Osteoarthritis (OA), though being treated via various methods and medicines, is still a major healthcare concern mostly due to the increase in diagnosis of these age-related diseases. The present study aimed at investigating the effects of oral and intra articular injection of grape seed oil on OA in male rat models.n = 10): C1, no treatment; C2, receiving 300 mg/day of Piascledine per os (PO); C3, 1 mg sodium hyaluronate intra-articularly in days 1, 7, 14; C4, 1 mg methyl-prednisolone acetate intra-articularly; E1, avocado and grape seed oil combination  PO; E2, 500 mg/day of grape seed oil PO; E3, 200 mg/day grape seed oil intra-articularly. After 10 weeks, the rats were anesthetized and evaluated radiologically and histopathologically. P value ≤ 0.05 was considered as statistically significant.Seventy male rats were selected and their anterior cruciate ligament was cut to induce OA. They were divided into 7 groups (P ≤ 0.05). E1 and E3 showed significantly better effects on medial femoral condyle, medial tibial condyle, joint space width, total osteophyte, and OA scores (P ≤ 0.04).All the groups made significant differences with C1 regarding all inspected radiological criteria (P ≤ 0.04). E1 and E3 had significantly superior results regarding joint surface, cell viability, and calcification (P ≤ 0.04).Joint surface, matrix, cell distribution, cell population viability, calcification, and subchondral bone in treatment groups had significantly better scores versus C1 (Grape seed oil has protective effects, both in injectable form and PO in combination with avocado, on OA in rats. Further clinical trials are necessary. Knee osteoarthritis (OA) is a degenerative joint disease and a dominant cause of pain and disability; OA is described as a slow development disease that is mostly diagnosable in its terminal phase . About 1Grape seed is well known for its variety of anti-oxidative derivatives such as oligomeric proanthocyanidins and catechins, as well as anti-inflammatory potentials, is one of the most popular remedies for the treatment of OA , 11. LitI)Tween 80 (as a nonionic emulsifier in the aqueous phase)/Span 20 (as an emulsifier of lipid-containing compounds)/stirring:In this study, different attempts were made to prepare emulsions of grape seed oil  with various combinations of the constituents. Out of these attempts, the following three series of emulsions were prepared with different hydrophilic-lipophilic balance (HLB) . Emulsification was performed using the following procedures. Initially, grape seed oil was transferred to the beaker and heated at 20 °C, then water, as the solvent, was added. Subsequently, the emulsifier was added under intense agitation at 500 rpm for 5 min. Then, in the case of groups I and II, as described below, the mixture was mixed for an extra 25 min; however, group III was completely digested by applying 10 min ultrasonic homogenizer. Finally, the mixture was sterilized by autoclave (121 °C for 15 min) and the emulsion was packaged under a laminar hood with aseptic conditions.A: 25 g grape seed oil + 23 g water 15% + 0.9375 Span 20 + 0.0625 of Tween 80B: 25 g grape seed oil + 23 g water 15% + 0.78 Span 20 + 0.22 of Tween 80C: 25 g grape seed oil + 23 g water 15% + 0.625 Span 20 + 0.375 of Tween 80II)Tween 80/Span 60/stirringD: 25 g grape seed oil + 23 g water 15% + 0.42 Span 20 + 0.53 of Tween 80E: 25 g grape seed oil + 22.5 g water 15% + 1.455 Span 60 + 1.04 of Tween 80F: 25 g grape seed oil + 22.5 g water 15% + 1.215 Span 60 + 1.285 of Tween 80G: 25 g grape seed oil + 22.5 g water 15% + 0.975 Span 60 + 1.525 of Tween 80III)Tween 80/Span 60/homogenizer/5% emulsifierH: 25 g grape seed oil + 22.5 g water 15% + 0.72 Span 60 + 1.77 of Tween 80I: 25 g grape seed oil + 22.5 g water 15% + 1.455 Span 60 + 1.04 of Tween 80J: 25 g grape seed oil + 22.5 g water 15% + 1.215 Span 60 + 1.285 of Tween 80K: 25 g grape seed oil + 22.5 g water 15% + 0.975 Span 60 + 1.525 of Tween 80IV)Tween 80/Span 60/homogenizer/2.5% emulsifierL: 25 g grape seed oil + 22.5 g water 15% + 0.72 Span 60 + 1.77 of Tween 80M: 25 g grape seed oil + 23.75 g water 15% + 0.7275 Span 60 + 0.52 of Tween 80N: 25 g grape seed oil + 23.75 g water 15% + 0.6075 Span 60 + 0.6425 of Tween 80O: 25 g grape seed oil + 23.75 g water 15% + 0.4875 Span 60 + 0.7625 of Tween 80Group III with the ratio of 40:60% with an emulsifier (5%) and group IV with the ratio of 40:60% with 2.5% emulsifier was evaluated for the monitoring of stability in a room (24 °C ± 1) and fridge (2 °C ± 1) temperatures.The 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH) is a stable radical with a maximum absorbance at 517 nm that can readily undergo reduction by an antioxidant. The scavenging effect on DPPH radical was determined by the method reported earlier with minor modifications . DiffereAvocado fruits were obtained from a local market. Two hundred and fifty gram avocado slices were blended and macerated in 500 ml of alcohol (70% ethanol) at room temperature. The product was then filtered and the alcoholic extract was collected (the first portion). Subsequently, another 500 ml of ethanol was added to the residues, boiled for about 2 h under reflux condenser in a water bath, and then filtered. The filtrate was collected and added to the first portion. Together, these two portions formed the plant residue. Afterward, 500 ml of distilled water was added to the plant residue and left at room temperature for 2 days, and then filtered and added to the previous crude extract. Another volume of water was added to the residue, boiled for about 2 h under reflux condenser and filtered. Both products were gathered to form the hydro-alcoholic crude extract. The solvents were evaporated under vacuum by rotary evaporator. The extract was obtained and stored in a freezer until use.Seventy Sprague-Dawley male rats were used in this study, obtained from the animal center of Shiraz University of Medical Sciences. Animals were kept at 23 ± 2°C and a 12 h light:12 h dark cycle, 1 week before the experiment. On day 1 of the study, OA was induced based on a previously reported method through left anterior cruciate ligament transection under general anesthesia induced by intraperitoneal injection of a mixture of acepromazine and ketamine  . For pren = 10): Group C1, negative control group with OA without any treatment; C2, positive control group receiving 300 mg/day of Piascledine dissolved in 1-2 cc distilled water  per os (PO) daily from day 1 for 10 weeks; C3, second positive controls receiving 1 mg of sodium hyaluronate  intra-articularly in the 1st, 7th, and 14th day; C4, positive control group receiving 1 mg methyl prednisolone acetate  intra-articularly in day 1, then in days 7 and 14; E1, the first experimental group receiving a combination of avocado and grape seed oil emulsion with a ratio of 2:1  PO daily from day 1 for 10 weeks; E2, 500 mg/day of grape seed oil emulsion PO daily from day 1 for 10 weeks; E3 group treated with 200 mg/day grape seed oil injected intra-articularly in days 1, 7, and 14. All animals were controlled during 10 weeks of the study. The dosages of the medicines were chosen according to previously conducted pilot experiments and published papers showing the best efficacy with the lowest amount of the agent.Post-operatively, all 70 rats were randomly assigned into seven groups , toluidine blue, and safranin O (for proteoglycan contents of the matrix). A blinded pathologist performed the evaluation by using a modified histological grading method provided . A scoriU tests were used through the SPSS software version 21.0 . A P value ≤ 0.05 was considered as statistically significant.Gathered data were analyzed by the GraphPad software version 6.0 . For histopathological and radiological evaluation, non-parametric Kruskal-Wallis and Mann-Whitney Regarding the composition of grape seed oil, in group I, samples A-D, each of the emulsions could not remain constant and two phases were produced. In the second experiment , the Span 60 was used instead of Span 20. After 1 day, the emulsions turned into two phases. In the third experiment , the homogenizer apparatus was used for mixing the lipid and aqueous layer. Interestingly, after 10 days, the emulsion did not turn into two phases. Conclusively, group III was chosen for further evaluations. Another critical factor that should be considered was sterilization. As a result, I-L was autoclaved at 121 °C for 15 min. The K and L samples became two phasic during sterilization and were removed. Finally, G and K with HLB:10 and ratio of 40:60% were chosen.Studying the stability of the emulsion included droplet size determinations and viscosity measurements for a period of 1 day to 3 months in room and fridge temperatures. None of the emulsions of 5% with HLB = 10 exhibited creaming or phase separation during the abovementioned time period. Finally, samples containing 5% emulsifier with a ratio of 40:60% (lipid: aqueous) were selected in order to have more stability for biological evaluations.The DPPH assay constitutes a quick and low-cost method that has frequently been used for evaluation of the anti-oxidative potential of various natural products. In the presence of antioxidant agents, the odd electron of DPPH becomes paired, resulting in the absorption loss. The absorbance change produced by this reaction is assessed to evaluate the antioxidant potential of the test sample. The results revealed that the IC50 scavenging effect of the avocado extract was 0.66 ± 0.1 mg/ml.P ≤ 0.05) regarding all inspected criteria in this study. Considering the findings of OA in the medial femoral condyle, joint space width, and total score for OA, groups E1 and E3 showed the lowest scores which significantly differed with the other groups (P ≤ 0.04). Scores of medial tibial condyle osteophytes were considerably lower in E1, E3, and C4 groups compared to the others (P ≤ 0.03), also, E1 and C3 groups depicted noticeably better results in medial fabella osteophytes (P ≤ 0.05). Regarding medial fabella osteophytes, total osteophyte, and total OA scores E1 group presented even better results than E3 ; E1 had considerably better results in regard to subchondral bone score compared to other PO treatments, and E3 showed noticeably lower score regarding cell distribution compared to other intra-articular treatments  which gives us the idea that the constituents of routinely used medications may change. Future in vivo research is required to investigate the potential preventive effects of grape seed oil in humans, as well as the optimal dosage, the application form, and possible side effects.


We also explore the roles of experiential avoidance and decentering as mechanisms of change.Bereaved participants who attended the ayahuasca center responded to an online survey that included the Texas Revised Inventory of Grief, Symptom Assessment-45, WHO Quality of Life-Bref, Acceptance and Action Questionnaire, and Decentering. Baseline assessment was completed by 50 individuals (T0). Of these, 39 completed the post-assessment at 15 days (T1), 31 at 3 months (T2), 29 at 6 months (T3), and 27 at 12 months (T4) after leaving the retreat. Pearson’s analysis was performed to examine the relationship between the severity of grief and mechanisms of change during the period of T0 and T1.d = 0.84; T2: Cohen’s d = 1.38; T3: Cohen’s d = 1.16; T4: Cohen’s d = 1.39). We found a relationship between experiential avoidance , decentering , and a reduction in the severity of grief.A significant decrease in Texas Revised Inventory was observed at all time points  contains supplementary material, which is available to authorized users. In the present article, only the data for the grief subsample has been included and analyzed.Participant enrollment was carried out between 2015 and 2017. During 2018, the follow-up period of 1 year was concluded. Participants were invited to participate in the study through the website of the Temple of the Way of Light , people who were taking certain medications , people with a heart condition, those with chronic high blood pressure, and pregnant women were not accepted  and 15 days (T1), 3 months (T2), 6 months (T3), and 12 months (T4) after leaving the retreat. Participants who were included in the study more than 2 months before attending the center were invited to respond to an additional assessment 2 months before entering the center (T-1) in order to control for the effect of time on outcomes in a subgroup.All questionnaires were administered online using LimeSurvey (version 1.92.), which allowed for the collection and preservation of the data on a secure server that is accessible only to the researchers via a password.This questionnaire is a self-report measure designed to quantify the severity of grief (Faschingbauer N = 1621) and clinical samples (N = 13.844) . The scale has been shown to have adequate psychometric properties with Cronbach’s coefficient alpha values ranging from 0.69 to 0.92.This questionnaire is a self-report clinical measure designed to assess psychiatric symptoms in 9 different symptom domains, as well as to provide a general measure of overall psychiatric distress (GSI)  combined with the leaves of P. viridis  for several hours. Analyses were carried out by Energy Control  using liquid chromatography-mass spectrometry (LC-MS). One ayahuasca sample contained 2 mg/ml N, N-DMT, 2 mg/ml of harmine, 0.37 mg/ml of harmaline, and 1 mg/ml of tetrahydroharmine. The other sample contained 2 mg/ml N, N-DMT, 2 mg/ml of harmine, 0.65 mg/ml of harmaline, and 2 mg/ml of tetrahydroharmine. No other psychoactive compounds were detected.We obtained samples of two different ayahuasca batches prepared by Shipibo shamans at the Temple of the Way of Light along the period of the study. The ayahuasca was prepared by boiling the stems of t test or the Wilcoxon paired test, as appropriate. Bonferroni corrections were applied to correct for multiple comparisons. A 95% CI for the mean of the primary outcome at all time point assessments was included to further clarify plausible values. Effect sizes were calculated using Cohen’s d. Effect sizes were defined as large up to 0.8 (d > 0.8)  Cohen . To examAdditional subgroup analysis was performed separately for patients who responded to T-1, T0, and T1 assessments. We used the chi-square test for categorical variables and Fisher-Snedecor for continuous variables to evaluate homogeneity between the group who completed the follow-up at T1, and a subgroup that completed an additional assessment at T-1. Subgroup analyses were conducted using repeated measures analysis of variance (ANOVAs) with post hoc Bonferroni-correction, in order to examine differences in primary and secondary outcomes between the periods of T-1 to T0 and T1 to T0.p values < 0.05 were considered statistically significant. Statistical analysis was performed using Statistical Package for the Social Sciences .In this study, Out of 117 participants who were assessed for eligibility, 79.49% participants signed the informed consent form. However, 30.1% participants were excluded because baseline data were not available, 12.91% were excluded because no follow-ups were available, and 3.23% expressed a desire to abandon the study. Finally, 50 participants were elegible for the analysis, having completed the baseline assessment and, at least, one follow-up assessment. Overall, the rate of participants not responding to the assessment at T1 was 22%, at T2, it was 38%, at T3, it was 42%, and 46% at T4  significant people in the past (range 2–10).The quality of the relationship with the deceased was positive for 68%, negative for 12%, and ambiguous for 20%. For 82% of participants, the death was considered a traumatic event and 14% were unsure. Among the sample, 68% had lost an average of 4.11 (n = 15), 40% drank ayahuasca less than 3 times, 40% between 3 and 7 times, and 20% more than 7 times.The main motivations for attending the retreat were therapeutic (34.1%), spiritual growth (34.1%), and personal development (31.7%). None of the participants affirmed a recreational purpose, such as drug tourism. Among the sample, 76% expected that the ayahuasca ceremonies at the center would improve their grief symptoms and 24% were not sure. Of the overall sample, 70% had never used ayahuasca before. Of the remaining 30% of the sample (SD = 8.7) days at the center (range 9–30), where they participated in an average of 6.62 (SD = 1.37) ayahuasca ceremonies (range 4–9). Of the 39 participants who responded at T1, 53.8% perceived ayahuasca as the most relevant factor in the evolution of their grief symptoms during their stay at the center, followed by the work of the Shipibo healers (35.9%), being with other guests (5.1%), the experience of being in the jungle (2.6%), and having attended other alternative activities, such as yoga classes (2.6%). Moreover, 78.4% described an ayahuasca experience that directly affected their grief process.Participants attended an average of 18.1  are presented in Table p < 0.001). This significant improvement in the severity of grief is shown graphically with confidence intervals (95% CI) , showing a high effect size at T1 = 1.11, 95% CI = 0.77–1.45; T2 = 1.06, 95% CI = 0.73–1.39; T3 = 0.90, 95% CI = 0.59–1.22; and T4 = 1.1, 95% CI = 0.68–1.52. Almost all of the psychopathological subscales of SA-45 showed a significant decrease after the retreat (T1: p ≤ 0.003), which were maintained over the follow-up assessments  with the exception of hostility at T3 (p = 0.451) and T4 (p = 0.087). The only subscale that did not show a significant decrease after the retreat was phobic anxiety (T1: p = 0.062), although a significant reduction in this symptom was observed over subsequent assessments .Similarly, SA-45 showed a significant improvement on the Global Severity Index (GSI) for all assessments . The improvements in physical health were maintained at T2, T3, and T4 (p < 0.001); in psychological health at T2, T3, and T4 (p < 0.001); and in social relationships at T2 (p = 0.003), T3 (p = .001), and T4 (p = 0.004). The effect size was larger for the psychological health subscale .Finally, the Symptom Assessment-45 Questionnaire (WHOQoL-BREF) questionnaire also showed significant improvements in physical health, psychological health, and social relationship scales after the retreat (T1: p < 0.001), maintaining these gains at all follow-up assessments . AAQ-II showed a high effect size for all assessments, as T1 = 0.90, 95% CI = 0.62–1.18; T2 = 1.09, 95% CI = 0.72–1.48; T3 = 1.14, 95% CI = 0.80–1.51; and T4 = 1.07, 95% CI = 0.57–1.53. EQ-Decentering also showed a high effect size for all assessments, as T1 = 0.96, 95% CI = 0.64–1.28; T2 = 0.99, 95% CI = 0.55–1.43; T3 = 0.93, 95% CI = 0.46–1.36; and T4 = 1.26, 95% CI = 0.86–1.66.Both mechanisms of change, experiential avoidance (AAQ-II) and decentering (EQ-Decentering), significantly improved after the retreat . In addition, the correlation between the significant improvement in decentering ability (EQ-Decentering) between T0 and T1 and the decrease in grief symptomatology (TRIG) between T0 and T1 (r = −0.47) was also significant (p < 0.01)  between T0 and T1 was found to be associated with the significant decrease in the severity of grief (TRIG) between T0 and T1 (01) Fig. .Fig. 3Scp > 0.05). The average number of days registered in the subsample between T-1 and T0 was 45.0 (SD = 3.87), and between T0 and T1, it was 47.67 (SD = 8.38). ANOVA revealed a significant main effect of time on the TRIG . Post hoc tests showed a significant improvement only between T1 and T0  and not between T0 and T-1 , indicating that the reduction in the severity of grief was only observed during participants’ stays at the center.Of the 39 participants who completed the follow-up assessment at T1, a subgroup of 9 participants who completed an additional assessment 2 months prior to the retreat (T-1) were elegible for the subgroup analysis (ANOVA). The homogeneity of the groups was tested by comparing key variables . No significant differences were found in baseline sample characteristics nor outcome variables  = 10.30; p = 0.006], only between T1 and T0  and not between T0 and T-1 , and with psychological health of the WHOQol-BREF  =12.28; p = 0.004), only between T1 and T0  and not between T0 and T-1 .Similarly, ANOVA revealed a significant main effect of time on secondary variables during participants’ stays at the center. This is the case with the GSI of the SA-45 questionnaire , only between T1 and T0  and not between T0 and T-1 , as well as EQ-Decentering , only between T1 and T0  and not between T0 and T-1 .Finally, AAQ-II also showed a significant main effect of time [g = 0.41) and at follow-up (g = 0.45) obtained in the last meta-analysis focused on the effectiveness of current treatments developed to treat complicated grief and prolonged grief disorder (PGD) . Studies that have obtained higher effects sizes have applied specific protocols of intervention, which involved conducting from 12 to 25 psychotherapeutic sessions during periods of 3 to 9 months, or even longer


Nigella sativa) seed extract has been shown to improve dermatological conditions, yet its beneficial effects for skin are not fully elucidated. Herein, Thymocid®, a chemically standardized black cumin seed extract, was investigated for its cosmeceutical potential including anti-aging properties associated with modulation of glycation, collagen cross-linking, and collagenase and elastase activities, as well as antimelanogenic effect in murine melanoma B16F10 cells. Thymocid®  inhibited the formation of advanced glycation end-products (by 16.7–70.7%), collagen cross-linking (by 45.1–93.3%), collagenase activity (by 10.4–92.4%), and elastases activities . In addition, Thymocid® (2.5–20 µg/mL) decreased melanin content in B16F10 cells by 42.5–61.6% and reduced cellular tyrosinase activity by 20.9% (at 20 µg/mL). Furthermore, Thymocid® (20 µg/mL for 72 h) markedly suppressed the mRNA expression levels of melanogenesis-related genes including microphthalmia-associated transcription factor (MITF), tyrosinase-related protein 1 (TYRP1), and TYRP2 to 78.9%, 0.3%, and 0.2%, respectively. Thymocid® (10 µg/mL) also suppressed the protein expression levels of MITF (by 15.2%) and TYRP1 (by 97.7%). Findings from this study support the anti-aging and antimelanogenic potential of Thymocid® as a bioactive cosmeceutical ingredient for skin care products.Black cumin ( Nigella sativa Linn. (family Ranunculaceae), commonly known as black seed, black cumin, or cumin noir, originated in regions of Eastern Europe, the Middle East, northern Africa, the Indian subcontinent, and the west and middle of Asia -acryloyl)-Leu-Gly-Pro-Ala (FALGPA) solution (100 µL) was then added to each well. The absorbance of each well was recorded at a wavelength of 345 nm with a kinetic mode for 30 min using a plate reader. The activity of collagenase was calculated as follow: collagenase activity = (∆ODc/∆T) × 0.2/(0.53 × V), where ∆ODc = difference of optical density (OD) reading from sample at different time points, 0.2 = reaction volume (mL), 0.53 = millimolar extinction coefficient of FALGPA, V = sample volume added into the reaction well (mL). The inhibition rate was calculated as inhibition% = 100 × (ActivityAn elastase inhibition assay was conducted using previously reported methods with minor modifications . BrieflyA tyrosinase inhibition assay was conducted as per previously reported methods with slight modifications . In brie2 and constant humidified atmosphere. Test samples were dissolved in DMSO as stock solution and then diluted with cell culture medium to the desired concentrations (DMSO < 0.1%).Murine melanoma B16F10 cells obtained from American Type Culture Collection  were cultured as recommended by ATCC. Briefly, B16F10 cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (Life Technologies) and 1% antibiotic solution . Cells were maintained at 37 °C in the presence of 5% CO3 cells per well and allowed to attach overnight. Next, the culture medium was replaced with fresh medium supplemented with various concentrations of Thymocid®  for 72 h. After the incubation, freshly prepared MTS reagent (20 µL) was added to each well and incubated at 37 °C for 30 min, and optical density of each well was measured at a wavelength of 490 nm using a plate reader.The viability of B16F10 cells was determined by the MTS assay as described previously with minor modifications . Briefly® was evaluated by the measurement of melanin content in B16F10 cells following previously reported method with modifications [3 cells per well and allowed to attach for 24 h. Cells were then treated with Thymocid® at concentrations of 2.5, 5, 10, and 20 µg/mL for 72 h. Then cells were lysed by adding sodium hydroxide solution , and lysed cells were centrifuged (3000× g for 5 min). The supernatant was decanted, and the cell pellet was exposed to sodium hydroxide (200 µL), followed by placing in a water bath at 80 °C for 1 h. Samples were then briefly mixed by vortex, and melanin content was transferred to a 96-well plate. The absorbance of each well was measured at a wavelength of 405 nm using a plate reader.The antimelanogenic effect of Thymocidications . Briefly4 cells per well in a 24-well plate and allowed to grow for 24 h prior to being treated with Thymocid®  for 72 h. Next, cells were harvested and washed twice with ice-cold PBS followed by centrifugation at 12,000× g for 10 min, and cells pellets were re-suspended in PBS containing Triton X-100 (1%). The cells were lysed by a freeze-and-thaw cycle to release tyrosinase from the melanosome membrane. Cellular tyrosinase was collected by centrifugation at 10,000× g at 4 °C for 30 min. A reaction mixture consisting of cellular tyrosinase (20 μg in 200 µL of PBS) and L-DOPA solution (1.25 mM) was incubated at 37 °C for 30 min. The formation of dopachrome was quantified by measuring the optical density at a wavelength of 495 nm using a plate reader.The cell-based tyrosinase assay was performed using a previously reported method with modifications . BrieflyMITF), tyrosinase (TYR), tyrosinase-related protein-1 (TYRP1), and tyrosinase-related protein-2 (TYRP2) were measured by a real-time polymerase chain reaction (RT-PCR) assay with a previously reported method [5 cells per well and allowed to grow for 24 h. Then cells were treated with Thymocid® (2.5 or 10 µg/mL) for 24, 48, or 72 h. Total RNA was isolated from cells using TRIzol reagent  according to the manufacturer’s instructions. The extracted genes were quantified by using a SYBR Green kit  and compared to levels of b2m rRNA as a reference housekeeping gene.The mRNA expression level of melanin-synthesis-related genes including microphthalmia-associated transcription factor  and cultured for 72 h. After washing with PBS, B16F10 cells were harvested, whole-cell lysates were prepared and quantified, and the protein expressions of MITF, TYR, TYRP1, and TYRP2 were quantified by Western blot assay, as described previously [B16F10 cells were seeded in 6-well plates at a density of 1.0 × 10eviously . Antibodt test or ANOVA with Tukey post-test was used for statistical analysis of the data using the GraphPad Prism software 6.0 or Office Excel 2010 software. Significance for all tests was defined as p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****).Data are presented as mean ± standard deviation (S.D.) of at least three replicated experiments. Two-tailed unpaired Student’s ®, was standardized to thymoquinone (TQ), as its major phytochemical marker. The level of TQ in Thymocid® was quantified using a standard curve based on the HPLC analysis  using a BSA model and protein cross-linking using a type I collagen model. Thymocid®  reduced the fructose-induced formation of AGEs by 16.7%, 32.5%, and 70.7%, respectively  inhibited methylglyoxal (MGO)-induced collagen cross-linking by 45.1%, 92.6%, and 93.3%, respectively , a known inhibitor ( employed as a positive control), was observed to decrease over 30 min, while Thymocid® (at 62.5–1000 µg/mL) decreased the OD345 values within 10–30 min, suggesting that both 1,10-phenanthroine and Thymocid® reduced the activity of collagenase (® (at 62.5–1000 µg/mL) reduced collagenase activity by 10.4–92.4%, respectively . Thymocid® (at concentrations of 62.5–1000 µg/mL) inhibited elastase activities in a concentration-dependent manner as it reduced the activity of type I and III elastase by 25.3–75.4% and 36.0–91.1%, respectively  by 18.0–30.0%, 16.1–30.2%, and 12.9–45.3%, at concentrations of 62.5, 125, 250, 500, and 1000 µg/mL, respectively  dose-dependently increased the activity of tyrosinase to 153.3–228.7% for L-tyrosine as substrate, and 113.7–146.3% for L-DOPA as substrate, respectively  on B16F10 cells was evaluated by measuring cell viability using the MTS assay. Thymocid® was nontoxic to B16F10 cells at concentrations ranging from 2.5 to 20 µg/mL, as it maintained the viability of B16F10 cells greater than 95.7%  suppressed the production of melanin   D.®’s suppression of melanogenesis  in B16F10 cells, its effects on the expression of melanogenesis-related genes including MITF, TYR, TYRP1, and TYRP2 were evaluated. As shown in ® (at 20 µg/mL) for 24, 48, or 72 h, suppressed the mRNA expression of MITF, TYR, TYRP1, and TYRP2 in a time-dependent manner. At 48 and 72 h, treatment with Thymocid® had a suppressive effect on the expression of MITF, TYR, TYRP1, and TYRP2 as it reduced their mRNA expression to 42.0%, 76.5%, 62.2%, and 61.2%, and 3.3%, 83.6%, 0.3%, and 0.2%, respectively, whereas it only reduced expression of MITF to 78.9% within 24 h  reduced suppressed the protein expression levels of TYRP1 (by 69.1–97.7%) and TYRP2 (by 9.4–9.3%), respectively   B.®. TQ has been reported to show several skin beneficial effects such as chemoprevention of skin tumorigenesis [®, other phytochemicals present in Thymocid® may also exert biological effects in an additive, complementary, and/or synergistic manner. In fact, TQ showed effects similar to those of Thymocid® in some bioassays, including anti-elastase and antityrosinase conducted in this study  and evaluate their biological activities are warranted. Moreover, the presence of other TQ derivatives including thymol, carvacrol, thymohydroquinone, and dithymoquinone, as well as alkaloids including nigellicine, nigellimine, nigellidine, and nigelanoid are also reported in published studies on the chemical composition of BCSEs [® in the current study, it has been reported that carvacrol and thymol exhibited skin-protective effects including inhibitory effects on collagenase and elastase [® may also exert biological effects that contribute to the overall skin beneficial effects of BCSE. Therefore, a “whole-food” approach was used to evaluate the biological effects of Thymocid®, rather than testing its individual compounds. Thymocid® showed anti-aging effects by maintaining protein structure against glycation (® on the formation of AGEs in a BSA–fructose model (® protected the structure of bovine type I collagen from glycation-induced cross-linking (® may attribute to its antioxidant effects [® on the structure of skin’s connective proteins were supported by its anti-collagenase (®’s effects on elastase in human skin fibroblast cells are warranted to support its antiwrinkle effects.Thymoquinone (TQ), a major bioactive compound in BCSE , was useigenesis  and antiigenesis . Althougis study ; howeverelastase . Therefolycation . The inhse model A was in -linking B. Given  effects . The prolagenase  and antilagenase . This is® promoted the activity of tyrosinase, which corroborates a previously reported study showing that BCSE was able to increase melanin production in melanophores isolated from wall lizard  [®, at nontoxic concentrations, suppressed the production of melanin (® are different (0.0356% vs. 5.12%). Therefore, further studies with more physiological relevant models including human melanocytes are warranted to confirm the modulatory effects of Thymocid® on melanogenesis. Mechanistically, our studies showed that Thymocid® inhibited cellular tyrosinase activity  . However melanin B,C while melanin D. This iactivity D and supactivity , which mma cells . In addi®, a chemically standardized and commercially available BCSE, protected the structure of BSA and type I collagen by inhibition of protein glycation and collagen cross-linking, respectively. Thymocid® also inhibited the activity of skin-wrinkle-related enzymes including collagenase and elastase. The inhibitory effects of Thymocid® on collagenase and elastase may contribute to its overall anti-aging effects. In addition, Thymocid® showed inhibitory effects on the production of melanin content in B16F10 cells. This antimelanogenic effect was associated with its modulation of cellular tyrosinase activity and expressions of melanogenesis-related genes and proteins including MITF, TYR, TYRP1, and TYRP2. Our findings highlight the potential of Thymocid® as a bioactive ingredient for cosmeceutical applications.In summary, Thymocid


An important hallmark of Alzheimer’s disease (AD) is the increase of Aβ1-42 burden and its accumulation to senile plaques, leading the reactive gliosis and neurodegeneration. The modulation of glia cell function represents an attractive therapeutic strategy, but is currently limited by an incomplete understanding of its relevance for AD. The chemotactic G-protein coupled formyl peptide receptor (FPR), which is known to modulate Aβ1-42 uptake and signal transduction, might be one candidate molecule regulating glia function in AD. Here, we investigate whether the modulation of FPR exerts beneficial effects in an AD preclinical model.To address this question, APP/PS1 double-transgenic AD mice were treated for 20 weeks with either the pro-inflammatory FPR agonist fMLF, the FPR1/2 antagonist Boc2 or the anti-inflammatory FPR2 agonist Ac2-26. Spatial learning and memory were evaluated using a Morris water maze test. Immunohistological staining, gene expression studies, and flow cytometry analyses were performed to study neuronal loss, gliosis, and Aß-load in the hippocampus and cortex, respectively.FPR antagonism by Boc2-treatment significantly improved spatial memory performance, reduced neuronal pathology, induced the expression of homeostatic growth factors, and ameliorated microglia, but not astrocyte, reactivity. Furthermore, the elevated levels of amyloid plaques in the hippocampus were reduced by Boc2-treatment, presumably by an induction of amyloid degradation.We suggest that the modulation of FPR signaling cascades might be considered as a promising therapeutic approach for alleviating the cognitive deficits associated with early AD. Additional studies are now needed to address the downstream effectors as well as the safety profile of Boc2. On the histopathological level, senile plaques and neurofibrillary tangles characterize Alzheimer’s disease (AD). An important component of the AD plaque is the accumulation of amyloid-β 1–42 (Aβ1-42), a 42 amino acid peptide fragment derived from sequential proteolytic cleavage of the amyloid precursor protein by beta- and gamma-secretase enzymatic activities  1:50 and CD45-FITC from eBioscience [11-0451-85] 1:50 in HBSS) was added to the pellet and incubated for 30 min on ice. After centrifugation at 250 g at 4 °C for 5 min, the cells were re-suspended in 200 μl PBS. Cell suspensions were analyzed using the cytometer LSRFortessaensities  . The Kolmogorov-Smirnow test was applied to test for Gaussian distribution of the data. For the non-parametric data, we used the Mann-Whitney or the Kruskal-Wallis test following Dunn’s multiple comparison test. For the normal-distributed data, the significance was calculated with the We have recently shown that the expression of FPR is induced in APP/PS1 double-transgenic mice by both, astrocytes, and microglia cells . To studtest trials), and their migration pathway was automatically analyzed. As demonstrated in Fig. p < 0.05; WT = 0.25 ± 0.01 vs. APP/PS1 = 0.32 ± 0.01 m), had a shorter integrated path length  and a shorter latency  which is the time it takes to find the platform. Furthermore, we determined the efficiency in searching the platform which is defined as the actual path length divided by the direct path length. There, WT mice showed a better efficiency in searching the platform compared to APP/PS1 double-transgenic mice .To analyze spatial memory performance, the animals were placed into the maze at the same starting position with a constant platform position , “corrected integrated path length” , and “latency”  between APP/PS1 and Boc2-treated APP/PS1 double-transgenic mice. Of note, a comparable protective effect was not observed in APP/PS1 double-transgenic mice treated with either fMLF or Ac2-26. In the next experiments, we therefore focused on the Boc2-treated mice.Next, we tested whether FPR modulation improves the spatial memory performance of our APP/PS1 double-transgenic mice. As demonstrated in Fig. p < 0.001 and p < 0.05; layer V 1465.43 ± 12.99 vs 1107.12 ± 23.45 cells/mm2, dentate gyrus 100 ± 7.9 vs. 67.18 ± 4.45 fluorescence intensity). In line with our finding on the behavioral level, neuronal densities were significantly higher in Boc-2 versus vehicle-treated APP/PS1 double-transgenic mice in both investigated brain regions .Neuronal degeneration is thought to contribute to deficits in spatial learning and memory , 38. To Gdnf), nerve growth factor (Ngf), and brain-derived neurotrophic factor (Bdnf) in the different treatment groups. Additionally, tropomyosin receptor kinase B (TrkB) expression levels were analyzed, also known as BDNF/NT-3 growth factor receptor. As demonstrated in Fig. TrkB in both, WT and APP/PS1 double-transgenic mice.Neurotrophic factors play key roles in the development and survival of neurons –43. Therp < 0.0001; hippocampus WT 96.25 ± 1.89 vs. APP/PS1 189.8 ± 5.93 cells/mm2; cortex WT 96.1 ± 1.54 vs. APP/PS1 159.9 ± 9.0 cells/mm2). This increase in microglia cell numbers was significantly ameliorated by Boc2 treatment . Another way to analyze microglia activation is to determine their morphology. Resting microglia have thin, ramified processes, and a relatively small cell body. Upon activation, these cells retract their processes and cell swelling occurs. To quantify this aspect, we measured a ramification index (RI), which was calculated by dividing the area around a cell, formed by its processes (Ap), by the internal cell area . As must be expected, the ramification index of IBA1+ cells was significantly decreased in APP/PS1 versus WT mice . Of note, the ramification index was significantly higher in Boc2-treated compared to vehicle-treated APP/PS1 double-transgenic mice .Growth factors are, among other cells, synthetized by astrocytes and can subsequently inhibit microglia reactivity . We, thup < 0.0001; hippocampus WT 105.7 ± 2.29 vs. APP/PS1 140.35 ± 3.36 cells/mm2; cortex WT 5.58 ± 1.0 vs. APP/PS1 68.37 ± 8.37 cells/mm2). In contrast to what we observed for microglia cells, astrocyte reactivity in APP/PS1 double-transgenic mice was not ameliorated by Boc2 treatment. Furthermore, Gfap expression levels were found to be increased in APP/PS1 double-transgenic versus WT mice, with no difference in Boc2-treated mice. In summary, Boc2 treatment modulated microglia but not astrocyte reactivity.Furthermore, astrocyte reactivity was analyzed in anti-GFAP stained sections. Comparable to what we observed for microglia cells, a significant increase in astrocyte cell numbers was observed in the hippocampus and cortex of APP/PS1 double-transgenic versus WT mice . To learn which plaque types  are preferentially reduced by Boc2 treatment, we quantified the numbers of small (> 75-125 μm2), medium (125 250 μm2), large (250 500 μm2), and very-large (> 500 μm2) sized plaques. As shown in Fig. p < 0.05; 9.75 ± 0.6 vs. 6.172 ± 1.0 plaques/mm2). Next, we analyzed microglia reactivity in the close vicinity of the Aβ1-42 plaques. First, as an internal reference, we analyzed peri-plaque microglia reactivity in fMLF-treated mice, which was shown to be not protective in our AD model  and neprilysin (Nep) in isolated brain tissues. As demonstrated in Fig. t test, p < 0.05; APP/PS1 25.84 ± 3.04 vs. APP/PS1 + Boc2 14.68 ± 2.77 %). Furthermore, expression levels of both degrading enzymes were reduced in APP/PS1 compared to WT mice .To analyze whether Aβ1-42 degradation or phagocytosis is responsible for the observed reduction in Aβ1-42 hippocampal plaque load, we measured, on the one hand, the phagocytic activity of microglia cells in Boc2-treated APP/P1 double-transgenic mice, and on the other hand, we determined the expression levels of the Aβ-degrading enzymes insulin-degrading enzyme  that different formyl peptides, such as fMLF, NfMA, or NfM can induce the release of the pro-inflammatory, inflammasome-associated cytokine IL-1β from macrophages , (ii) thdeficits , which hStreptococcus pneumoniae infection, treatment with Ac2-26 clearly showed protective effects (manuscript in preparation). In contrast to APP/PS1 double-transgenic mice, the blood-brain barrier is severely impaired in meningitis  of the flagged trials were recorded for each animal. There were no differences regarding the condition between the groups (two-way ANOVA with Turkey’s post hoc test). The data represent mean + SEM, n>11. Fig. S2. Illustration of Morris water maze training trials To investigate long-term memory we used the Morris water maze test. To analyze the learning process we used the latency time [s] of the training trials (1-12). Learning curves are separated for a better overview in A) WT group and B) in APP/PS1 mice group. The findings demonstrated that the animals improved their performance over one day . Data represent mean + SEM; n > 11. Fig. S3. Quantification of amyloid-β 1-42 phagocytosis of microglial cells by flow cytometry. Cells were isolated from adult mice brains 3h after intraperitoneal injection of methoxy-04. Exemplary graphs of each group shows the evaluation of the phagocytosis rate (Q2). Fig. S4. FPR modulation does not affect astrocytes in APP/PS1 mice A) GFAP positive cells/mm² in the hippocampus where increased from WT control to APP/PS1 control mice.B) Also in the cortex we could see the same increased amount of GFAP positive cells in APP/PS1 control mice compared to WT control (n>15) C) Exemplary anti-GFAP staining’s of WT, APP/PS1 and APP/PS1+Boc2 mice in the cortex. D) Relative expression of Gfap mRNA in the hippocampus showed no differences but E) in the cortex we detected an increased Gfap mRNA expression in APP/PS1 control mice . Scale bar c 50 μm. Shown are the mean values of each group with SEM. Two-way ANOVA with turkey test *p <0.05 **p<0.01 ***p<0.001 ****p<0.0001. Table S1. Used primer pairs with sequences, specific annealing temperature and supplier information.


Taken together, our study identified novel regulatory functions of BATF2 in regulating MDSCs recruitment, providing a prognostic value in terms of the number of BATF2+ plEVs in glioma stage.The basic leucine zipper ATF-like transcription factor 2 (BATF2) has been implicated in inflammatory responses and anti-tumour effects. Little, however, is known regarding its extracellular role in maintaining a non-supportive cancer microenvironment. Here, we show that BATF2 inhibits glioma growth and myeloid-derived suppressor cells (MDSCs) recruitment. Interestingly, extracellular vesicles (EVs) from BATF2-overexpressing glioma cell lines (BATF2-EVs) inhibited MDSCs chemotaxis in vitro. Moreover, BATF2 inhibited intracellular SDF-1α and contributes to decreased SDF-1α in EVs. In addition, BATF2 downregulation-induced MDSCs recruitment were reversed by blocking SDF-1α/CXCR4 signalling upon AMD3100 treatment. Specifically, detection of EVs in 24 pairs of gliomas and healthy donors at different stages revealed that the abundance of BATF2-positive EVs in plasma (BATF2 Glioblastoma multiform (GBM) is the most common and lethal type of human primary brain tumour , 2. The Extracellular vesicles (EVs) include small Extracellular vesicles (sEVs) with a size range of approximately 40–160 nm, and microparticles, microvesicles, large vesicles with diameters ranging from 50 nm to 1 μm , 18. EVsCCN1 promoter activity by inhibiting AP-1 binding [+ T-cell activation, which affects CD8α+ dendritic cell development and B lymphoma cell killing [Basic leucine zipper ATF-like transcription factor 2 (BATF2), also known as suppressor of AP-1 regulated by interferon (SARI), reduces  binding , 29. Rec binding . In pati binding , 32. A p killing . A previ killing . However+ EVs can constructively distinguish stage III–IV glioma from stage I–II glioma and healthy donors. Of note, our novel findings established the regulatory functions of BATF2 and BATF2-EVs in regulating MDSCs and proposed the potential of BATF2+ plEVs as a biomarker to reflect glioma stage.In this study, we found that BATF2 prevents GBM growth by inhibiting MDSCs recruitment, and EVs from BATF2-overexpressing cell lines inhibit MDSCs recruitment in vitro. Furthermore, digital EVs detection in 24 pairs of glioma plasmas at different stages indicated that plasma BATF2BATF2 (sh-1068 and sh-554) or control shRNA (sh-NC). BATF2 expression after viral transduction was confirmed by western blotting and qPCR analyses  were measured at the end point (p < 0.001)    and five distinct glioma cell lines . The results revealed diminished BATF2 expression in U251 cells and elevated expression in U87-MG cells Fig. . Thus, wses Fig. . Next, wwth Fig. . 3D videwth Fig. . Five wewth Fig. . Tumour 01) Fig. . Further01) Fig. . In cont01) Fig. . Tumour 01) Fig. . Collect+, mainly including MDSCs and macrophages) recruitment into tumour . Furthermore, we observed a significant decrease in the percentage of Gr-1+CD11b+ MDSCs gated on CD45+cells in GL261-BATF2 compared to GL261-Ctrl (p < 0.05). However, there was no significant change in F480+CD11b+ TAMs  and MDSCs recruitment in human glioma cell line U251-BATF2 compared to U251-Ctrl (p < 0.001)  Fig. . In addi01) Fig. . Moreove01) Fig. . In huma+ EVs in plasma bone marrow of U251-BATF2 tumour-bearing mice by using the Exo-counter platform. We observed that when BATF2 was overexpressed in U251 cells, BATF2+EVs were detected in the plasma and bone marrow of U251-BATF2-injected mice over time   but not CXCR7+ MDSCs (non-significant)  , with fewer in U251-BATF2 (14.2%) (p < 0.05)  in tumours. Our data showed that SDF-1α levels in intracranial tumours were significantly decreased in U251-BATF2 tumour tissues 01) Fig. . However01) Fig. . In part01) Fig. . In addi01) Fig. . To furt01) Fig. . Since Cnt) Fig. . Immunofurs Fig. , and verurs Fig. . To demo01) Fig. . ELISA r01) Fig. . HIF-1α 01) Fig. . To prov01) Fig. . Western01) Fig. . Further01) Fig. . Previou01) Fig. . In addi01) Fig. . SDF-1α 01) Fig. . Further05) Fig. . These d+Gr-1+ MDSCs in AMD3100-treated tumours   Fig. . The incce) Fig. . Gr-1 ance) Fig. . Furtherce) Fig. , suggestce) Fig. . Taken tn = 50) for BATF2, SDF-1α, HIF-1α (hypoxia marker), CD14 (Mo-MDSCs marker), and CD33 (MDSCs marker). IHC staining showed that BATF2 protein expression was inversely correlated with the expression of SDF-1α , CD33 , HIF-1α , and CD14    analysis showed that BATF2+EV detection in plasma showed an Ander Under the Curve (AUC) of 0.8576 (95% CI: 0.7450–0.9703) in stage I–II GBM vs. healthy donors, an AUC of 0.9653 (95% CI: 0.9147–1.016) in stage III–IV GBM vs. healthy donors, and an AUC of 0.7708 (95% CI: 0.6357–0.9059) in stage I–II GBM vs. stage III–IV GBM  Fig. . These o Fig. . T01) Fig. . Strikinnts Fig. . In concGBM Fig. . CollectCrosstalk between tumour cells and the tumour microenvironment has attracted great attention from researchers in the field , 42. BAThttps://www.proteinatlas.org show that BATF2 is located in both the nuclear membrane and nucleolus. In our study, we were the first to identify that BATF2 could be detected in EVs, and that BATF2 protein on the EVs surface could be labelled with gold-conjugated anti-BATF2 antibody and scanned by TEM  levels confer poor prognosis in GBM patients with low BATF2 expression levels. In addition to the decrease in MDSCs infiltration, low MMP-9, VEGFA, and MMP2 expression was observed in the BATF2 group. These cytokines are the main MDSCs-secreted factors that promote angiogenesis, tumour growth, and invasion [A previous study reported that high CD33 levels confer a poor prognosis for GBM patients . In our invasion , 44.It is possible that soluble factors, including CCL2, SDF-1α, M-CSF, GM-CSF, and G-CSF, are secreted by tumour cells , 10, 45.+cells, mainly including MDSCs and TAMs). However, the effects of BATF2 on other cellular components of the brain microenvironment, such as astrocytes, neurons, and endothelial cells, are worth studying.There are certain limitations to our study. The TME in GBM is complex due to native extrinsic components as well as tumour intrinsic mechanisms , 14. We +plEVs as a biomarker to reflect glioma stage.In conclusion, we found that BATF2 inhibits MDSCs infiltration and glioma growth. EVs from BATF2 upregulated cells could inhibit MDSCs recruitment in vitro. Moreover, BATF2 inhibited intracellular SDF-1α, and also contributed to decreased SDF-1α loading on EVs. Thus, by combining AMD3100 to block SDF-1α/CXCR4 signalling, the promoting effects of BATF2 downregulation on MDSCs recruitment were rescued. Furthermore, by calculating the numbers of BATF2-positive EVs in plasma, we proposed the clinical use of BATF2All glioma cell lines  and human astrocytes were purchased from American Type Culture Collection  and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS.g for 10 min at 4 °C, then the supernatant was removed and discarded. After suspension in PBS, a second round of centrifugation at 2500 × g for 20 min at 4 °C to remove cell debris was performed. The supernatant was filtered through a 0.22 μm filter to remove larger microvesicles and then ultracentrifuged at 135,000 × g for 120 min  using a Beckman Ti70 fixed-angle rotor. The EV pellet was washed with PBS followed by a second step of ultracentrifugation at 135,000 × g for 2 h at 4 °C, as recommended by MISEV2018 [EVs from cell culture supernatants were isolated by ultracentrifugation. Briefly, cells were grown in DMEM medium with exosome-depleted 5% FBS (Gibco Thermo Fisher Scientific). The media (~100 mL) was collected and pre-cleared by centrifugation at 500 × ISEV2018 .EVs were evaluated for morphology by TEM. EVs were resuspended in PBS and 50 μL of exosomes was absorbed onto Formvar  carbon-coated nickel grids for 1 h. Then, the grids were sequentially washed with 0.1 M sodium cacodylate, pH 7.6, fixed in 2% para-formaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate and contrasted with 2% uranyl acetate in 0.1 M sodium cacodylate for 15 min. After another wash, grids were incubated with 0.13% methyl cellulose and negatively stained with 0.4% uranyl acetate for 10 min, air-dried, and visualised under a JEM-2200FS transmission electron microscope operated at 100 kV.7−5 × 109 particles/mL. Samples were manually injected into the sample chamber at ambient temperature. Each sample was configured with a blue 405 nm laser and a high-sensitivity scientific complementary metal-oxide semiconductor (sCMOS) camera. At least 200 completed tracks were analysed per video. Particles were tracked, quantitated, and their sizes determined using NTA software v.3.0.A NanoSight NS300 system  was applied to determine the size and concentration of particles and confirm that their sizes were equivalent to those of EVs. A total of five videos, 30 s each, were recorded for individual samples. EVs were resuspended in PBS at a concentration of 2 μg/μL (1:100 dilution in particle-free PBS) to achieve 5 × 106 particles) of Ctrl-EVs and BATF2-EVs derived from each cell line were mixed in 150 µL of PBS. Fifty microlitres of the EV mix  was injected intratumourally into U251-bearing mice.Thirty micrograms . After 5 weeks, the animals were euthanised, dissected, and subjected to MRI, micro-CT, and H&E staining. All animal experiments were approved in accordance with the institutional guidelines of the Ethics Committee of West China Hospital, Sichuan University.Antibodies used in this study were as follows: BATF2 ; BATF2 ; BATF2 ; CD63 ; CD9 ; TSG101 ; SDF-1α ; SDF-1α ; HIF-1α ; CXCR4 ; VEGF ; CD33 ; CD14 ; Gr-1 ; CD11b ; CXCR7 ; SV40 Large T Antigen ; MMP-9 ; 10 nm gold-conjugated goat anti-rabbit secondary antibody , GAPDH , and β-Actin .3 Ctrl-EV and BATF2-EV/target MDSC which was found effective in preliminary experiments performed with different EV numbers ranging from 5 × 103 to 1 × 104. Invasive MDSCs accumulated at the lower chambers were collected after 16 h and counted using a cytometer.To examine the chemotactic ability of glioma cells, freshly isolated splenic MDSCs were seeded on the upper chambers of 24-well transwell inserts (Merck Millipore). In the bottom chambers, tumour-derived EVs from cell supernatant were added. A total of 7 × 10t test in either pairwise or multiple comparisons; p < 0.05 was considered statistically significant. For quantification of IHC staining, Mann–Whitney U tests were used to calculate p values. Spearman rank-order correlation coefficient analysis was performed to assess relationships between different factors. All calculations were performed using SPSS v.17.0 software  or Prism GraphPad 7.0.All experiments were repeated 3–5 times, and error bars are presented as means ± s.d. Statistical significances were calculated by using Student’s movieS1Supplementary Figures


Xenopus embryos, Rspo2 is a negative feedback inhibitor in the BMP4 synexpression group and regulates dorsoventral axis formation. We conclude that R-spondins are bifunctional ligands, which activate WNT- and inhibit BMP signaling via ZNRF3, with implications for development and cancer.BMP signaling plays key roles in development, stem cells, adult tissue homeostasis, and disease. How BMP receptors are extracellularly modulated and in which physiological context, is therefore of prime importance. R-spondins (RSPOs) are a small family of secreted proteins that co-activate WNT signaling and function as potent stem cell effectors and oncogenes. Evidence is mounting that RSPOs act WNT-independently but how and in which physiological processes remains enigmatic. Here we show that RSPO2 and RSPO3 also act as BMP antagonists. RSPO2 is a high affinity ligand for the type I BMP receptor BMPR1A/ALK3, and it engages ZNRF3 to trigger internalization and degradation of BMPR1A. In early  Xenopus embryonic development as BMP antagonists by targeting BMP receptor 1A for degradation.R-spondins are known modulators of Wnt signaling. Here, the authors demonstrate that R-spondins function in  Due to their accessibility, extracellular components of the BMP pathway are of particular interest as therapeutic targets6 and mechanistic understanding of receptor modulation should improve the ability to manipulate BMP-dependent processes.Bone Morphogenetic Proteins (BMPs) are a subfamily of TGFβ growth factors that exert a plethora of crucial functions in embryonic development, adult tissue homeostasis, as well as regeneration, and they underlie human pathology such as skeletal disorders, cancer, and fibrosis in multiple organs7. Ligands and receptors combine in a combinatorial fashion8 and phosphorylate SMAD1, 5, and 8, which enter the nucleus with SMAD4 to regulate target gene expression10. There exists a multitude of extracellular modulators of TGFβ signaling, either soluble or membrane-associated proteins that control ligand availability, processing, ligand–receptor interaction, and receptor activation11. However, only two BMP receptor antagonists are known, which directly bind and inhibit receptor function, the TGFβ-family proteins BMP3 and Inhibin13.BMPs signal through a tetrameric receptor kinase complex composed of type I  and type II receptors 20. RSPOs are a key ingredient to maintain organoid cultures where they stimulate stem cell growth22. They amplify WNT signaling by preventing Frizzled/LRP5/6 receptor ubiquitination and degradation via transmembrane E3 ubiquitin ligases ring finger 43 (RNF43) and zinc and ring finger 3 (ZNRF3), thereby sensitizing cells to WNT ligands25. RSPOs bind to ZNRF3/RNF43 and to the stem cell marker Leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), and two related proteins, LGR4 and LGR6, leading to the internalization of the RSPO-LGR-ZNRF3/RNF43 complex and lysosomal degradation26. RSPOs harbor two furin-like repeats  domains that bind to ZNRF3/RNF43 and LGRs, respectively27. In addition, they contain a thrombospondin 1 (TSP1) domain, which possess about 40% overall sequence homology28. The TSP1 domain is not essential for WNT/LRP6 signaling but it binds to HSPGs (Heparan Sulfate Proteoglycans) and thereby promotes WNT5A/PCP (planar cell polarity) signaling29.R-spondins (RSPO1-4) are a family of four secreted ~30 kDa proteins implicated in development and cancer30. Moreover, WNT and RSPO ligands are functionally non-equivalent since e.g., WNT ligand overexpression cannot induce crypt expansion in contrast to RSPO2 or RSPO331 and RSPO2 and WNT1 have distinct effects on mammary epithelial cell growth32 and cochlea development33. Hence, these inconsistencies in our current understanding raise the questions: do RSPOs possess WNT-independent functions? Do they engage other receptors? If so, in which physiological processes is this relevant?Unexpectedly, recent studies showed that RSPO2 and RSPO3 can potentiate WNT signaling in the absence of all three LGRs in vitro and in vivoXenopus. By gain-of-function and loss-of-function experiments rspo2 cooperates with Spemann organizer effectors to regulate the BMP morphogen gradient, which controls dorsoventral axis formation. Our study reveals R-spondins as BMP receptor antagonists in development, inviting re-interpretation of the mode of action of R-spondins and ZNRF3 in stem cell and cancer biology.Here we show that RSPO2 and RSPO3 are high affinity ligands for the BMP receptor BMPR1A/ALK3. RSPO2 forms a ternary complex between BMPR1A and the E3 ligase ZNRF3, which triggers endocytosis and degradation of the BMP receptor. We show that Rspo2 antagonizes BMP signaling during embryonic axis formation in rspo2 overexpression affected BMP signaling in Xenopus embryos20. We tested if RSPO2 could suppress BMP signaling in human cells. To this end, we utilized human hepatocellular carcinoma (HEPG2) cells, which express very low levels of RSPOs 20 increased endogenous BMP signaling, and this was unaffected by lrp6 Mo   Fig. , as welled) Fig. . Typicalrspo2 morpholino data, we used a previously established guide RNA (gRNA)27 to generate Crispr-Cas9-mediated Xenopus rspo2 knockout (KO) embryos  and noggin (nog)  expression, similar to knockouts of chordin or noggin 41. Interestingly, while induction of the direct BMP4 targets sizzled and vent1 by bmp4 was unaffected by CHX, rspo2 induction was inhibited . Interestingly, RSPO2 and RSPO3 treatment specifically inhibited BMPR1AQD but not ACVR1QD or BMPR1BQD, while RSPO1 and RSPO4 had no effect to any of the constitutively active receptors   possessing the TSP1 domain of RSPO2  of BMPR1A Fig. . RSPO2 snM) Fig. , comparaPO2 Fig. . R1-TSPRing Fig.  and inteing Fig. . HoweverXenopus, where we took advantage of the fact that the TSP1-domain is encoded by a distinct exon in the 3′-end of the rspo2 gene. We generated a rspo2 Mo (rspo2∆TSP Mo), which specifically abolished TSP1-domain splicing, yielding 3’ truncated rspo2 mRNA lacking the TSP1 domain but retaining the FU domains  rescued these defects  explants, where endogenous rspo2, bmpr1a and bmp4 are coexpressed, showed that ablation of rspo2 by Mo injection results in significant increase of Bmpr1a-EYFP plasma membrane levels 26 26 Fig.  prevente it Fig. . Moreove3∆R Fig. . Altoget27 , which eliminated inhibition of BMP signaling by RSPO2 Fig. . In addiEA1 Fig.  and lysoEA1 Fig. , suggestEA1 Fig.  and induXenopus, rspo2 is a negative feedback inhibitor of the BMP4 synexpression group, which cooperates with Spemann organizer effectors to inhibit BMP signaling during axis formation. Given the importance of RSPOs and BMPs as developmental regulators, as well as growth factors of normal and malignant stem cells, these conclusions have implications for development and cancer.The three main findings of our study are (i) the discovery R-spondins as BMP receptor antagonists, (ii) that RSPO2 depletes BMPR1A/ALK3 by engaging ZNRF3 for internalization and lysosomal degradation, and (iii) that in 22 and their rational use requires an understanding of their mechanism of action. For example, the fact that R-spondins inhibit BMP signaling may explain the reported non-equivalence of WNT and RSPO ligands in stem cells and development33. It may also explain their potency as stem cell growth factors, as e.g., intestinal stem cells requires both, WNT activation and BMP inhibition22.With regard to stem cells, R-spondins are a key ingredient of the culture media, which have made the organoid revolution possible11. Two extracellular BMP receptor antagonists are known, BMP3 and Inhibin13. Both are TGFβ family members, whose unproductive binding to type II receptors prevents signal transmission. Relatedly, the BMP antagonist BAMBI is a BMP pseudoreceptor lacking kinase activity, which also leads to formation of a dead-end complex with BMP receptors44. In contrast, RSPO2 and RSPO3 share no sequence homology with TGFβ family members, they inhibit type I instead of type II BMP receptors, and they do so by engaging the ZNRF3 E3 transmembrane ubiquitin ligase to internalize BMPR1A. RSPO2 thereby routes BMPR1A to clathrin-mediated endocytosis for lysosomal degradation. This mode of action resembles the function of the Spastic Paraplegia related gene NIPA1, a transmembrane antagonist, which promotes BMP receptor type II endocytosis and lysosomal degradation45.TGFβ growth factors play an eminent role in biology and medicine, and their receptor signaling is exquisitely regulated extracellularly with over 20 TGFβ antagonists, most of which antagonize signaling by ligand sequestration 7. However, we found that RSPO2 specifically binds to BMPR1A but not to ACVR1 or BMPR1B . Functional redundancy between BMP antagonists is a characteristic feature observed in fish, frog, and mouse embryos56. We note that the mouse Rspo2 expression pattern at E9.5 mimics that of mouse Bmp4, including forebrain, midbrain/hindbrain junction, branchial arches and limb apical ectodermal ridge58. Thus, although Rspo2 deficient mouse embryos gastrulate normally59, it may be fruitful to analyze compound mutants between Rspo2 and BMP antagonists for axial defects. The fact that R-spondins are bifunctional ligands, which activate WNT signaling and inhibit BMP signaling has implications for development, stem cell biology, and cancer. Mechanistically, the general picture emerging is that R-spondins function as adapters, which escort client extracellular proteins for ZNRF3/RNF43-mediated degradation, e.g., LGR4-6 and BMPR1A. Our results assign a key role to the largely ignored TSP1 domain of R-spondins in providing target specificity. The substantial sequence variability between TSP1 domains of RSPO1-4 invites screening for additional RSPO receptor targets beyond BMPR1A.We established that HEK293T and HEPG2 cells (ATCC) were maintained in DMEM High glucose (Gibco 11960) supplemented with 10% FBS (Capricorn FBS-12A), 1% penicillin-streptomycin (Sigma P0781), and 2 mM L-glutamine (Sigma G7513). H1581 cells (gift from Dr. R. Thomas) were maintained in RPMI (Gibco 21875) with 10% FBS, 1% penicillin-streptomycin, 2 mM L-glutamine and 1 mM sodium pyruvate (Sigma S8636). Mycoplasma contamination was negative in all cell lines used.Xenopus laevis frogs were obtained from Nasco. Xenopus tropicalis frogs were obtained from Nasco, National Xenopus Resource (NXR) and European Xenopus Resource Centre (EXRC).X.laevis and X.tropicalis experiments were approved by the state review board of Baden-Württemberg, Germany (permit number 35-9185.81/G-141/18 (Regierungspräsidium Karlsruhe)) and executed according to federal and institutional guidelines and regulations. Developmental stages of the embryos were determined according to Nieuwkoop and Faber (Xenbase). No statistical analysis was done to adjust sample size before the experiments. No randomization of injection order was used during the experiments.All ∆C-AP-pCDNA3, RSPO2∆C-AP-pCDNA3, RSPO2∆C-AP-pCS2+, RSPO3∆C-AP-pCDNA3, murine RSPO4∆C-pCDNA3) were generated by replacing the C-terminal domain (∆C) by AP and used to produce conditioned media. Human RSPO2 wild-type (RSPO2), the Furin1 and the Furin2 domain deletion mutants (RSPO2∆FU1/2), and the TSP1 domain deletion mutant (RSPO2∆TSP) are ORFs lacking the C-terminal domain, C-terminally tagged with a Flag-tag and subcloned into pCS2+20. R1-TSPR2, R1-TSPR2-AP and R1-TSPR2-Flag plasmids were cloned in pCS2+. Human RSPO2∆FU1 (deletion of amino acids encompassing the 6 cystines in the FU1 domain) and RSPO2∆FU2 mutants (deletion of amino acids encompassing the 8 cystines in the FU2 domain) were cloned in Flag-tag or AP-tag pCS2+. Human RSPO1TSP1 and RSPO2TSP1-HA were cloned in Streptag-HA-flag-pCS2+. Xenopus Rspo2∆FU1 (deletion of amino acids encompassing the 6 cystines in the FU1 domain) and Rspo2F107E mutants were cloned in Myc-tag or AP-tag pCS2+. All constructs were confirmed by sequencing. Conditioned media from all RSPO constructs were adjusted to equal concentration by western blot and AP activity measurement, and further validated by WNT reporter assay using HEK293T cells. The extracellular domain of BMPR1A (BMPR1AECD) was subcloned in AP-pCS2+ for generating conditioned medium and used in in vitro binding assays. Constitutively active forms of ACVR1, BMPR1A, and BMPR1B (QD) were generated by Gln-Asp mutations as described60. HA-tagged BMPR1A/ALK3 was a gift from Dr. D. Koinuma61.Alkaline phosphatase (AP) fusions with RSPOs 26, and ORFs were further subcloned in flag-pCS2+ for in vitro transcription.For For HEPG2 and H1581 cells, siRNAs and plasmids were transfected using DharmaFECT 1 transfection reagent (Dharmacon T-2001) and Lipofectamine 3000 (Invitrogen L3000), respectively, according to the manufacturer protocols. For HEK293T cells, X-tremeGENE 9 DNA transfection reagent (Roche 6365787001) was used, according to the manufacturer protocols.ECD-AP, DKK1 or WNT surrogate plasmids. After 24 h, media were changed with fresh DMEM, 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin and cultured 6 days at 32 °C. Conditioned media were harvested three times every two days, centrifuged and validated by TOPFlash assay or western blot analyses. Mouse WNT3A conditioned medium was produced from mouse L-cells stably transfected with WNT3A (ATCC CRL-2647) following the manufacturer’s instruction. For human RSPO2ΔFU1, RSPO2ΔFU2, Xenopus Rspo2ΔFU1, and Rspo2F107E mutants conditioned media, HEK293T cells were seeded in 12 well culture plates and transfected with 500 μg of each plasmid, and harvested three times every two days. Production of the media was validated with western blot analyses and AP activity analyses.HEK293T cells were seeded in 15 cm culture dishes and transiently transfected with RSPOs-AP, RSPOs-flag, BMPR1A−1 of HEPG2 cells in 24-well plates. PGL3-BRE-Luciferase (500 ng ml−1) and pRL-TK-Renilla plasmids (50 ng ml−1) were transfected using Lipofectamine 3000. After 24 h, cells were serum starved 2 h and stimulated 14–16 h with 80 ng ml−1 recombinant human BMP4 protein (R&D systems 314-BP) along with RSPO1-4 conditioned medium. TOPFlash luciferase assays were executed in HEK293T cells using 10,000 cells per well in 96-well plates. TOPFlash-Luciferase (5 ng) and pRL-TK-Renilla plasmids (1 ng) per well were transfected using X-tremeGENE9 transfection reagent (Roche 06365787001). After 48 h, cells were stimulated 24 h with WNT3A conditioned medium along with RSPO conditioned medium. Luciferase activity was measured with the Dual luciferase reporter assay system (Promega E1960). Firefly luminescence (BRE or TOPFlash) was normalized to Renilla. Statistical analyses were made with Graphpad PRISM7 software.BRE luciferase assays were executed using 300,000 ml3VO4) or RIPA buffer with cOmplete Protease Inhibitor Cocktail (Roche 11697498001). Lysates were mixed with Laemmli buffer containing β-mercaptoethanol and boiled at 95 °C for 5 min to prepare SDS-PAGE samples. Western blot images were acquired with SuperSignal West pico ECL (ThermoFisher 34580) or Clarity Western ECL (Biorad 1705061) using LAS-3000 system (FujiFilm). Quantification of blots was done using Image J v1.51k software.Cultured cells were rinsed with cold PBS and lysed in Triton lysis buffer  at 4 °C for 30 min. The reaction was quenched by 10 mM Monoethanolamine and cells were harvested and lysed with Triton X-100 lysis buffer. 200–300 μg of lysate was incubated with 20 μl streptavidin agarose (ThermoFisher 20359) to pull-down biotinylated surface proteins and subjected to Western blot.H1581 cells were seeded in 6 cm culture dishes and transfected with 50 nM of indicated siRNAs for 3 days and 2 μg of BMPR1A-HA DNA for 2 days. Surface proteins were biotinylated with 0.25 mg ml−1 sulfo-NHS-SS-Biotin (ThermoFisher 21331) at 4 °C for 30 min. After quenching excessive biotin with 10 mM monoethanolamine, pre-warmed control medium or RSPO2 conditioned medium was added at 37 °C to induce internalization. After 20 min stimulation, remaining surface-biotin was removed by 50 mM MesNa  in MesNa reaction buffer  at 4 °C for 30 min and MesNa protected-biotinylated proteins  were analyzed. Cells were harvested, and lysed with RIPA buffer  supplemented with complete protease inhibitor. Five hundred microgram of lysate was incubated with 20 μl streptavidin agarose (ThermoFisher 20359) to pull-down biotinylated proteins and subjected to Western blot.H1581 cells were seeded in 15 cm culture dish and transfected with 10 μg of BMPR1A-HA DNA for 2 days, and then split to 6 cm culture dishes. After 24 h, surface proteins were biotinylated with 0.5 mg mlXenopus embryos were performed using digoxigenin (DIG)-labeled probes according to the standard protocol (https://www.xenbase.org)62. Antisense RNA probes against rspo2 and bmp4 were generated by in vitro transcription with T7 RNA polymerase (Promega P2075)20. Probes against bmpr1a and znrf3 were prepared using full-size Xenopus bmpr1a ORF or znrf3 ORF as a template, linearized with Xho I (NEB R0146S) and transcribed with T7 RNA polymerase. Mo and mRNA injected embryos were collected at stage 11 (gastrula) or 32 (tadpole) for in situ hybridization. Images were obtained using AxioCam MRc 5 microscope (Zeiss). Embryos in each image were selected using Magnetic Lasso tool or Magic Wand tool of Adobe Photoshop CS6 software, and pasted into the uniform background color for presentation.Whole-mount in situ hybridizations of Xenopus embryos were performed according to the standard protocol (https://www.xenbase.org). X.laevis embryos were microinjected with reporter DNAs, in vitro transcribed mRNAs or antisense morpholino oligonucleotide (Mo) using Harvard Apparatus microinjection system. Mos for rspo220, lrp639, chordin, bmp438, znrf343 and standard control were purchased from GeneTools. rspo2∆TSP Mo was designed based on rspo2 sequence  and noggin (NM_001171898.1) were searched for guide RNA (gRNA) targeting sites using an online prediction tool (https://crispr.cos.uni-heidelberg.de). Primers for rspo2, noggin and chordin were designed or chosen27 for PCR-based gRNA template assembly (Supplementary Table 63. A primer lacking any target sequences was used as control gRNA. PCR reactions were performed with Phusion Hot Start Flex DNA Polymerase (NEB M0535), followed by in vitro transcription using MEGAscript T7 Transcription Kit (Invitrogen AM1334). Embryos were microinjected at one to two-cell stages with a mixture of 50 pg of gRNA and 1 ng of recombinant Cas9 protein (Toolgen) per embryo. Injected embryos were cultured until stage 30, fixed with MEMFA for phenotypical analysis. Scoring of phenotypes was executed at stage 30 with blinding from two individuals, and data are representative images from three independent experiments. Defects were categorized by the severity of ventralization. ‘Severe’ showed small head, enlarged ventral tissues and short body axis. ‘Mild’ showed one or two of the defects described above. ‘Normal’ showed no visible differences to the uninjected control.The 5′ region of genomic sequences from n NM_00112657.1 anpreprolactin (PPL) mRNA, control gRNA or standard control Mo. Per embryo; Fig. bmp4, rspo2 and rspo2 mutants mRNA; Fig. rspo2 Mo and 5 ng of lrp6 Mo, 300 pg of reporter DNA; Fig. bmp4 Mo, 2, 5, or 10 ng of rspo2 Mo, 300 pg of reporter DNA; Fig. bmp4 Mo and 5 ng of rspo2 Mo; Fig. bmp4 mRNA, 2 ng of lrp6 Mo, 1 ng of Cas9 protein; Fig. bmp4; Fig. rspo2 Mo and rspo2ΔTSP Mo, 300 pg of reporter DNA; Fig. chd Mo, 20 ng of rspo2ΔTSP Mo and 200 pg of bmpr1aDN; Fig. bmp4 and bmpr1a-EYFP, 250 pg of membrane-RFP, rspo2, and rspo2 deletion mutants mRNA; Fig. bmpr1a-EYFP, 250 pg of rspo2, and rspo2 deletion mutants, and gfp; Fig. rspo2 Mo and rspo2ΔTSP Mo; Fig. znrf3 Mo, 100 pg and 200 pg of rspo2 mRNA, and 300 pg of reporter DNA; Fig. bmp4 and bmpr1a-EYFP, 250 pg of membrane-RFP, 250 pg of rspo2, 100 pg of znrf3DN mRNA; Fig. bmp4 and bmpr1a-EYFP, 250 pg of membrane-RFP, rspo2, and rspo2 mutants mRNA; Supplementary Fig. bmp4 Mo and 5 ng of rspo2 Mo; Supplementary Fig. bmp4 mRNA, 2 ng of lrp6 Mo, 1 ng of Cas9 protein; Supplementary Fig. bmp4 and rspo3 mRNA; Supplementary Fig. rspo2ΔTSP Mo, 150 ng and 250 ng of rspo2 mRNA; Supplementary Fig. rspo2 Mo and 50 pg bmpr1aDN; Supplementary Fig. znrf3 Mo, 200 pg of ZNRF3 mRNA; Supplementary Fig. bmpr1a-EYFP.Equal amounts of total RNA or Mo were injected by adjustment with 63. For primer sequences, see Supplementary Table To validate CRISPR/Cas9-mediated genome editing, three embryos of each injection set were lysed at stage 30 for genotyping PCR reactions as describedXenopus embryos were harvested at stage 15 to 18, homogenized in NP-40 lysis buffer  with a volume of 20 μl per embryo. Lysates were cleared with CFC-113 (Honeywell 34874), followed by centrifugation , boiling at 95 °C for 5 min with NuPAGE Sample Buffer. 0.5–1 embryos per lane were loaded for SDS-PAGE analysis.Injected Xenopus laevis animal caps were dissected at stage 8 and treated with 30 μg ml−1 of cycloheximide (CHX) (Sigma C7698) until control embryos reached stage 10. CHX treatment was validated since cell division was retarded compared to untreated control. In situ hybridization and qRT-PCR were performed with same methods used in whole embryos.−1 of recombinant human RSPO1 (Peprotech 120-38), RSPO2 (Peprotech 120-43), RSPO3 (Peprotech 120-44), RSPO4 (R&D systems 4575-RS) or FGF8b (Peprotech 100-25) recombinant protein reconstituted in bicarbonate coating buffer  overnight at 4 °C. Coated wells were washed three times with TBST  and blocked with 5% BSA in TBST for 1 h at room temperature. 1.5 U ml−1 of BMPR1AECD-AP or control conditioned medium was incubated overnight at 4 °C. Wells were washed six times with TBST and bound AP activity was measured by the chemiluminescent SEAP Reporter Gene Assay kit (Abcam ab133077) or AquaSpark AP substrate (Serva 42593.01). For ZNRF3-BMPR1A binding assay, plates were coated with recombinant human ZNRF3 Fc Chimera protein (R&D systems 7994-RF). RSPO2-flag, RSPO2 deletion mutants-flag conditioned medium, or recombinant RSPO protein was preincubated 4–6 h with ZNRF3 prior to BMPR1AECD-AP treatment. Control conditioned medium and vesicles were used as control. Data show average chemiluminescent activities with SD from experimental triplicates. Statistical analyses show unpaired t-tests. The Kd was obtained with In vitro binding assay using RSPO2 recombinant protein (Peprotech 120-43) and BMPR1AECD-AP24.High binding 96-well plates (Greiner M5811) were coated with 2 μg mlΔC-HRP conditioned media 3 h at 37 °C. Cells were washed with Hanks’ balanced salt solution (HBSS) and fixed for 30 min with 0.5 mM dithiobis (succinimidyl) propionate (DSP) (Thermo 22585) crosslinker in HBSS supplemented with 10 mM HEPES, followed by permeabilization with 0.1% saponin in TSA buffer . The TSA reaction was executed for 30 min in dark with the 30 μM tyramide-Rhodamine in TSA buffer supplemented with 0.003% H2O2. Cells were washed and further stained with anti-HA and anti-Flag antibodies (1:250)39. Quantification was done using Image J v1.51k software.150,000 H1581 cells were grown on coverslips in 12-well plates, followed by siRNA and DNA transfection. After 48 h cells were fixed in 4% PFA for 10 min. Cells were treated with primary antibodies (1:250) overnight at 4 °C, and secondary antibodies (1:500) and Hoechst dye (1:500) were applied for 2 h at room temperature. For Tyramide Signal Amplification to detect RSPOs, H1581 cells were treated with RSPO1-3X. laevis embryos, bmpr1a-EYFP and membrane-RFP mRNAs were coinjected with the indicated mRNAs or Mos. Embryos were dissected for animal or ventrolateral explants at stage 9 or stage 11.5, respectively. Explants were immediately fixed with 4% PFA for 2 h and mounted with Fluoromount-G (ThermoFisher 00495802). Images were obtained using LSM 700 (Zeiss). Data are representative images from two independent experiments. For quantification, Pearson’s correlation coefficient for EYFP and RFP was analyzed using 16–30 random areas harboring 10 cells chosen from 6–10 embryos per each set.For Xenopus tropicalis LGR4 DNA were transfected in HEK293T cells and incubated with 1.5 U ml−1 conditioned media for 3 h on ice. After several washes with PBS and crosslinking with DSP, cells were treated with 2 mM Levamisole for 20 min to inactivate endogenous AP activities and developed with BM-Purple (Sigma 11442074001). Cells were mounted with Fluoromount G. Images were obtained using LEICA DMIL microscope/Canon DS126311 camera.Two hundred and fifty nanogram of human BMPR1A-HA and 64. For Xenopus laevis, animal cap explants were harvested at stage 10 and lysed in 1 ml of TRizol (Ambion 18914101). Extraction and precipitation of RNA were performed following the manufacturer’s instruction. Reverse transcription was executed with 1 μg RNA using SuperScript II reverse transcriptase and random primers (Invitrogen P/N58875). PCR amplification with the obtained cDNA was performed using UPL , #140 (sizzled), #145 (vent1)) probes and corresponding primers, further analyzed by LightCycle 480. Primers used in this study are listed in Supplementary Table Cultured cells were lysed in Macherey-Nagel RA1 buffer containing 1% β-mercaptoethanol and total RNAs were isolated using NucleoSpin RNA isolation kit (Macherey-Nagel 740955). Reverse transcription and PCR amplification were performed as described beforeP values in the analyses are as follows. (Left to right of the graph) Fig. a), <0.0001, 0.0007, <0.0001, 0.0008; (b), <0.0001, 0.0007, 0.0002, 0.003, 0.0013; (e), 0.0064, 0.0009; (f), 0.0012, 0.0005; (h), 0.0006, <0.0001; (j), 0.0347, 0.0132, 0.0040, 0.1778, 0.9918, 0.0541, 0.9925, 0.9082, 0.3433, 0.9920, 0.9991, 0.8893. Figure c), 0.0219, 0.1122, 0.0407, 0.0259; (d), 0.0153, 0.0458, 0.0233, 0.0001, 0.0096; (h), 0.0007, 0.0650, <0.0001, 0.0035. Figure c), 0.0039, 0.0085, <0.0001, 0.7836, 0.0036, 0.3596. Figure a), 0.0013, 0.0020, 0.0007; (b), 0.1193, 0.2732, 0.2591, 0.1096, 0.7246, 0.6867, 0.8089, 0.5742; (c), 0.9290, 0.9059, 0.1857, 0.0216, 0.4426, 0.9921, 0.9994, 0.9995; (e), 0.6542, 0.0007, 0.0033, 0.7650, 0.2872; (h), 0.0047; (j), 0.0006, 0.2410, 0.8921; (k), 0.0044, 0.0005, 0.0036. Figure b), 0.0766, <0.0001; (c), <0.0001, 0.0002; (e), 0.0006, <0.0001, 0.0005; (g), <0.0001, 0.0019, <0.0001, <0.0001, <0.0001. Figure e), <0.0001, 0.1072, 0.0523; (h), 0.0020, 0.0005. Figure a), 0.0011, 0.0104, 0.0043, 0.0031, 0.0001, 0.9979, 0.9973, 0.9997, 0.9938, 0.2378, 0.2508, 0.9775, 0.9894; (b), 0.6982, 0.0614, 0.0034, 0.0030; (c), <0.0001, 0.1144; (e), <0.0001, 0.5172, <0.0001; (f), 0.0188, 0.0005, 0.8546, 0.9962; (g), 0.0001, 0.0003, 0.0016, 0.0465, 0.0014; (i), <0.0001, 0.5588, <0.0001, <0.0001. Figure a), <0.0001, <0.0001, <0.0001; (b), 0.0002, 0.0026, 0.0004; (i), <0.0001, 0.0643, 0.8264; (m), 0.0011, 0.0015; (o), 0.0137, 0.0004, 0.2874, 0.1482; (q), <0.0001; (s), <0.0001. For all P values of Supplementary Figures, see Supplementary Information.All exact Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Chloroflexota is poorly studied. We applied cryo-focused ion beam milling and cryo-electron tomography to study the ultrastructural organization of thermophilic Roseiflexus castenholzii and Chloroflexus aggregans, and mesophilic “Ca. Viridilinea mediisalina.” These species represent the three main lineages within a group of multicellular filamentous anoxygenic phototrophic Chloroflexota bacteria belonging to the Chloroflexales order. We found surprising structural complexity in the Chloroflexales. As with filamentous cyanobacteria, cells of C. aggregans and “Ca. Viridilinea mediisalina” share the outer membrane-like layers of their intricate multilayer cell envelope. Additionally, cells of R. castenholzii and “Ca. Viridilinea mediisalina” are connected by septal channels that resemble cyanobacterial septal junctions. All three strains possess long pili anchored close to cell-to-cell junctions, a morphological feature comparable to that observed in cyanobacteria. The cytoplasm of the Chloroflexales bacteria is crowded with intracellular organelles such as different types of storage granules, membrane vesicles, chlorosomes, gas vesicles, chemoreceptor-like arrays, and cytoplasmic filaments. We observed a higher level of complexity in the mesophilic strain compared to the thermophilic strains with regards to the composition of intracellular bodies and the organization of the cell envelope. The ultrastructural details that we describe in these Chloroflexales bacteria will motivate further cell biological studies, given that the function and evolution of the many discovered morphological traits remain enigmatic in this diverse and widespread bacterial group.The cell biology of  Cyanobacteria and Chloroflexota (Chloroflexi) phyla, respectively. Despite green non-sulfur bacteria and filamentous cyanobacteria being phylogenetically distant lineages that have many differences in their biology, their phenotypes often share many significant similarities that are adapted to specific ecological niches. Their multicellular filaments often form a dense “fabric” of cyanobacterial mats or biofilms   , b. AlthRoseiflexus castenholzii, thermophilic Chloroflexus aggregans, and the recently described mesophilic bacterium “Ca. Viridilinea mediisalina”  is an electron microscopy technique that allows macromolecular structures to be studied at a resolution of approximately 4 nm and in 3D, while also preserving the sample in a near-native state . Cryo-ETisalina” . We chosoflexota , the mecoflexota , and finR. castenholzii DSM 13941 was grown in liquid medium consisting of the following components (g l–1): 0.5 KH2PO4, 0.5 NH4Cl, 0.3 MgCl2⋅6H2O, 0.5 KCl, 0.5 NaCl, 0.5 Na2SO4, 0.2 Na2S⋅9H2O, 0.3 NaHCO3, 2.0 yeast extract, 3.0 HEPES. The medium was completed by Wolfe’s vitamin (10 ml l–1) and trace element (10 ml l–1) solutions from ATCC. The pH of the medium was adjusted to 7.7. The culture was maintained in a glass tube with screw caps and rubber septa at a temperature of 50.5°C under constant light (incandescent light bulb).Culture of C. aggregans DSM 9485 was grown in liquid medium consisting of the following components (g l–1): 0.5 KH2PO4, 0.5 NH4Cl, 0.3 MgCl2⋅6H2O, 0.5 KCl, 0.5 NaCl, 0.5 Na2SO4, 0.5 Na2S⋅9H2O, 0.3 NaHCO3, 1.5 yeast extract, 3.0 HEPES. The medium was complemented with the 1000x trace element solution (1 ml l–1) and iron (III) citrate (0.006 g l–1) as described for BG-11 medium : 0.2 KH2PO4, 0.2 NH4Cl, 0.2 MgCl2⋅6H2O, 0.3 KCl, 25.0 NaCl, 0.3 Na2SO4, 0.3 Na2S2O3 0.7 Na2S⋅9H2O, 0.01 CaCl2⋅2H2O, 0.6 NaHCO3, 0.1 sodium acetate. The medium was supplied by 100x MEM vitamins 10 ml l–1, the 1000x trace element solution (1 ml l–1) and iron (III) citrate (0.006 g l–1) as described for BG-11 medium  was checked under a light microscope Zeiss Axiovert 200M, with 10X–63X NA 1.4 DIC objectives and images were collected on a Hamamatsu ORCA-ER CR4742-95 .Plunge freezing was performed as described by R. castenholzii and “Ca. Viridilinea mediisalina” that could then be imaged by cryo-ET. “Ca. Viridilinea mediisalina” and R. castenholzii were covered by an electron-dense extracellular matrix that dramatically decreased the contrast of the images. For this reason, their cells were pre-processed by cryo-FIB, a sample-thinning technique that essentially does not affect the native preservation of the sample  equipped with a Quantum LS imaging filter and K2 Summit (Gatan). The microscope was operated at 300kV and the imaging filters with a 30 eV slit width. The pixel size at the specimen level was 4.34 Å. Tilt series covered an angular range from −60° to +60° with 2° increments and −10 to −8 μm defocus. The total dose of a tilt series was 110–140 e-/Å2. Tilt series and 2D projection images were acquired automatically using SerialEM  were extracted from a tomogram of “Ca. Viridilinea mediisalina” using PEET. The final average had a box size of 48 pixels in × and z, and 68 pixels in y for the final step on data binned by 2 . A single particle was chosen as a first reference. A cylindrical mask was applied. Missing wedge compensation was activated. An initial average was performed on data that were binned by 4 (“1k reconstruction”), and the final motive lists were then translated and used to perform a new round of subtomogram averaging on tomograms that were binned by 2 (2k reconstructions). C6 symmetry was imposed based on the evident hexagonal arrangement of the S-layer and of the non-symmetrized average. The Fourier shell correlation curves were calculated in PEET to estimate resolution. The model for visualization was generated from the averages and overlaid in tomographic slices in UCSF Chimera.The tomogram used for sub-tomogram averaging was not corrected for the contrast transfer function, as it was not deemed necessary for the targeted final resolution. Individual particles were identified visually and manually modeled with open contours in 3dmod . The manCa. Viridilinea mediisalina” Kir15-3F), EMD-10806 (C. aggregans DSM 9485), EMD-10807 (R. castenholzii DSM 13941). The S-layer average has been deposited to the EMDB under number EMD-10804.A representative tomogram for each of the three strains has been deposited to the EMDB: EMD-10808  . Phylogeon 0.3.3 . Maximumon 0.3.3  using moon 0.3.3  and branon 0.3.3 . The lison 0.3.3 . The algCa. Viridilinea mediisalina,” C. aggregans, and R. castenholzii, showed a typical filamentous multicellular morphology when observed under an optical microscope  have not been detected in C. aurantiacus and O. trichoides . In the absence of homologs, we propose the name septal channels to be adopted for these structures. The septal channels displayed a straight passage through the cell wall that created an indentation at the cytoplasmic membrane of both cells  pili biogenesis, such as rcpC-tadZABC, tadEEG, and flp. Indeed, most of the crucial genes for the type IV pilus system are absent, such as for example pilA, pilC, and pilG. Most bacteria rely on the type IV pilus system for pili-based motility   and were designated as polyphosphate granules  than polyhydroxyalkanoate granules  . We idensalina”) , similarsalina”) . The abilymerase , in the es order . The secgranules , in accogranules . Genomese kinase , which ie kinase . We idengranules . Additiogranules . We hypogranules , based o genomes  and on pantiacus . In R. cgranules . This thC. aggregans and “Ca. Viridilinea mediisalina” has been detected previously by negative stain TEM  can be found in the article/VAG and RK performed the experimental work, processed the cryo-ET data, and wrote the manuscript. DG performed the comparative genomic analyses and phylogenetic analyses. MP, VAG, and VMG coordinated the project. All authors discussed the results and approved the submitted version of the manuscript.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.