In the updated WHO classification, SM is divided into different subgroups: Indolent SM (ISM), smoldering SM (SSM), SM-AHN (SM with an associated hematologic [non-MC lineage] neoplasm), aggressive SM (ASM), mast cell leukemia (MCL) according to the burden of disease, percent of mast cell infiltration, accompanying signs of organ damage Table . MCL is The prognosis, symptoms, and treatment vary among patients depending on the disease variant, the presence of an additional hematologic neoplasm as well as the presence of comorbidities .In this study, we aimed to assess the current data and clinical findings of 104 adult patients diagnosed with cutaneous and/or systemic mastocytosis in our clinic, Istanbul University, Faculty of Medicine which is a center of excellence of the European Competence Network on Mastocytosis.This study was approved by the institutional ethics committee . The procedures followed were in accordance with the Declaration of Helsinki 1975, as revised in 2000, and patients had provided written informed consent. The patients and the data were collected retrospectively at the Istanbul Faculty of Medicine, Division of Hematology. The primary objective of the study was to evaluate the demographic features, clinical and laboratory findings at the diagnosis, treatments and complications during the follow up in our center. The measurement of tryptase was made by the Fluoro-Enzymatic Immunoassay (FEIA) method. KIT D816V mutation was analyzed with a highly sensitive real time polymerase chain reaction (RT-PCR) assay . Patient3/μL, hemoglobin ≤11 g/dL, platelets ≤100 × 103/μL and skin involvement are independent prognostic factors for overall survival. Patients who have no risk factors are grouped in advanced systemic mastocytosis 1 (AdvSM-1), those with one risk factor in AdvSM-2, individuals with two or three risk factors in AdvSM-3, and patients with four or five risk factors in AdvSM-4 subgroup [In IPSM scoring, the nonadvanced group was classified as intermediate risk group 1 and 2 according to age (> 60) and serum alkaline phosphatase (> 100 u/l) value. Patients without these risk factors were defined as the low risk group. In patients with advanced systemic mastocytosis, age ≥ 60 years, tryptase ≥125 ng/mL, leukocytes ≥16 × 10p-values of less than 0.05 were considered statistically significant.All analyses were performed on SPSS v21 . Shapiro-Wilk test was used to determine whether variables are normally distributed. Normally distributed variables were analyzed with the independent samples t test or one-way analysis of variances (ANOVA) depending on count numbers of groups. Pairwise comparisons were performed with the Tukey test or Tamhane test depending on homogeneity of variances. Non-normally distributed variables were analyzed with the Mann Whitney U test or Kruskal Wallis test depending on the count numbers of groups. Pairwise comparisons were performed with the Bonferroni correction method. Categorical variables were analyzed with the chi-square test or Fisher’s exact test. Survival times were calculated with the Kaplan-Meier method. Cox regression analysis  was performed to determine significant prognostic factors of the mortality. Two-tailed We included 104 patients  into our study. Median age at the start of symptoms was 35 (range 14–86) and median age at diagnosis was 41 (range 18–87). Mean follow-up period was 50.87 ± 49.43 (range 1–256) months and 16 (15.38%) patients died due to different reasons (Table Eighteen (17.3%) patients had CM and 86 (82.7%) patients were diagnosed with systemic mastocytosis. In this systemic mastocytosis group, 55 (52.9%) patients had ISM, 14 (13.5%) patients had SM-AHN, 6 (5.8%) patients had SSM, 6 (5.8%) patients had ASM, and 5 (4.8%) patients had MCL.In our series, the most common first symptom was skin lesions (63.5%).The other common first symptom was anaphylaxis (19.2%). The most common symptoms were skin symptoms (76%) , hepatomegaly (32.7%), anaphylaxis (28.9%) and bone symptoms (28.9%). Seventy three 79.4%) patients had normal x-ray and 18 (19.4%) patients had normal DEXA. Different complications occurred in 18 (17.5%) patients (Table % patientp = 0.026) and splenomegaly (p = 0.006) percentages were significantly higher in the patients with positive BM CD30 than in patients with negative BM CD30. There were no significant differences between patients with positive and negative BM CD30 regarding gender, age at the start of the symptoms, age at diagnosis, x-ray findings, DEXA results, skin involvement, lymphadenomegaly (LAM), cardiovascular symptoms, GI tract symptoms, skin symptoms, ascites, portal hypertension, anaphylaxis, B symptoms, bone symptoms or mortality.Hepatomegaly associated with mast cell disease (The most common treatments were H1 antagonists (94.23%) and H2 blockers (92.30%). Gastrointestinal complaints of 61 patients (69.32%) regressed with H2 blocker treatment.We observed partial response in 2 patients (22.2%) and CI in 3 patients (33.3%) with cladribine treatment.No response was obtained with IFN in 6 patients (5.77%).Imatinib treatment was given to 5 (4.81%) patients. Four of these patients did not have the D816V KIT mutation and adequate response could not be obtained in all of them. One patient received imatinib due to the diagnosis of concomitant CML.n = 2) without success.We applied dasatinib treatment to a limited number of patients (We administered azacitidine to a patient who was diagnosed with SM-AHN (MCL-MDS) and observed no response.Seventeen (16.4%) patients received midostaurin. CI was achieved in 9 patients and PR was achieved in 8 patients. Cytopenia and constitutional symptoms improved with midostaurin treatment. This treatment was discontinued in 3 patients due to intolerance or adverse reactions (two patients had refractory nausea and 1 patient toxic hepatitis). Since tissue biopsy was not repeated in all patients who received treatment, the presence of complete response could not be evaluated. Currently, 5 patients with partial response are still on follow-up.We applied avapritinib treatment in one patient who had toxic hepatitis with midostaurin. In the third month of the treatment, response was observed in skin lesions due to mast cell infiltration and constitutional symptoms decreased. The patient is still receiving avapritinib therapy.Allogeneic stem cell transplantation was applied to 4 (3.9%) patients. Three of these patients were diagnosed with SM-AHN  in 1 patient). One patient with the diagnosis of ASM was responsive to midostaurin treatment and an allogeneic stem cell transplantation was performed due to transformation to AML, but died due to relapsing leukemia. The patient with ASM-CMML died due to infection in the early period. The other patient with ASM-CMML died due to leukemia, too. One patient with ASM-MF is still on follow-up Table .Table 5Sp < 0.001)  Table . As expe1) Table . Surviva Table . p < 0.001)  ISM, 523/2985 (17.5%) CM, 63/2985 (2.1%) SSM, 91/2985 (3%) ASM, 34/2985 (1.1%) MCL and 229/2985 (7.6%) SM-AHN [Clonal mast cells typically carry somatic activating mutations that cause mast cells and their progenitors to gain function . The mosIn contrary to SM, CM is usually diagnosed in childhood and whereas systemic symptoms may be seen due to release of mediators the specific infiltration of mast cells is only limited to skin in this type of mastocytosis . MorpholISM is the most common form of systemic mastocytosis. In contrast to CM, ISM usually develops in adults. Bone marrow is always involved, but in typical ISM, the degree of bone marrow infiltration is very low. Bone marrow mastocytosis (BMM), a newly defined ISM subtype, also describes isolated marrow involvement without skin involvement . In thesWhile some of the SSM patients have a silent course, some may transform to aggressive mastocytosis or SM-AHN or MCL. The risk of progression depends on the type of SM. The cumulative probability of disease progression in ISM ranged from 1.7 ± 1.2% at 5–10 years to 8.4 ± 5% at 20–25 years . In anotDiagnosis of SM-AHN can coexist with myeloid malignancies such as myeloproliferative neoplasms (MPNs), MDS/MPNs such as chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia, BCR-ABL1 negative, MDS/MPN unclassifiable, or AML, and less frequently accompanied by lymphoid malignancies  . DiagnosWhile the main symptoms of the disease occur due to mast cell degranulation in ISM patients, complaints may be observed due to mast cell infiltration and organ dysfunction in ASM. Physical and psychological stress factors, certain drugs and foods, insect bites, radiology contrast agents can cause mast cell degranulation. Allergic reactions, skin rashes are the most common symptoms of the indolent form .In advanced systemic mastocytosis (advSM), cytopenia and organ dysfunctions may be the first form of presentation. While less skin findings are seen in advSM, hepatomegaly due to mast cell infiltration, lymphadenomegaly, GI system and cardiovascular system findings, ascites and portal hypertension are more common in this group. Various complications can be seen due to involvement of different organs. Portal hypertension, fragility fractures, duodenal perforation and gastrointestinal bleeding, pleural effusion, portal vein thrombosis, and solid organ malignancies can be observed during follow-up. The most common complications in the patients we followed were portal hypertension and malignancy Table .It is known in the literature that there is an increase in the frequency of solid organ malignancies in patients with SM (for solid cancers the hazard ratio was 2.4), and this risk is higher especially in ASM . In a reManifestations of bone disease include osteopenia with or without lytic lesions, osteoporosis with or without atraumatic fracture, osteosclerosis with increased bone density, and isolated lytic lesions. In the updates of the diagnostic criteria, it is stated that an osteolytic finding greater than 2 cm should be evaluated as a C finding . OsteopoCD30 is a transmembrane receptor, normally not expressed by mast cells. Recent data suggest that CD30 expression in MC is strongly associated with SM but is not found in other myeloid neoplasms , 26. TheCurrently the management of ISM, SSM and cutaneous mastocytosis is symptomatic therapy with H1 and H2 blockers, leukotriene receptor antagonist drugs and mast cell stabilizers. Patients with increased risk of anaphylaxis should be advised to carry a self-injectable epinephrine. In advanced forms, cytoreductive treatment should be used, aiming to reduce mast cell burden. With cytoreductive therapy, regression in organ infiltration can be achieved. We observed in our patients that spesific cutaneous lesions disappeared with cytoreductive therapy, especially with midostaurin and cladribine, in patients with advanced systemic mastocytosis.The patients with ASM have been treated with cladribine or interferon-alpha ± steroids prior to the approval of midostaurin. Good response rates are observed with the use of avapritinib , 28. ImaSeveral limitations of this study should be addressed. Firstly, all analyzes were made from retrospective data. Therefore, all data could not be obtained. Secondly, the genetic status of the patients could not be obtained. Most of the patients were tested for c-kit mutation, but other prognostic molecular markers  could not be obtained. CD2, CD25 and CD30 could not be measured with flow cytometry in all patients. The type of cytoreductive therapy applied was not defined according to the disease subtype. These constitute the main limitations. However, it is very important to have a multidisciplinary approach in a rare disease.p < 0.001), in the presence of concomitant dysplasia  and when the infiltration rate of mast cells in the bone marrow was above 25%  (Table p < 0.001) (Table Patients with ISM have a nearly normal life expectancy. The disease progression rate is also very low, %1.7 in 5 years . Risk faIn conclusion, mastocytosis is a rare group of diseases that require a comprehensive evaluation and can show multisystem involvement. The wide range of complaints may cause patients to consult various clinics, with resulting mis- or underdiagnosis. Therefore, cooperation with different branches in an excellence center plays an important role in the diagnosis and treatment of the disease.


Spinactaletes boneti mitochondrial genome was assembled and annotated. It has 14,766 bp in length, all 37 genes are present and the gene order is the same as the Pancrustacean ancestral gene order. Phylogenetic analyses based on maximum likelihood placed the species as a sister group of the remaining Entomobryomorpha, not closely related to the Isotomoidea superfamily, contradicting the actual systematics of the group. Spinactaletes boneti . Representatives of this family can be distinguished from the other entomobryids by the presence of a tracheal system, abdomen III reduced, abdomen IV and V fused, abdomen VI reduced to the anal valves, abdomen IV with five sutures , and 3–4 trichobothria. Spinactaletes is a genus of obligatory marine littoral springtails, mostly associated with calcareous rocks  by Yun BU and José Palacios-Vargas on 10 November 2019. Twenty specimens were deposited at Shanghai Natural History Museum . One individual (voucher number MX-ZH-2019005) was used for DNA extraction and whole-genome amplification. All laboratory experiments including library construction and sequencing were performed by Shanghai Yaoen Biotechnology Co., Ltd, China. Illumina NovaSeq platform was used for sequencing paired-end reads with 150 bp length, producing approximately 10 G of data. The mitogenome was assembled de novo using NovoPlasty v3.8.3 . The identity and position of the 13 PCGs, 22 tRNA, and 2 rRNA genes were determined using MitoZ v2.4-alpha  was included and the final dataset comprised 22 species. Nucleotide sequences of all 13 protein coding genes were aligned using MAGUS (Smirnov and Warnow https://tree.bio.ed.ac.uk/software/figtree/). The resulting topology based on ML . Bayesian analyses placed the new mitogenome in the same position but with a bit lower posterior probability support (0.63). Our result is in agreement with the study made by D’Haese  were downloaded from GenBank. All accession numbers are listed in ed on ML  suggeste D’Haese , based o


Meibomian glands (MGs) are modified sebaceous glands within the eyelids, which synthesize and secrete meibum into the tear film, forming the outer lipid layer to delay the evaporation of aqueous tear . MeibomiDemodex by coagulative necrosis, and provide long-term effectiveness (Intense pulsed light (IPL) is an effective treatment for MGD and has been used in ophthalmology. IPL is a broad-spectrum incoherent light with wavelengths of 500–1,200 nm, which is produced by high-output xenon flash lamps , 7. The tiveness –12. Althtiveness . The reptiveness , 15, dectiveness , 17, andtiveness . We havetiveness . Given tIn clinical treatment, IPL is usually applied to the skin below the lower eyelids and the eyes are protected by eye shields. It is worth noting that although MGs are not directly exposed to light, MG scores and MG secretion functions were improved in both the upper and lower eyelids , 20. Thi–/–ApoE mice as the model to observe the clinical and pathological changes after IPL treatment. Previous studies have reported that apolipoprotein E knockout (–/–ApoE) mice begin to show morphological and functional abnormalities of MGs at 5 months of age, which is attributed to hyperlipidemia  and raised in the animal center of Peking University First Hospital. All mice were fed a standard-fat diet and were divided into three subgroups: the wild-type (WT) group (C57BL/6 mice), model group (–/–ApoE mice) and IPL-treated group (–/–ApoE mice received three IPL treatments). Clinical parameters of all the mice were examined 2 weeks after each IPL treatment.Male C57BL/6 mice and After the mice were anesthetized, the hair on both sides of their face was shaved. Prior to treatment, a cooling ultrasound gel was administered to the irradiation area and eyes were shielded from the light stimulus.2 and 1 pulse was applied to both sides with three treatment sessions at 2-week intervals. IPL therapy was administered to the skin 3 mm far from the lid margin of the lower eyelid and the irradiation area was 1 square centimeter of the mouse face. The irradiance and frequency of radiation were based on clinical application and the data from our preliminary experiments.An Eyesis IPL system (MDC (TianJin) Co., Ltd. China) was used for treatment. The intensity of the IPL treatment was 12 J/cmClinical parameters, including plugged orifice numbers, lid margin irregularity, lid margin thickening, and corneal fluorescein staining scores, were measured under a stereoscope  at 2 weeks after each IPL treatment. All parameters were blindly assessed by a single experienced ophthalmologist (WD).To determine plugged orifice numbers, eight orifices in the center of the upper and lower eyelid were selected respectively, and plugged orifices were counted based on previously reported criteria . A swollEyelids were harvested after the mice were sacrificed. Then, eyelid tissues were embedded in optimal cutting temperature compound, immediately frozen in liquid nitrogen, sectioned at 6 μm and kept at −80°C. Frozen sections were used for hematoxylin and eosin (H&E), oil red O (ORO), and immunofluorescence staining. Additional eyelids were immersed in formalin for fixation and then processed into paraffin blocks until sectioned  for immunohistochemical staining.Frozen sections were fixed with 4% paraformaldehyde for 10 min and rinsed with phosphate buffered saline (PBS). Sections were then immersed in 60% isopropanol for 30 s and stained with freshly prepared ORO solution for 15 min, followed by 60% isopropanol for 5 s. Thereafter, the sections were stained with hematoxylin and mounted with glycerol gelatin.Briefly, frozen sections were dried at room temperature for 15 min and washed with PBS. Then, the sections were incubated with anti-Ki67 antibody  and anti-Keratin 10 (K10)  for 30 min at 37°C. After washing with PBS, the sections were covered with Alexa Fluor 488-conjugated donkey anti-rabbit IgG  for 30 min at 37°C. Then, nuclear identification was performed with DAPI . Fluorescent images were taken under a fluorescence microscope . The number of Ki67-positive cells and the fluorescence intensity of K10 were quantified with ImageJ 1.52a software.Paraffin sections were routinely deparaffinized and hydrated. Following antigen retrieval, the sections were washed and incubated with endogenous peroxidase blocker for 10 min. After washing with PBS and incubating with 2% bovine serum albumin (BSA) for 1 h at room temperature, the sections were subsequently incubated with anti-NADPH oxidase 4 (NOX-4) antibody  at 4°C overnight. The next day, the sections were washed and covered with enzyme-conjugated goat anti-rabbit IgG  for 1 h at room temperature, followed by visualization with a diaminobenzidine (DAB) kit . Afterward, the sections were counterstained with hematoxylin, dehydrated with ethanol and mounted in neutral gum. The staining was evaluated under a microscope.After the mice were euthanized, MG tissues were immediately placed in 3% glutaraldehyde at 4°C overnight. After washing with PBS, they were fixed in 1% osmium tetroxide for 2 h. The specimens were then dehydrated in graded acetone, embedded in Epon, sectioned into 70–90 nm thick ultrathin sections and stained with 5% uranyl acetate and lead citrate. The sections were photographed with a transmission electron microscope .The eyelids were harvested and imaged after the mice were euthanized. Then, the palpebral conjunctiva was scraped off the eyelids, and the tarsal plates (TPs) were isolated from the overlying muscle and surrounding tissues. The TPs were then immediately frozen with liquid nitrogen.The TPs were meticulously isolated and then placed into lysis buffer containing cocktails of protease and phosphatase inhibitor. There were 4 samples in each group, and each sample consisted of TPs from both upper eyelids of the same mouse. The same amount of protein was loaded on 12% SDS-PAGE gels for electrophoretic separation, followed by being electronically transferred to a PVDF membrane. After blocking with 5% skimmed milk, the membrane was incubated with rabbit anti-caspase-3 antibody , rabbit anti- nuclear factor kappa B (NF-κB) antibody , rabbit anti-phospho (p)-NF-κB antibody , and mouse anti-β-actin antibody  at 4°C overnight. Then, the blots were incubated with HRP-labeled goat anti-mouse IgG  or anti-rabbit IgG  for 1 h at room temperature. BeyoECL Star  was used to visualize the specific bands which were imaged using a chemiluminescence imaging system . Staining intensities were quantified with ImageJ 1.52a software.–ΔΔCT method with β-actin as the reference gene.The TPs were isolated from both upper eyelids and combined as one sample, and five samples in WT and IPL-treated group and 6 samples in model group were used for Quantitative Real-Time PCR (qRT-PCR). TRIzol reagent  was used for extracting RNA and its purity and concentration were measured by spectrophotometry. RNA (2 μg) was reverse transcribed into cDNA using a reverse transcription kit . cDNA (10.0 ng) was added to 20 μl total reaction volume containing reverse transcription master mix  and specific primers. The sequences of the primers are shown in post-hoc test was performed for data analysis. GraphPad Prism version 7.0  was used for statistical analysis and graphing. Any p-value < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS 26.0 statistical software . All data are presented as means ± SD. One-way ANOVA with LSD –/–ApoE mice showed toothpaste-like plugs at MG orifices that protruded from the margin, which were different from the orifices observed in age-matched WT mice, while the degree of orifice obstruction in both the upper and lower eyelids improved from the first IPL treatment. We counted the number of plugged orifices and found that the difference in the number of plugged orifices in the upper eyelids among the three groups at T1 and T3 time points was statistically significant (p < 0.05); post-hoc analysis found that at T1 time point, the number of plugged orifices in the IPL-treated group was lower than that in ApoE–/– mice (p < 0.05), and –/–ApoE mice had more plugged orifices than WT mice and IPL-treated mice at T3 time point  (–/–ApoE mice was more than that of WT mice (p < 0.05), while the number of plugged orifices of IPL-treated mice was reduced at each time point compared with ApoE–/– mice (p < 0.05) . As shown in < 0.001) . In addi < 0.05) .–/–ApoE mice tended to increase compared with WT mice, while the scores in –/–ApoE mice treated with IPL irradiation showed a trend of decrease, although the difference was not statistically significant . At each time point, the corneal fluorescein staining scores of –/–ApoE mice was higher than that of WT mice (p < 0.05), and the scores of IPL-treated group was lower than that of –/–ApoE mice (p < 0.05)  .–/–ApoE mice.In short, these results suggested that three IPL treatments alleviated the degree of clinical MGD-like changes and attenuated corneal epithelial damage in –/–ApoE mice, and IPL-treated mice. Images showed that –/–ApoE mice had great morphological hypertrophy in partial MGs with orifice obstruction. However, after IPL treatment, the plugged orifices reopened and MG hypertrophy was reduced , and the levels approached those observed in WT mice (–/–ApoE mice (p < 0.05)  . The res–/–ApoE mice were greatly reduced, while after IPL treatment Ki67-positive cells increased and were densely arranged at the edge of acinus (P < 0.01)  . Althoug–/– mice .–/–ApoE mice were elevated (p < 0.001). Similar to that in WT mice, the expression of K10 in the IPL-treated group was reduced (p < 0.01) (–/–ApoE mice was decreased after IPL treatment (p < 0.01)  . qRT-PCR < 0.01) .–/–ApoE mice, but at approximately the same level as WT mice (–/–ApoE mice (p < 0.01)  . The nuc WT mice . However WT mice . Caspase < 0.01) , indicat–/–ApoE mice as our model, which developed hyperlipidemia that induced the occurrence and pathological course of MGD. Our results show that indirect irradiation via IPL to MGs reduces the severity of orifice plugging in –/–ApoE mice. In addition, our results demonstrate the anti-inflammatory effect of IPL, which may delay the progression of MGD. Indirect IPL irradiation also improves the biological function of MGs, including increased proliferation of MG basal cells and decreased oxidative stress  inflammasome and its downstream inflammatory factors—caspase-1, IL-1β, and IL-18 were upregulated in dry eye patients (Studies have shown that localized radiation therapy in patients with metastatic cancer sometimes leads to diminution of metastases far from the irradiation site, this abscopal effect thought to be mediated by systemic cytokines and/or immune responses . Interespatients  and in dpatients . These fpatients . In futuIn this study, clinical and histological observations showed that IPL treatment significantly reduced toothpaste-like meibum in MG orifices and ductal dilation, while K10 expression was downregulated in MGs. The melting point of MG secretions from MGD eyes was 3°C higher than that from normal eyes , and theIn addition to anti-inflammatory effect of indirect IPL irradiation, we also found that IPL can influence the biological function of MG epithelial cells. We found the increased proliferation rate of acinar basal cells and the improvements in lipid droplets in IPL-treated mice. This result might explain why MG dropout showed improvements in IPL-treated patients –55. It h–/–ApoE mice whose meibocytes were subjected to oxidative stress. In addition, the morphology and function of mitochondria improved and apoptosis decreased. In the UVB-induced photoaged phenotype of skin cells, IPL also reduced intracellular oxidative stress, which is consistent with our results (Oxidative stress has been reported to be involved in the pathophysiological changes in MGD . The exc results . Althoug results . In the  results , 62. MorIn conclusion, the present study demonstrates that IPL irradiation can improve the structure and function of MGs and modify the disease progression of MGD in a mouse model, which may be related to the indirect effects of PBM.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Laboratory Animal Ethics Committee of Peking University First Hospital.XY and SY conceived and designed the study, and reviewed and revised the manuscript. LX and WS performed the experiment and wrote the manuscript. WD assessed the clinical parameters. YL, SC, XS, MH, YC, and YG were involved in data analysis and interpretation. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Campus health systems can provide timely and accessible resources for students with co-occurring substance use and mental illness, but little is known about the degree to which students use these systems. This study examined mental health service utilization among students with symptoms of anxiety or depression, stratified by substance use.N = 65,969), stratified by substance use type . We performed a series of weighted logistic regressions to assess the adjusted association of substance use type with past year use of campus, off-campus outpatient, emergency department, and hospital mental health services.This cross-sectional study used data came from the 2017–2020 Healthy Minds Study. Mental health service use was examined among students with clinically significant anxiety or depression  and off-campus outpatient mental health service utilization . Other drug use was associated with increased odds of off-campus outpatient , emergency department  and hospital service utilization .Universities should consider screening for substance use and common mental illnesses to support the health of high-risk students. AlcoholThe increasing prevalence of substance use among young adults is particularly concerning given a concomitant increase in the prevalence of anxiety and depression in this population . One-thiA large body of research has examined the role of alcohol and marijuana use among students with co-occurring mental illness with respect to mental health service use, but relatively little research has examined the role of other drugs. Numerous studies have found that students with mental illness and comorbid hazardous alcohol or marijuana use more often seek out mental health services than those without . HoweverUniversity health systems have the potential to serve as an entry point for students struggling with co-occurring mental illness and substance use. Examining patterns of mental health service utilization among students with symptoms of common mental health disorders and substance use is critical to identify opportunities for early intervention and identify treatment pathways for this understudied, high-risk, and growing population. As such, population-level research is needed to fill gaps in knowledge and to inform the optimization of campus health service delivery systems. This study examines the role of substance use type in mental health service use in a national sample of university students with clinically significant symptoms of generalized anxiety or major depression, the most common mental health disorders affecting this population. Specifically, we examined the association of substance use type with campus mental health service use and use of mental health services in the community.22.1This cross-sectional study used three years of data collected during the school year from the Healthy Minds Study (HMS): Fall 2017-Spring 2018, Fall 2018-Spring 2019, and Fall 2019-Winter 2020 . The HMSDue to the COVID-19 pandemic and subsequent disruption in the delivery of mental health services, HMS data from the 2021 academic year was not included. The COVID-19 pandemic resulted in the closure of universities across the U.S., making campus mental health systems difficult or impossible for students to access . The dis2.2N = 65,969, 36.0% of all respondents during the study period). We elected to limit the sample to those with clinically significant symptoms of depression or anxiety to examine the association between substance use and mental health service utilization among those with symptoms of common mental health disorders, a high-risk and growing population of students. The Patient Health Questionnaire-9 (PHQ-9)   was used (PHQ-9)  was used (PHQ-9) .2.32.3.1The primary independent variable was substance use type. We opted to categorize by type of substances used for several reasons. First, students infrequently reported use of certain substances , and small cell sizes prevented these substances from being evaluated individually in adjusted models. Second, the HMS survey did not collect data on substance use frequency or evaluate the presence of SUDs during the study period. For these reasons, we opted to categorize substance use by types of substances used.Categorization of substance use was based on responses to multiple survey questions inquiring about past two week use of alcohol, and past month use of tobacco, marijuana, stimulants, cocaine, heroin, ecstasy, opioids, hallucinogens, and methamphetamines (see Appendix A for survey questions). These survey questions specifically inquire about non-prescribed use of substances (i.e. non-prescribed used of benzodiazepines or opioids). Use of other substances could be noted in a free-text response. We generated binary indicator variables for use illicit use of other drugs  and electronic cigarette use or other tobacco use through this item, which were frequently reported in this manner.Specifically, we measured substance use as follows: Non-substance users reported no use of any substances; alcohol and tobacco users reported only use of either or both substances; marijuana users reported use of marijuana exclusively or in addition to tobacco and alcohol use; other drug users reported illicit or non-prescribed use of stimulants, cocaine, opioids, ecstasy, hallucinogens, or methamphetamines exclusively or in addition to tobacco, alcohol, and/or marijuana use. Other drug and tobacco indicator variables generated from free-text responses contributed to counts of each substance, respectively. We opted to evaluate marijuana separately due to the changing regulatory landscape across the U.S. and the higher frequency of use among young adults (34.8%) in comparison to the other illicit substances, which remain illegal across states in the U.S. and are used less frequently .Substance use was coded as a factor variable, where non-substance users were coded as “0,” alcohol or tobacco users were coded as “1,” marijuana users were coded as “2,” and other drug users were coded as “3.” This method assigned respondents to the highest level of substance use they reported. For example, a student who reported use of alcohol and cocaine would be considered an other drug user, and a student who reported use of alcohol and marijuana would be considered a marijuana user.2.3.2From which of the following places did you receive counseling or therapy? Respondents could endorse use of multiple service types in response to this question (i.e. use of emergency department and outpatient services). All outcomes were measured as binary variables, where “0″ indicated no past-year use of a given service and “1″ indicated past-year use of a given service (see Appendix A for survey questions).The primary outcome variables were any past-year use of campus mental health services, off-campus outpatient services , emergency department, and hospital mental health services . Evaluation of past-year mental health service use was based on response to one survey question: 2.3.3We also evaluated barriers to accessing mental health services in the past year. Barriers included financial limitations, not having enough time, unsure of where to seek care, difficulty finding an available appointment, and preferring to deal with issues on own or with friends (see Appendix A for survey questions). Barriers to mental health services were also evaluated as binary variables, taking a value of “0″ if a barrier was not endorsed and “1″ if it was.2.3.4We examined respondent characteristics that were included in all waves of the HMS survey during the study period. Demographic characteristics included age, gender , race , sexual orientation , degree program (undergraduate vs. graduate degree program), health insurance type , grade point average  and living place . In addition to these variables, we also included PHQ-9 and GAD-7 scores as covariates to assess differences in the severity of depression and anxiety symptoms. We also included a binary measure of perceived need for help with mental health problems in the past year.2.4t-tests for continuous variables and chi-square tests for categorical variables. All bivariate estimates are presented as weighted percentages or means with standard deviations. We performed a series of weighted logistic regressions to assess the association between past-year mental health service utilization by type and substance use group. Logistic regressions were adjusted for age, gender, race, sexual orientation, degree program, living place, insurance type, PHQ-9 score, GAD-7 score, and perceived need for mental health services. Due to small cell sizes, we reduced the levels of race and health insurance in adjusted analyses (see Appendix B). Adjusted models also included university fixed effects to account for time-invariant differences between universities, and year fixed effects to control for secular trends. To account for correlation among errors, we used robust standard errors in all models. All model estimates are presented as odds ratios (OR) with 95% confidence intervals (CI). Additionally, the marginal effect (ME) of substance use group was calculated for each model, which expresses the change in the probability that the outcome occurs  when substance use type changes while holding other covariates constant  than students using marijuana , tobacco or alcohol , or no substances . In this sample of students with clinically significant anxiety or depression, students reporting no substance use less frequently perceived a need for mental health services (80.9%) than alcohol or tobacco users (83.8%), marijuana users (89.0%), and other drug users (89.9%).Sample characteristics stratified by substance use group are presented in Substance use frequency between groups is presented in 3.2N = 26,259) used any mental health services in the past year. Past year utilization of any mental health services was highest among other drug users (49.1%) and marijuana users (49.1%), while a smaller proportion of alcohol or tobacco (39.6%) and non-users (38.2%) reported use of any services (p < 0.001)  of the sample utilized campus mental health services, utilization was significantly higher among marijuana (25.8%) and other drug users (24.2%) in comparison to alcohol or tobacco users (21.0%) and non-users (21.0%) (p < 0.001). Use of off-campus outpatient mental health services was 23.8%  across the entire sample. Other drug and marijuana users utilized off-campus outpatient mental health services at higher levels, 29.3% and 29.1%, respectively, than alcohol or tobacco (22.8%) and non-users (21.6%) (p < 0.001). Across the entire sample, 1.4% (N = 843) reported use of the emergency department for mental health needs. Other drug users reported past year utilization of the emergency department for mental health needs nearly twice as often (3.2%) as marijuana users (1.7%), and nearly three times as often alcohol or tobacco users (1.1%) and non-users (1.2%) (p < 0.001). Use of hospital mental health services was 1.8% (N = 1189) across the entire sample; 3.5% of students reporting use of other drugs utilized hospital mental health services in comparison to only 2.3% of marijuana users, 1.4% of alcohol or tobacco users, and 1.7% of non-users (p < 0.001).Across the entire sample, 37.8% (< 0.001) . While o3.3Adjusted results examining mental health service utilization outcomes are presented in After adjustment, marijuana use was associated with increased odds of both campus  and off-campus outpatient mental health service utilization . Holding all other covariates constant at their observed values, this translates to a 1.6 percentage point increase in the probability of campus mental health service utilization  and a 4.1 percentage-point increase in the probability of off-campus mental health service utilization . Marijuana use was not associated with either utilization of the emergency department for mental health needs or hospital services after adjustment.p > 0.30). Several demographic and psychological characteristics were also associated with mental health service use, and the full results are presented in Other drug use was associated with increased odds of off-campus mental health service utilization , translating to a 4.4 percentage point increase in the probability of utilization . Other drug was also associated with increased odds of both emergency department  and hospital mental health service utilization , respectively translating to a 1.6  and 1.1 (CI 0.4. 1.8) percentage-point increase in the probability of using emergency department and hospital services. However, other drug use was not associated with campus mental health service utilization after adjustment. Hosmer–Lemeshow goodness-of-fit tests indicated that the fit of all models was satisfactory (i.e. 3.4p < 0.001). Other drug users also more often reported that they preferred deal with mental health issues on their own or to seek help from family or friends (36.0%) in comparison to marijuana users (33.9%), alcohol or tobacco users (30.0%), and non-users (31.1%) (p < 0.001). Having not enough time for mental health services was the most commonly reported barrier by other drug users (43.9%), and these students more often reported this as a barrier to care then students who used marijuana (40.6%), alcohol or tobacco (36.5%), or non-substance users (32.6%) (p < 0.001).Other drug users more often reported barriers to mental health services than all other substance use groups . Student4This is the first large-scale study to have examined mental health service utilization among university students with co-occurring substance use and symptoms of common mental health disorders. In this study of students with clinically significant symptoms of depression or anxiety, 84% of students reported need for mental health services but only 38.2% of students accessed any services. We found that students who use other drugs  and have symptoms of depression or anxiety, utilize emergency department and hospital services more often than students who exclusively use alcohol, tobacco, or marijuana with these symptoms. These findings demonstrate a critical need to identify and provide appropriate services to students with co-occurring mental illness and other drug use, which carries risk for lifetime development of SUDs . While mStudents with common mental health disorders more frequently use substances than those without, which suggests that some may be self-medicating symptoms with substances . This isWhile the campus mental health system may be ideally positioned to provide outpatient mental health care, previous research has demonstrated that university students frequently seek support from nonprofessional sources , 2012. IThe COVID-19 has spurred increasing rates of both mental illness and substance use among young adults . Many st5While this study is strengthened by random selection of students and the multisite methodology of HMS, there are several elements that limit the generalizability of findings. First, while the survey employs weights to account for non-response bias, low response rates are a notable limitation . While non-response weights can adjust for known characteristics, students who elected not to complete HMS may differ from those that did in important ways that influence mental health service use . Second, schools elect to participate in HMS, and while schools are diverse in composition and geographic location, they are not randomly selected. Additionally, students who elect to complete the survey may different than those who do not. However, we apply non-response weights to mitigate this selection bias. Third, past-month substance use may not accurately capture patterns or the intensity of substance use. Additionally, we are unable to determine if substance use precedes mental health service utilization. While these limitations warrant consideration, the HMS is a valuable data source to examine national trends in substance use and mental health service utilization that would otherwise be difficult to assess.6Decreasing the gap between need , access, and utilization for students with co-occurring illness and substance use is critically important to avoid unnecessary morbidity. Future research should examine the role of stigma among students, which may impact access to mental health care. Institutions should consider methods to identify and support these students, which may include increased use of routine screening and investment in care coordination with off-campus resources.The authors have nothing to disclose.This work was supported by NIDA P30DA035772 , NIDA 5T32DA041898-03 (Auty), NIMH 1K01MH121515 (Lipson), William T Grant Foundation Scholars Program (Lipson).Samantha G. Auty: Conceptualization, Formal analysis, Data curation, Writing – original draft, Writing – review & editing, Visualization, Funding acquisition. Sarah K. Lipson: Conceptualization, Formal analysis, Writing – original draft, Writing – review & editing. Michael D. Stein: Conceptualization, Writing – review & editing, Supervision. Sharon Reif: Conceptualization, Writing – review & editing, Supervision.The authors have no conflicts of interest to disclose.


The evolutionary origins of neurons remain unknown. Although recent genome data of extant early-branching animals have shown that neural genes existed in the common ancestor of animals, the physiological and genetic properties of neurons in the early evolutionary phase are still unclear. Here, we performed a mass spectrometry-based comprehensive survey of short peptides from early-branching lineages Cnidaria, Porifera and Ctenophora. We identified a number of mature ctenophore neuropeptides that are expressed in neurons associated with sensory, muscular and digestive systems. The ctenophore peptides are stored in vesicles in cell bodies and neurites, suggesting volume transmission similar to that of cnidarian and bilaterian peptidergic systems. A comparison of genetic characteristics revealed that the peptide-expressing cells of Cnidaria and Ctenophora express the vast majority of genes that have pivotal roles in maturation, secretion and degradation of neuropeptides in Bilateria. Functional analysis of neuropeptides and prediction of receptors with machine learning demonstrated peptide regulation of a wide range of target effector cells, including cells of muscular systems. The striking parallels between the peptidergic neuronal properties of Cnidaria and Bilateria and those of Ctenophora, the most basal neuron-bearing animals, suggest a common evolutionary origin of metazoan peptidergic nervous systems. Biochemical identification of neuropeptides in Cnidaria and Ctenophora, followed by analyses of their expression, suggests that peptidergic neurons were present at early stages of nervous system evolution. Comparative genomic studies of the basal metazoans and their closest sister unicellular lineage Choanoflagellata have revealed that the origins of major neural components, including ion channels and synaptic proteins, predate the emergence of neurons8. The premetazoan origin of neural genes suggests that the functional neuron was established by a combination of protein modules with different evolutionary origins10. One of the key open questions is what genes or modules were deployed in the first functional neuron. To answer this, the nature of the neurons in the earliest divergent animal groups needs to be clarified. One of the extant groups is Ctenophora (comb jellies), which is the most basal lineage bearing neuron-like cells with morphologically recognizable long processes (neurites) and presynaptic characteristics12. The genetic and physiological features of ctenophore neurons, however, largely remain to be experimentally examined. A deep single-cell-level inspection of neural genes in the ctenophore did not successfully provide a genetic signature characterizing the ctenophore neurons13. The lack of definitive neural markers in Ctenophora, together with the uncertainty of the phylogenetic position of this lineage14, is regarded as support for the independent evolutionary origin of the ctenophore nervous system11.Understanding the origins of neurons remains one of the most intractable challenges in evolutionary biology. In the early phase of animal evolution, the transmission of cellular activity to specific target cells that are spatially separated in the macroscopic multicellular body should have an essential role in the behavioural and physiological responses of animals. However, it is still unclear which of the diverse physiological and genetic features of the complex nervous systems of extant animals are functionally deployed in ancestral neurons. There has been increasing interest in recent years in the extent to which the neural genes required for the functioning of the nervous system in Bilateria are present in the genomes of the basal metazoans Ctenophora, Porifera, Placozoa and Cnidaria16. Neuropeptides are the most diverse group of neuromodulators, with multiple crucial roles in the neural control of behaviours in virtually all multicellular animals17, and are thought to act as the major neuronal messengers in Cnidaria, the lineage that diverged before Bilateria 1. Recently, a new search engine implemented with machine learning technology predicted a number of peptide gene candidates12. However, peptide prediction is still largely based on the structural features of bilaterian peptide precursors, which limits the reliability of in silico predictions especially in basal metazoans.A key to identification and characterization of ctenophore neurons is identification of the neurotransmitters they use. Among neurotransmitter systems commonly functioning in bilaterians, the peptide system has been assumed to be related to the ancestral nervous systemria Fig. 18–20. GeNematostella vectensis (Cnidaria), Ephydatia fluviatilis (Porifera) and Bolinopsis mikado (Ctenophora), with a special focus on short peptides with amidated carboxyl termini, the major structural characteristic of neuropeptides among the metazoans tested22. We identified mature peptides encoded in 13 and 15 precursors in B. mikado and N. vectensis, respectively , on the other hand, no amidated endogenous peptides were identified in this study. Furthermore, we did not detect in B. mikado the FMRFa-like and vasopressin-like amidated peptides that were predicted in Pleurobrachia pileus (Ctenophora)23. A cluster analysis of peptide precursor sequences did not detect similarity of ctenophore peptides to cnidarian and bilaterian peptides  were predicted as peptide precursor candidates12. Among them, we confirmed that the mature amidated short peptides NPWa, FGLa, RWFa, VWYa and WTGa were encoded by five genes, ML215411a, ML030511a, ML233326a, ML02736a and ML02212a, respectively. Other peptide genes identified in this study, NVFa/NIFa1 (ML40299a), NVFa/NIFa2 (ML16906a), TFQa (ML030215a), LNSa (ML218828a) and PARa (ML1541126a), have not been predicted elsewhere  cells and to clarify their physiological and genetic features. This is key to understanding the detailed characteristics of the ancestral nervous system and clarifying its origin(s), including whether it evolved multiple times. To obtain an experimentally validated overview of the mature structures and repertoire of peptides in early-branching metazoans, we performed mass spectrometry-based peptide identification for ado Fig. . RecentlB. mikado, and against RFa, PRGa, QWa and HIRa of N. vectensis, and analysed the morphology and localization of the peptide-expressing cells , indicating that these amidated short peptides are functional in neurons   neurons extended multiple neurites and formed part of the polygonal SNN structure, confirming that the tubulin-positive network structure in ctenophore species consists of neurons  of lls Fig. . Immunosons Fig. . Our staNN) Fig. . VWYa-exons Fig. 26. In adves Fig. .Fig. 2MoB. mikado. The neuropeptides  and other short amidated peptides  were expressed in cells with no visible neurite at the apical organ, tentacles or pharynx  distributions in M. leidyi12. This was not surprising, because both M. leidyi and B. mikado belong to the order Lobata and are members of the genera most closely related to each other27. To obtain a wider view of the anatomical and physiological features of ctenophore nervous systems, we performed immunostaining of adult combless benthic ctenophore Vallicula multiformis (order Platyctenida) using antibodies against NPWa, VWYa, FGLa and WTGa . We confirmed that a neural network of VWYa+ neurons also existed in this benthic ctenophore  or unknown (M. leidyi). The neuronal clusters of N. vectensis were divided into peptidergic and uncharacterized neurons   expressed the VWYa, NPWa and WTGa neuropeptides. Immunostaining confirmed that these peptides were expressed in cells including neurite-bearing neurons; therefore, these four cell clusters consist at least in part of peptidergic neurons. A comprehensive survey of protein components responsible for functions of neuropeptide-expressing neurons in Bilateria29 showed a rich repertoire of gene homologues for neuropeptide signalling molecules with different expression levels and patterns  and neprilysin, which are required for synthesis and degradation of active neuropeptides, were abundantly expressed. Among these enzymes, PAMs are of particular interest as they are critical for peptide amidation and have been identified in metazoans including Cnidaria and Porifera30. Our structural and phylogenetic analyses of ctenophore PAM proteins demonstrated that ctenophore PAM genes harbour domains required for peptide amidation , which have been identified in M. leidyi12 32. Neurexin, a core presynaptic membrane protein coordinating synaptic transmission, and PDZ domain-containing postsynaptic proteins were not abundantly expressed in the peptidergic cells, but they were enriched in the other cell clusters of M. leidyi  to that found for key molecules in peptide signalling. Highly expressed homologues of TF families, including bHLHA, SoxB, and Lim- and Paired (PRD)-class homeobox genes, were enriched in the peptidergic clusters of the basal metazoans  vesicular transporters, which are required for the accumulation of glutamate in secretory vesicles, were not enriched in the peptidergic clusters but were abundantly expressed in other cell clusters  but not its reverse peptide (control) induced adradial canal contraction and mouth opening , on the other hand, triggered expansion of the oral epithelium, where VWYa+ neurons form the subepithelial network, resulting in an increase in body volume -incorporated support vector machine (SVM), a machine-learning-based method for prediction of peptide–G-protein coupled receptor (GPCR) pairs38 types by single-cell transcriptomics41.We found highly conserved machinery for peptide biosynthesis, release and degradation, which are essential for peptide signalling. The unexpected level of conservation of the genetic signature of peptide-expressing cells among metazoans is strongly suggestive of a common and single evolutionary origin of peptidergic cells. The localization of some of the identified peptides revealed by immunostaining cannot be explained by the cell cluster classification of 42. However, the chemical substances that are generally called ‘neurotransmitters’ are widely used for intercellular signalling in non-neural cells in metazoans, as well as in plants and microorganisms. Therefore, when considering the most ancestral functionality of the neuron, it is unclear to what extent of the placement of particular chemicals may fulfil the ‘neuronality’ of the cell. More information about the Ctenophora nervous system is needed to further elucidate which features or combinations of features found in modern neurons can provide insight into the most ancestral state of neurons.The origin of neurons has long been debated in view of the various characteristics of neurons . It has been proposed that neurons evolved independently from secretory cell types multiple times, given the huge diversity of neuronal physiological characteristics, including chemical transmitter compositionN. vectensis and M. leidyi  were cultured as previously described47 with few modifications. Adult animals were kept at 18 °C and maintained at a salinity of one-third artificial seawater  and fed with freshly hatched artemia two times per week. Spawning induction was performed at 26 °C under light for at least 11 h. B. mikado (Ctenophora) were cultured as previously published48 with slight modifications. Adult individuals collected at Kamo Bay , Hakkeijima , Nanao Bay  and Tabira Bay  in Japan were maintained in a 60 l aquarium with slow water circulation at Shimoda Marine Research Center  and OIST Marine Science Station (Okinawa Japan). Individuals were fed artemia in the morning and evening, with two to three feedings of frozen copepod (Pacific Trading and Kyorin) in between. The adult stage of the animals was used for mass spectrometry analysis. Isolated and H2O2-treated gemmules of E. fluviatilis (Porifera) were provided by Prof. Noriko Funayama (Kyoto University). Gemmules were cultured in M-medium  at 23–24 °C. To obtain large colonies for mass spectrometry analysis, we cultured the newly hatched juvenile sponges (~1 mm in diameter) with phytoplankton (50 µl l−1 freshwater Chlorella sp. (Nikkai Center) and 1 ml l−1 PhytoChrome (Brightwell Aquatics)) for 2–3 months. V. multiformis (Ctenophora) were collected at Kiyoshi-Hiroshi sea-grape farm in Ginoza . They were maintained in 1.5 l seawater and fed with freshly hatched artemia two times per week.of N. vectensis, E. fluviatilis and B. mikado were extracted by homogenization in acidified methanol solution  with a bead homogenizer , followed by removal of debris and large proteins by centrifugation . The supernatants were vacuum dried with a GeneVac EZ-2 Elite (SP Scientific) and then subjected to solid-phase extraction with an Oasis HLB 1 cc cartridge (Waters). The eluents were vacuum dried and stored at −80 °C for liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) analysis. The sample preparation procedure was performed independently four times for each animal species, and the resulting samples were subjected to the following analysis separately. For each animal species, one of the four extract replicates was subjected to fractionation to reduce sample complexity before LC-MS/MS analysis. Fractionation was performed according to the high-pH reversed-phase protocol49 with slight modification. The details of peptide extraction, solid-phase extraction and fractionation are described in the Endogenous peptide fractions −1. Fragment ion spectra of peptides were acquired by a data-dependent approach by using CHarge Ordered Parallel Ion aNalysis with slight modification50. Details of the parameter settings used for mass spectrometry, the liquid chromatography conditions, acquisition method and overall workflow are described in the The peptide samples from each animal species were analysed by LC-MS/MS analysis as follows. Dried samples were resuspended and analysed with a nanoflow liquid chromatography system  coupled with an Orbitrap mass spectrometer . The samples were loaded using an autosampler into a trap column  and subsequently into an analytical column . Separation of peptides was performed with a 65 min gradient  with a flow rate of 500 nl min51). The same search parameters for mass tolerance and variable posttranslational modification were used for Mascot. Searches were performed against amino acid (AA) sequences translated in six frames from the transcriptome data of the respective animal species. The results of peptide-to-spectrum matching were processed to select neuropeptide-related endogenous peptides as follows. HMMER52 v.3.2.1 was used to scan precursor proteins of detected peptides for Pfam53 (version 33.1) motifs. All peptides whose precursor protein had any Pfam motifs except for neuropeptide-related motifs  was performed using ResPep SL (Intavis). The synthesized peptides were subjected to LC-MS/MS analysis using the same settings as described above. Fragment spectra acquired from the synthetic peptides were compared with the spectra of endogenously detected peptides for validation. Raw LC-MS/MS data of endogenous and synthetic peptides, peptide-to-spectrum matching results, AA sequences used for peptide-to-spectrum matching, raw output of search engines showing unfiltered lists of identified peptides with their precursors and other files related to peptide identification have been deposited in the ProteomeXchange Consortium via jPOST54 with the data set identifier PXD030145. A complete list of identified peptides is provided in Supplementary Tables Peptide-to-spectrum matching was performed using PEAKS X software  and Mascot . The raw data of the acquired MS/MS spectra were processed with PEAKS X software; PEAKS DB algorithms were used to perform peptide-to-spectrum matching. The search parameters were as follows: 5 ppm for precursor tolerance, 0.3 Da for fragment tolerance, no fixed modifications, oxidation of methionine (15.99491 Da), pyroglutamation from glutamic acid (−18.01057 Da) or glutamine (−17.02655 Da) and amidation at peptide C termini (−0.98402 Da). For Mascot, fragment ion spectra were extracted in mgf file format from the raw data using MS convert in ProteoWizard package 3.0.20139 56 with an e value cutoff of 1 × 10−5.Similarities between metazoan neuropeptide precursors, including newly identified ones, were visualized using the same approach as JékelyHomo sapiens, Drosophila melanogaster (Arthropoda) and Lineus longissimus (Nemertea). N-terminal and C-terminal cleavage sites (2 AA) and flanking regions (6 AA) of the collected neuropeptides in their precursors were extracted and subjected to composition analysis or sequence logo analysis with WebLogo57. The sources of peptide information for each animal species and the details of the cleavage site analysis are described in the To assess actual cleavage sites in neuropeptide precursors in an unbiased way, we focused on neuropeptide structures that had been identified by mass spectrometry-based systematic peptide identification. AA sequences of neuropeptides were acquired from B. mikado was extracted from whole bodies of adult individuals collected from Hakkeijima , Japan. RNA sequencing was performed using a HiSeq 2500 (Illumina) with 150-bp paired-end reads in two lanes. A total of 157.1 million reads were obtained and trimmed using libngs (https://github.com/sylvainforet/libngs) with minimum quality of 25 and minimum size of 70 nucleotides. High-quality paired reads were retained for de novo assembly with Trinity58 v.2.3.2. Reads from different locations were assembled independently.Total RNA of B. mikado, for which sequences were identified from transcriptome data generated by Jokura et al.48. The identification of PAM sequences was performed by blastp search with default e value cutoff, followed by protein classification based on RPS-BLAST against the NCBI Conserved Domain Database59. For phylogenetic tree reconstruction, PAM sequences containing both PHM and PAL domains were aligned by ClustalW60, with careful manual examination before trimming. The alignment of PAM is available on request. Owing to uncertainties regarding the functions, causes and origins of the incomplete PAM homologues, ‘short’ gene models were not included in the phylogenetic analyses. The best substitution model was estimated by SMS61 as WAG+I+G+F. Phylogenetic trees were built using PhyML62 v.3.0 for maximum-likelihood analysis and MrBayes63 v.3.2.3 for Bayesian inference. For maximum-likelihood analysis, 100 bootstraps were calculated. For Bayesian inference, two runs (four chains) each of 10 million generations were calculated, with the initial 25% discarded as burn-in. The average standard deviation of split frequencies between runs converged to less than 0.002, and the average potential scale reduction factor was 1.000.Sequences of PAM genes were collected from GenBank, except in the case of N. vectensis and M. leidyi were used for the gene expression analysis28. Raw unique molecule identifier (UMI) counts were retrieved from http://compgenomics.weizmann.ac.il/tanay/?page_id=724. A custom Python script was written to generate gene–cell-cluster expression matrices. Cell clusters were assigned a cell type identity according to the original papers, which generated the scRNA-seq data, with few modifications. In M. leidyi, clusters 52 and 53 were renamed as colloblasts and cluster 54 as tentacle/venom64. A global-scaling normalization method was used to normalize the gene expression measurements for each cell by the total expression, multiplied by a scale factor . Genes with total UMI counts of less than 10 were removed from the analysis. N. vectensis66 and M. leidyi2 protein models were annotated by blastp alignment to the Caenorhabditis elegans and human UniProt/NCBInr databases with an e value cutoff of 1 × 10−5. We also performed reciprocal blastp against the N. vectensis and M. leidyi databases using C. elegans DCV proteins as queries. All putative homologues were manually queried with the NCBI BLAST webpage to further confirm high-quality alignment and identity. Lists of annotated genes are provided in Supplementary Tables of N. vectensis and colloblasts of M. leidyi). The code used is available at https://github.com/oist/scrna-counts.Previously generated single-cell RNA sequencing (scRNA-seq) data of adult B. mikado; and Cys-HIRamide, Cys-PRGamide, Cys-QWamide and Cys-RFamide for N. vectensis) were used to immunize rabbits with Freund’s Complete Adjuvant for the first injection or Freund’s Incomplete Adjuvant for boosting (Scrum Inc.). After checking the immunoreactivity by ELISA, each antiserum was affinity purified using peptide-conjugated beads.The C-terminal structures of amidated peptides  and juvenile polyps (10 days postfertilization) were performed as previously described47 with slight modifications. Before fixation, polyps were relaxed by adding 0.3 M MgCl2 solution dropwise. Animals were fixed with Zamboni’s fixative  in phosphate-buffered saline (PBS), 0.1 M phosphate buffer pH 7.2) overnight at 4 °C. After washing three times with PBS-Tween-20 (0.2%), specimens were incubated in 0.1 M glycine (pH 7.0) in PBS for 15 min at room temperature followed by washing twice with PBS-Triton (0.2%). The specimens were then incubated in blocking solution , 5% normal goat serum, 0.1% sodium azide in PBS-Tween-20 (0.2%)) for 1 h at room temperature and then incubated with the anti-neuropeptide primary antibody (1:200 in blocking solution) overnight at 4 °C. The specimens were washed twice with PBS-Triton (0.2%) and then incubated with the Alexa-488-conjugated anti-rabbit secondary antibody (1:500 in blocking solution)  for 1 h at room temperature. The specimens were washed three times with PBS-Triton (0.2%) and mounted on a slide glass with SlowFade antifade reagent  under a cover glass. Specimens were imaged at multiple planes covering an entire z axis using a fluorescence microscope  equipped with a Ds-Ri2 (Nikon). Acquired cross-sections at different depths were mathematically deblurred using a blind deconvolution algorithm with 30 iterations . Deconvolved sections were then stacked for maximum intensity projection.Immunostaining for B. mikado larvae (1–2 days old) was performed according to the method of Pang and Martindale35 with slight modifications. Specimens were collected in a 3-cm plastic dish. An equal volume of 6.5% MgCl2 solution was added and specimens were left to rest for 15–20 min until the tentacles extended. Larvae were transferred to a fixation buffer propanesulfonic acid) and fixed for 1 h at 4 °C. Samples were washed with PBS containing 0.05% Tween-20 (five times for 5 min each time). Samples were transferred to a blocking solution , and blocking was performed for 1 h at room temperature. After blocking, larvae were transferred to the primary antibody solution (1:500 dilution with blocking solution) and reacted at 4 °C overnight. Samples were washed with PBS containing 0.05% Tween-20 (six times for 15 min each time) then incubated with Alexa-488-conjugated anti-rabbit secondary antibody  for 3 h at room temperature. Samples were washed with PBS containing 0.05% Tween-20 (three times for 15 min each time), mounted in PBS containing 0.05% Tween-20 and observed by confocal microscopy (Olympus Fluoview FV10i).Immunostaining for V. multiformis was performed as for B. mikado with slight modifications. Specimens were placed on cover glasses in culture wells filled with seawater. After animals had adhered to the glass, seawater was removed and the animals were fixed with chilled 4% PFA in PBS at 4 °C overnight. After washing with PBS containing 0.1% Triton X100 for 15 min three times at room temperature and blocking with 1% BSA in PBS for 2 h at room temperature, samples were incubated with primary antibody at 4 °C for overnight in 1% BSA in PBS using the following dilutions: anti-VWYa, 1:100; anti-NPWa, 1:250; anti-FGLa, 1:300; anti-WTGa, 1:250 and anti-tyrosinated tubulin 1:500 . Then, samples were washed with PBS containing 0.1% Triton X100 (three times for 15 min each time) at room temperature and incubated overnight at 4 °C with 14 µM 4′,6-diamidino-2-phenylindole (DAPI) in 1% BSA in PBS and the following secondary antibodies: Alexa-488 conjugated goat anti-rabbit IgG (1:500) (Jackson ImmunoResearch); Alexa-555 conjugated goat anti-mouse IgG (1:500)  and Phalloidin-iFluor 555 conjugate (1:200) . After washing with PBS containing 0.1% Triton X100 (three times for 15 min each time), the samples were mounted on slide glasses with SlowFade Gold antifade reagent . For double staining of VWYa and NPWa, we used Zenon Rabbit IgG Labeling Kits  for direct labeling of anti-VWYa and anti-NPWa antibodies with Alexa-488 and Alexa-647, respectively, according to the manufacturer’s instructions. After the blocking steps described above, 1 µg of each of the labelled antibodies was prepared and applied to the samples for 2 h at room temperature. After washing with PBST (three times for 15 min each time), samples were postfixed with 4% PFA in PBS at 4 °C overnight. Then, they were washed with PBS containing 0.1% Triton X100 (three times for 15 min each time) at room temperature, incubated with 1% BSA in PBS containing Phalloidin-iFluor 555 conjugate and 14 µM of DAPI for 2 h at room temperature. After washing with PBS containing 0.1% Triton X100 (three times for 15 min each time), they were mounted on slide glasses with Slowfade Gold antifade reagent. The fluorescence of the samples was observed and images were recorded with a confocal microscopy system  and Metamorph v.7.10.1.161 (Molecular Devices). Images were edited with ImageJ (v.2.5.30/1.53f).Immunostaining for B. mikado was transferred with seawater on to a glass slide with two spacers in parallel and covered with a coverslip. Seawater in the chamber was exchanged by draining seawater from one side with filter paper and adding fresh seawater from the other side. For NPWa functional analysis, the larva in the chamber was filmed under a microscope . The filming of B. mikado larvae was started 3 min before the start of the experiment. The medium was then replaced with seawater, seawater with the reverse peptide or seawater with the NPWa peptide with 3-min intervals, and finally filmed for 5 min after addition of seawater with the NPWa peptide. The concentration of the peptides ranged from 1 µM to 100 µM in tenfold increments. Adradial canal length was measured using ImageJ software. For each concentration (n = 5 for each), the average value from 10 frames (approximately 25 s) after each change of the medium was calculated and normalized by that obtained under seawater conditions. For VWYa functional analysis, a larva in the chamber was recorded 1 min before the start of the experiment, then 1.5 min after the change of medium to seawater, seawater with the reverse peptide or seawater with VWYa peptide. The concentrations of the peptides were 10 µM and 100 µM. The length of the oral half of the larvae was measured five times, and the average value was used for comparison. The shape of the oral half of the larvae was considered to be a hemisphere, and its volume was calculated and compared. The value was standardized by the average value obtained under seawater conditions.A cydippid larva of 38 prediction model, constructed with 2,467 compound–protein interactions (CPIs). The CPIs were collected from the IUPHAR database (10.1111/bph.15538), UniProtKB (10.1093/nar/gku989) and the literature  z scale descriptors37. For neuropeptide–GPCR prediction, AA sequences of GPCRs were acquired by scanning M. leidyi protein models with HMMER52 (v.3.2.1) to detect GPCR-related Pfam motifs  with an e value cut off of 0.01  Supplementary_Data_2.docx, structures of N. vectensis neuropeptide precursors and their homologues in Cnidaria; (3) Supplementary_Data_3.pdf, dotplots of gene homologues involved in neuropeptide signalling and (4) Supplementary_Data_4.fasta, AA sequences of neuropeptide precursors used for cluster analysis.Zip file containing four supplementary data files: (1) Supplementary_Data_1.docx, structures of