We aimed to determine the optimal bipolar electrocoagulation power for laparoscopic surgery and to investigate which method, bipolar electrocoagulation, advanced bipolar, or ultrasonic technique was more reliable.Goat mesenteric vessels (210 in vivo samples) with diameters of 3.03–5.44 mm were selected. Bipolar electrocoagulation with 80 W, 75 W, 70 W, 65 W, 60 W, 55 W, and 50 W, and advanced bipolar and ultrasonic techniques were performed on mesenteric vessels. The thermal damage width, hemostatic effect, and burst pressure of these tissues were recorded. SPSS version 13.0 was used for all data analysis.The results showed that 60 W was the optimal for bipolar electrocoagulation based on the thermal damage width, hemostatic effect, and burst pressure. In contrast, the thermal damage width of advanced bipolar and ultrasonic techniques was smaller than that of bipolar electrocoagulation, and advanced bipolartechnique had the highest successful rate for hemostasis and highest burst pressure.Bipolar electrocoagulation was optimally performed with 60 W of power. Compared with ultrasonic and bipolar electrocoagulation techniques, advanced bipolar use was more reliable for mesenteric vessels in laparoscopic surgery; however, bipolar electrocoagulation with optimal power can be used for its simplicity of operation and low cost. Laparoscopic surgery is widely used in gynecological surgery. Hemostasis is a fundamental principle of surgery. Owing to the quest for more efficient and safer hemostatic techniques in laparoscopic surgery, several coagulating techniques and devices have been developed, including bipolar electrocoagulation, advanced bipolar (like LigaSure) and ultrasonic device (such as Ultracision) .LBipolar electrocoagulation is still widely used in laparoscopic surgery because of its simplicity of operation and low cost , 3. SeveThe present study investigated thermal damage to mesenteric vessels and hemostatic effects in goats using bipolar electrocoagulation at different power levels. The thermal damage and hemostatic effects of bipolar electrocoagulation, advanced bipolar (LigaSure) and ultrasonic device (Ultracision) were compared. We aimed to determine suitable bipolar electrocoagulation power for use in laparoscopic surgery, as well as to investigate which method, bipolar electrocoagulation, advanced bipolar, or ultrasonic technique, was more reliable.Twelve healthy adult goats  weighing 35–41 kg were obtained from the animal experimental center of Hebei Medical University. Mesenteric vessels with diameters of 3.03–5.44 mm were selected using Vernier calipers, and 210 in vivo samples were used for bipolar electrocoagulation ; in addition, 80 samples were treated using the LigaSure device and 80 were treated using the Ultracision device. The bipolar electrocoagulation can provide high-frequency electric energy for tissues through the two clamps of the bipolar forceps to dehydrate and solidify the tissue so as to stop bleeding. The working principle of Ultracision is to convert electrical energy into mechanical energy through a special conversion device. After high-frequency ultrasonic vibration, the water in the tissue cells is vaporized, the protein hydrogen bond is broken, and the tissue is cut after solidification. The figures for three devices are shown in Fig. The study was approved by the Medical Ethic Committee of the Third Hospital of Hebei Medical University.The goats were fixed on an operating table in supine position, and intravenous anesthesia was performed with 35 mg/kg ketamine, continuously administered as needed. A midline abdominal incision was made and mesenteric vessels with a diameter of 3.03–5.44 mm were selected. Vessels were divided using LigaSure, Ultracision, and bipolar electrocoagulation techniques, and the cut ends, together with 20 mm × 20 mm tissue sections, were excised. A high-frequency electrosurgical system  was set to electrocoagulation mode, and tissues were treated using different power levels . LigaSure was set at 3/5, and Ultracision was set at 3. For the bipolar coagulation ‘Bipolar Soft Coag’ was used. If the hemostatic effect was inadequate, electrocoagulation was repeated. Operations were performed by the same person, using consistent operative technique. Goats were sacrificed after surgery with 30 mg kg-1 of 5% solution of sodium pentobarbital .Electrocoagulation with bipolar forceps  was performed until no thermal bubbling was generated, or until the sensor system automatically terminated operation with use of LigaSure (with 5 mm (diameter) vessel closure forceps; Valleylab, American) and Ultracision (with 5 mm (diameter) tool head; Johnson & Johnson, American) devices. Absence of bleeding after incision was considered successful electrocoagulation. If bleeding persisted, the need for repeated electrocoagulation was considered failure of a single electrocoagulation treatment.Using an optical microscope equipped with a standard micromeasurement instrument, and with the lateral margin of the electrocoagulation forceps as the starting point, measurement of thermal damage distance was made by the same pathologist in all cases. The width of thermal damage was the vertical distance from the region with pathological changes of thermal damage to the broken ends. The recording unit was mm, and the measurement was accurate to 0.01 mm.Burst pressure was also measured. First, a catheter was inserted into the open end of the vessel segment and secured. Second, normal saline was infused into the arterial lumen at a fixed rate , and the pressure was recorded using a pressure transducer . Then, the maximum pressure (in mmHg) before leakage at the sealing site was defined as burst pressure. All burst pressure measurements were performed by two researchers, blinded for the respective study groups.P < 0.05 was considered a significant difference. The Kruskal-Wallis test was used to analyze the diameter of mesenteric vessels divided with the 3 different devices. One-way analysis of variance was used for comparison of thermal damage width, P < 0.05 was considered a significant difference, and the least significant difference test was used for pairwise comparison.SPSS version 13.0  was used for all data analysis. The chi-square test was used to compare the hemostatic effect (success rate of single vessel closure) of single electrocoagulation treatments at various specific power levels , and The mesenteric vessels showed acute coagulation necrosis. The outer layer of thermal damage showed carbonization or solidification, and the carbonization zone was relatively thin, with loss of normal cell morphology. The solidification zone was located in the carbonization zone, with a number of tissue cell layers arranged closely and oriented in one direction, showing cell elongation, cell membrane damage, patchy cytoplasm, vacuole formation, and eosinophilic enhancement. The nuclei were ruptured and the nuclear material was condensed, dissolved, and fragmented. Chromatin was blurred and indistinct, or fused with cytoplasm, without epithelial stripping, and epithelial tissue remained intact Fig. .Fig. 2P2 = 0.901, P = 0.999).Goat mesenteric vessels (3.03–5.44 mm in diameter) with diameter comparable to human uterine arteries [P < 0.001), and the thermal damage width of mesenteric vessels was maximal at 75 W and 80 W. However, there was no statistically significant difference between treatment at 75 W and 80 W (P = 1.000). There were statistically significant differences in thermal damage width between vessels treated at 55 W and 60 W in comparison with other wattages (P < 0.001), and the thermal damage width of mesenteric vessels was minimal at 55 W and 60 W with bipolar electrocoagulation. However, there was no significant difference between treatments at 55 W and 60 W (P = 1.000). In addition, the difference was not statistically significant at 50 W compared with 65 W and 70 W (P = 1.000), and thermal damage width was similar for these 3 groups .The thermal damage width of mesenteric vessels using different electric devices was as follows: bipolar electrocoagulation group (5.091 ± 0.332 mm) > LigaSure group (4.470 ± 0.693) > Ultracision group (4.089 ± 0.762); (χP < 0.05). Significant differences were observed between 60, 55, and 50 W (P < 0.05), and the success rate was higher at 60 W. No statistically significant difference was observed in the success rate for vessel closure after single electrocoagulation treatments at 65 W and 60 W, but there were statistically significant differences in thermal damage width between 65 W and 60 W (P < 0.05). Thus, 60 W was the optimal power for bipolar electrocoagulation because of the lower thermal damage width and better hemostatic effect.The hemostatic effect of single bipolar electrocoagulation treatments at different powers and using different electric devices is shown in Table P < 0.05). No significant difference was found between Ultracision and bipolar electrocoagulation (P > 0.05) techniques.In addition, LigaSure had the highest single success rate for hemostasis and showed significant differences with Ultracision and bipolar electrocoagulation (P > 0.05), and these four groups had higher burst pressures. Furthermore, there was no significant difference for pairwise comparisons of 60 W, 55 W, and 50 W (P > 0.05), and these three groups had lower burst pressures. Significant differences were found between 80 W, 75 W, 70 W, 65 W, and 60 W, 55 W, 50 W .The comparison of burst pressure for bipolar electrocoagulation at different powers is shown in Table 2 = 158.205, P < 0.001 for pairwise comparison).In addition, the comparison of burst pressure for bipolar electrocoagulation, LigaSure, and Ultracision techniques was as follows: LigaSure (1033.575 ± 82.025 mmHg) > Ultracision (673.538 ± 50.454) > bipolar electrocoagulation (553.567 ± 37.058) , because the success rate of vessel closure after single electrocoagulation treatment decreased (53.33%) after power reduction. Furthermore, repeated electrocoagulation led to heat accumulation, and the range of thermal damage was bound to increase.Ying et al.  reportedP < 0.05), and the success rate was higher at 60 W. No statistically significant difference was observed for the success rate of vessel closure after single electric coagulation treatment between 65 W and 60 W (P < 0.05), but there was statistically significant difference for thermal damage width between 65 W and 60 W (P > 0.05). Furthermore, no statistically significant difference was observed for burst pressure between 60 W and 65 W. Thus, 60 W was a suitable power for bipolar electrocoagulation, based on the thermal damage width, hemostatic effect, and burst pressure. In laparoscopic hysterectomy, the power of bipolar electrocoagulation can be adjusted on the basis of 60 W to achieve an optimal hemostatic effect. In addition, the success rate of vessel closure after single bipolar electrocoagulation was not 100.00% at 60 W; thus, suturing and ligation of vessel ends is safe and reliable after dividing uterine vessels with electrocoagulation.The success rate of vessel closure after single bipolar electrocoagulation treatment was 100.00% with power between 70 W and 80 W, suggesting that if the blood vessel was fully exposed and the uterine vessel or ovarian suspensory ligament was not in contact with the ureter, it was appropriate to choose high power. However, high power will lead to an increase in secondary injury because of thermal damage. Therefore, we studied the hemostatic effect of single bipolar electrocoagulation at several specific powers . Significant differences were observed between 60 W and 55 W as well as 50 W . No significant difference was found between ultrasonic device and bipolar electrocoagulation (P > 0.05). The findings indicated that advanced bipolar device was the safest way to close a vessel, and the hemostatic effect was equal to that of a vascular clamp and suture line [2 = 158.205, P < 0.001 for pairwise comparisons). The vascular coagulation effect of advanced bipolar device is clear, but the repeated use of an advanced bipolar device knife head causes dulling and the possibility of poor results. Therefore, surgeons should not blindly trust devices, and should individualize hemostatic techniques based on operative conditions.Advanced bipolar device had the highest single-treatment success rate for hemostasis and showed a significant difference compared with ultrasonic device and bipolar electrocoagulation (ure line . In addiThere are several limitations in this study. First, testing alternative modes and generators were not done. Additionally, this study is an animal experiment, and we will deepen the study to investigate the differences of thermal injuries of more different electrosurgical instruments. Further clinical studies are still needed.In conclusion, 60 W was an optimal power setting for bipolar electrocoagulation based on the thermal damage width, hemostatic effect, and burst pressure. Bipolar electrocoagulation had the largest thermal damage width of the mesenteric vessels, advanced bipolar device the second largest, and ultrasonic device the least. The advanced bipolar technique had the highest single success rate of hemostasis compared with ultrasonic device and bipolar electrocoagulation. Furthermore, the burst pressure of mesenteric vessels among all three techniques varied as advanced bipolar > ultrasonic device > bipolar electrocoagulation. Thus, compared with ultrasonic device and bipolar electrocoagulation techniques, advanced bipolar device is more reliable for mesenteric vessels in laparoscopic surgery when considering first attempt sealing alone without regard to cost; however, bipolar electrocoagulation with optimal power can be used because of its simple operation and low cost; when considering both cost and first attempt sealing, a higher energy setting is required with bipolar electrocoagulation.


Zygnema sp., a mat‐forming alga thriving in extreme habitats, was collected from a field site in Svalbard, where the bottom layers are protected by the top layers. The two layers were investigated by a metatranscriptomic approach and GC–MS‐based metabolite profiling. In the top layer, 6569 genes were significantly upregulated and 149 were downregulated. Upregulated genes coded for components of the photosynthetic apparatus, chlorophyll synthesis, early light‐inducible proteins, cell wall and carbohydrate metabolism, including starch‐degrading enzymes. An increase in maltose in the top layer and degraded starch grains at the ultrastructural levels corroborated these findings. Genes involved in amino acid, redox metabolism and DNA repair were upregulated. A total of 29 differentially accumulated metabolites (out of 173 identified ones) confirmed higher metabolic turnover in the top layer. For several of these metabolites, differential accumulation matched the transcriptional changes of enzymes involved in associated pathways. In summary, the findings support the hypothesis that in a Zygnema mat the top layer shields the bottom layers from abiotic stress factors such as excessive irradiation.Within streptophyte green algae Zygnematophyceae are the sister group to the land plants that inherited several traits conferring stress protection.  Zygnema are commonly found in Polar ecosystems, forming extensive mats in shallow pools, meltwater streams or on moist soil surfaces  and photosynthetically active radiation (PAR) , incurring damage to key macromolecules, such as proteins, lipids and DNA, photobleaching and photodestruction  between the two samples, mean rETRmax values of the bottom layer were higher than for the top layer  contained almost exclusively freshly divided cells with two stellate chloroplasts containing large vacuoles, whereas individual filaments in the top layer were pale and contained more storage compounds  were analysed and the results indicated a high relative abundance (>85%) of tum Fig. . AdditioThe sequencing runs produced 26 845 Mbp for the reference library and 98 462 Mbp in total for the sample libraries. The quality‐filtered reads of the reference library were assembled to 122 089 transcripts with an N50 of 764 bp and a total base count of 79 578 347.Zygnema sp. (1KP project) and Z. circumcarinatum  orthology (KO) terms were assigned to the transcripts and subsequently mapped onto the KEGG metabolic pathway map (ko01100). The major pathways, such as carbohydrate metabolism, amino acid metabolism, fatty acid metabolism, nucleotide metabolism and respiration, were well covered, confirming the high quality of the assembled transcriptome (The benchmarking single copy orthologues (BUSCO) analysis of the assembled sequences found 46.8% of the orthologues to be complete, 5.3% to be fragmented and 47.9% missing  pathways for ribosome, oxidative phosphorylation, carbon metabolism, proteasome, protein processing in endoplasmic reticulum, carbon fixation in photosynthetic organisms, pyruvate metabolism, citrate cycle and phagosome showed upregulation. Downregulated pathways were not observed.Figure a oxygenase, chlorophyll synthase and ELIPs. Enhanced transcription of gene products involved in carbohydrate metabolism including starch degrading enzymes, e.g. ɑ‐amylase, isoamylase and 4‐alpha‐glucanotransferase, and trehalose‐phosphate phosphatase. Amino acid metabolism and cell wall biosynthesis were upregulated in the top layer when compared with the bottom layer of the mat. The top layer of the Zygnema mat showed elevated transcript levels associated with carotenoid and vitamin B6 biosynthesis, ascorbate and thioredoxin metabolism, and other ROS scavengers. Other stress‐related transcripts, such as chaperones and heat shock proteins (Hsps), and genes involved in DNA repair were also upregulated in the top layers. In summary, photosynthesis, carbohydrate and amino acid metabolism, cell wall biosynthesis and antioxidant protection were upregulated in the top layer compared with the bottom layer.Table GC–MS based metabolite profiling was used to further investigate the composition of the different layers of the mat. Out of a total of 173 detected molecules, mostly comprising primary metabolites, 43 exhibited a differential accumulation or depletion Table . AnnotatZygnema sp., from Svalbard, Norway. As all samples for the present study were collected from one field site, the investigated species was previously characterized by rbcL sequences as Zygnema sp. V, belonging to the Z. circumcarinatum clade  in various Charophytes including Zygnema sp.  cycle to generate energy  but not in old cells (1 year). Even novel transglycosylation activities were described between xyloglucan and xylan, xyloglucan and mannan, illustrating the importance of cell wall modifying enzymes in several charophytes including Z. circumcarinatum  peroxiredoxin Q, thioredoxin H and M as well as the thioredoxin reductase (TrxR) exhibited enhanced expression. Catalase and peroxiredoxin Q also participate in the degradation of hydrogen peroxide. Oxidized peroxiredoxin is subsequently reduced by the TrxR and thioredoxin  are also involved in photoprotection and minimizing oxidative damage in photosynthetic organisms  consisted of different layers. Similar to other algae, the top layer, which was transcriptionally very active , appeared to act as a sunshade for bottom layers. In addition, metabolic turnover was generally higher in the top layer. The upregulation of protection mechanisms in the top layer, likely to be an immediate response to stress while the mat is still fully submerged in water may also confer enhanced protection against future abiotic stress factors, such as desiccation, an environmental factor regularly experienced by Zygnema when their habitats dry out, contributing to the evolutionary success of the species.The −1; pH 7.3). Dark green filaments were collected from the fully submerged center and bottom of the mat (termed ‘bottom layer’) and light green filaments were taken from the top layer and the margins of the mat (termed ‘top layer’). Both samples contained young vegetative cells and belonged to genotype V, previously described by Pichrtová et al.  was added to 1 mL concentrated filaments. Filaments were cleaned from debris mechanically using a stereo microscope. For the metabolite profile, 2.5 mL sample was frozen in liquid nitrogen.et al., et al.  as previously described , including TransDecoder 2.1 (http://transdecoder.github.io/), NCBI BLAST+ 2.3.0 , 1KP  and Z. circumcarinatum . Then, 10 mg of each homogenate was suspended in 1 mL ice‐cold (−20 °C) water:acetonitrile:isopropanol (2:3:3) containing 4 μg mL−1 13C6‐Sorbitol  and extracted for 10 min at 4 °C with continuous shaking at 1400 rpm . Insoluble material was removed by centrifugation at 20 000g for 5 min. A volume of 25 μL of the supernatant was collected and dried for 3 h in a vacuum centrifuge . The same steps were performed on a blank sample for quality control. Vacuum‐dried samples were re‐suspended in 10 μL of pyridine  amended with 20 mg mL−1 methoxyamine‐hydrochloride (Sigma‐Aldrich) and incubated at 28 °C for 90 min, with continuous shaking in a thermomixer . Ninety microliters of N‐methyl‐N‐trimethylsilyl‐trifluoroacetamide  were then added and the reaction continued for 30 min at 37 °C. After cooling, the content of each tube was transferred to a 2 mL clear glass autosampler vial with micro insert  for injection. Samples were injected between 2 and 24 h after derivatization.Chemical derivatization and GC–MS metabolite profiling analysis were performed according to Fiehn et al. . Freeze‐−1 and at a constant injector temperature of 250 °C. The glass liner  was changed before each series of 25 sample injections. A 30 m long, 0.25 mm internal diameter Rxi‐5Sil MS from Restek with 0.25 μm Crossbond 1,4‐bis(dimethylsiloxy)phenylene dimethyl polysiloxane film and an additional 10 m integrated guard column was used . The oven temperature was held at 70 °C for 7 min then ramped at 10 °C min−1 to 330 °C, and held constant for 7 min. The transfer line temperature between the gas chromatograph and mass spectrometer was set to 300 °C. Electron impact ionization was employed at 70 eV with an ion source temperature of 330 °C. A mix of alkanes dissolved at 2 mg L−1 in hexane was injected in the middle of the queue to allow for the conversion of retention times into Kováts' alkane‐based retention indices  v2.71 software . Before and after each injection, the syringe was washed three times with 5‐μL hexane and three times with 5 μL ethyl acetate. The injector was operated in splitless mode, opening the split vent after 4 min, with a constant flow of helium at 1 mL min Kováts, . Mass spe Stein, . Deconvogenesis algorithm. All peak integrations were subsequently assessed using the Xcalibur Quan browser. Missing values were replaced with the manually integrated background level at the expected peak retention time. Relative values of metabolite contents were determined by normalizing the peak areas of each metabolite to that of the internal 13C6‐Sorbitol standard and to the sample dry weights.Identifications were only considered valid given a match of the spectrum  and retention index (±3 U difference from the in‐house library) with library data. The most prominent unidentified compounds are reported as unknowns with their retention index and a characteristic fragment. A specific fragment was selected for the relative quantification of each compound based on the data generated with AMDIS and the corresponding peak areas were then determined at the expected compound retention times using the Xcalibur v2.2 processing software (Thermo Scientific) with the R (R Core Team (p‐values were corrected according to Benjamini and Hochberg (Statistical evaluation of the data was performed with ore Team . The datore Team . The remore Team . AdjusteHochberg .Fig S1. rETR curves of top and bottom layer. Top: ETRmax = 29.8; α = 0:325; β = −0:003; IK = 91.5. Bottom: ETRmax = 46.7; α = 0:299; β = −0:01; IK = 156.3.Click here for additional data file.Table S1. Supporting InformationClick here for additional data file.Table S2. Supporting InformationClick here for additional data file.Table S3. Supporting InformationClick here for additional data file.Table S4. Supporting InformationClick here for additional data file.


Solenopsis invicta maintains its two types of societies: colonies with one (monogyne) or many (polygyne) queens, yet the underlying genetic mechanism is poorly understood. This behavior is controlled by two supergene alleles, SB and Sb, with ~600 genes. Polygyne workers, having either the SB/SB or SB/Sb genotype, accept additional SB/Sb queens into their colonies but kill SB/SB queens. In contrast, monogyne workers, all SB/SB, reject all additional queens regardless of genotype. Because the SB and Sb alleles have suppressed recombination, determining which genes within the supergene mediate this differential worker behavior is difficult. We hypothesized that the alternate worker genotypes sense queens differently because of the evolution of differential expression of key genes in their main sensory organ, the antennae. To identify such genes, we sequenced RNA from four replicates of pooled antennae from three classes of workers: monogyne SB/SB, polygyne SB/SB, and polygyne SB/Sb. We identified 81 differentially expressed protein‐coding genes with 13 encoding potential chemical metabolism or perception proteins. We focused on the two odorant perception genes: an odorant receptor SiOR463 and an odorant‐binding protein SiOBP12. We found that SiOR463 has been lost in the Sb genome. In contrast, SiOBP12 has an Sb‐specific duplication, SiOBP12b′, which is expressed in the SB/Sb worker antennae, while both paralogs are expressed in the body. Comparisons with another fire ant species revealed that SiOBP12b′ antennal expression is specific to S. invicta and suggests that queen discrimination may have evolved, in part, through expression neofunctionalization.Queen discrimination behavior in the fire ant  Solenopsis invicta maintains its two types of societies: colonies with one (monogyne) or many (polygyne) queens, yet the underlying genetic mechanism is poorly understood. We hypothesized that the alternate worker genotypes sense queens differently because of the evolution of differential expression of key genes in their main sensory organ, the antennae. We identified 81 differentially expressed genes using RNA sequencing, and follow‐up experiments revealed that antennal expression of an odorant binding protein, SiOBP12b', is specific to S. invicta and suggests that queen discrimination may have evolved, in part, through expression neofunctionalization.Queen discrimination behavior in the fire ant  Society complexity ranges from simple cooperative pairs  accept only one SB/SB reproductive queen in their colonies, the polygyne workers (a mix of SB/SB and SB/Sb genotypes) accept multiple SB/Sb reproductive queens and kill all SB/SB queens. The queen discrimination behavior by the SB/Sb workers is presumably based on differences in cuticular chemical profiles between SB/SB and SB/Sb queens  to profile gene expression in the antennae of three worker classes: monogyne 2Detailed methods are provided in the supplementary materials Appendix .2.1S. invicta were collected from Taoyuan County, Taiwan. For Gp‐9 genotyping, an PCR/RFLP assay was conducted on pooled DNA from at least ten workers  workers.All S. invicta, we sampled different body parts from three classes of workers for each bioreplicate , polygyne SB/SB (P_BB), and polygyne SB/Sb (P_Bb). For RNA sequencing, we extracted total RNA from 36 to 55 pairs of antennae for four bioreplicates . For qRT‐PCR experiments, we extracted total RNA from 20 to 24 antennal pairs or 15 units of a body part . For sequence trace analysis, we extracted total RNA from the antennae, heads, or thoraces–abdomens of 10–11 SB/Sb workers. Different experiments used different colony pairs.For e Figure : monogynS. geminata  were from 20 and 26 individuals. Antennae samples were from 99 and 108 individuals .Body part samples of 2.3We extracted RNA using the Direct‐zol RNA MiniPrep Kit  following a modification of the manufacturer's instructions  with novel transcripts within our RNA‐Seq data as follows. We first controlled the quality of the sequence reads of the 12 NuGEN amplified antennal cDNA libraries by retaining reads passing a base‐quality threshold of 20 and minimum length of 30 bases using cutadapt v1.16 Martin, . Next, wTo identify differentially expressed genes (DEGs) on the full dataset of 12 samples, we first mapped the quality‐controlled reads onto OGS‐plus with Bowtie v2.2.6 , we amplified partial cDNAs of the two paralogs using shared PCR primers and Sanger‐sequenced the PCR products without purification.To obtain the cDNA sequence of the expressed 2.6SiOBP12B, SiOBP12b, SiOBP12b′, and SgOBP12 may be potentially functionally important, we compared their predicted protein sequences to Gp‐9 (NCBI RefSeq Q8WP90) and the pheromone‐binding protein (PBP) in Bombyx mori (NCBI RefSeq NP_001037494). SiOBP12b′ was translated from RACE cDNA sequence obtained in this study (MN193778). SiOBP12B  or social (monogyne vs. polygyne) effects on antennal gene expression differences, we examined three worker classes: monogyne SB/SB, polygyne SB/SB, and polygyne SB/Sb , possibly suggesting regulation by both genotype and social form. However, visual inspection and the Tukey HSD test suggested that genotype is the main factor regulating these two genes  >95%; range 1.2‐ to 168‐fold difference; 46 with ≥2‐fold difference; Figure s Figure A, B. Defs Figure .p‐value = .0002, Fisher's exact one‐tailed test). In contrast to a previous study on whole worker bodies, which found more genes regulated by social environment than by genotype  and genetic effects  on the worker antennae .The queen discrimination behavior in fire ants is linked to the social supergene  compared to SB/SB (M_BB and P_BB) samples.Inspection of the putative functions of all DEGs revealed 13 genes potentially involved in chemical metabolism  and odorant perception . Of these, eight genes were likely socially regulated with six and two genes being more and less expressed, respectively, in monogyne (M_BB) compared to polygyne (P_BB and P_Bb) samples. Five genes were regulated by genotype with four more highly expressed in SINVm1_gene_00166) genes being more and less expressed, respectively, in M_BB and P_BB compared to P_Bb samples.Besides these two classes of genes, 16 transposable elements (TEs) were differentially expressed among the samples. Of these, the expression of six TEs was associated with social environment, with two and four TEs being more and less expressed, respectively, in M_BB samples compared to P_BB and P_Bb samples. Another ten TEs were differentially expressed by genotype, with two and eight  assays on independent antennal RNA samples for four (of the seven) DEGs that are located in the supergene. We found the upregulation of all four genes in P_Bb samples compared to both M_BB and P_BB samples, confirming the RNA‐Seq analysis results Figure . We were3.2 and an OBP , based on a new PacBio assembly of the SB genome. SiOR463 is located between SiOR163 and an unannotated OR, SiOR462 (SINVm1_gene_13698). SiOR463 has been deleted in the Sb genome  but rather from a duplicated  copy of SiOBP12, which we call SiOBP12b′  nor Sb alleles (Table SiOBP12 and found that the expressed transcript has an extension of ~170 bp of the 5’‐UTR compared to SiOBP12B (NCBI HQ853360). This extension mapped to a different genomic region (contig 000102F in the Sb PacBio genome). Third, copy number variation analysis indicated that there is twice the number of copies of SiOBP12 in the Sb compared to the SB genome  in the genome of Solenopsis geminata, an outgroup species to the South American socially polymorphic clade of fire ants that includes S. invicta  was partially degenerated  is conserved in the two species . However, the SiOBP12b′ region is a complex duplication of ~30 kb that is absent in the S. geminata genome while its 5′ and 3′ adjacent regions are conserved and separated by only a ~2 kb unconserved fragment  or very low expression of this allele  by SiOBP12b′ in which case the protein sequence of the parental SiOBP12 and SiOBP12b′ should be highly similar. Alternatively, amino acid changes at functionally important residues may indicate neofunctionalization. Although both SiOBP12B and SiOBP12b have premature stop codons, their degeneration appears to be recent because both have high nucleotide and amino acid similarities to the ortholog SgOBP12 and paralog SiOBP12b′. Thus, mutations in SiOBP12b′ can be inferred based on the patterns of substitutions in comparison among these sequences. We compared the four OBP12s to Gp‐9 (NCBI RefSeq Q8WP90) and the solved structure of the B. mori pheromone‐binding protein (NCBI RefSeq NP_001037494) and found 14 changes (8% of full‐length protein) in OBP12b′ relative to the presumptive ancestral sequence  was also differentially expressed in the worker heads (without antennae), thoraces, and abdomens using qRT‐PCR assays. Similar to the well‐known Gp‐9, we found no expression differences between the three worker classes . Interestingly, the expression pattern of SgOBP12 was similar to those of SiOBP12B—low expression in the antennae and at comparable levels relative to the internal reference genes in the heads, thoraces, and abdomens . We found a similar expression level of both SiOBP12B and SiOBP12b′ in the heads and bodies while, again, the antennae showed predominantly (or even pure) signal from SiOBP12b′ . Globally, our results are consistent with previous studies of gene expression comparing different social supergene genotypes on whole bodies of queens, workers, and males, in that we also found an over‐representation of DEGs located in the supergene  and odorant perception   Table . WorkersR) Table . TherefoR) Table . For exaR) Table . SimilarR) Table . While i4.2Gp‐9 is a classic, although disputed, candidate that may have this function with regard to regulating social form differences  before being transferred to an OR Leal, . The OBPs Figure . PreviouSiOBP12b′ may be the strongest candidate among these to explain the sensitivity of SB/Sb workers to introduced queens . Additionally, because SiOBP12b′ is only present in SB/Sb individuals, its nonantennal functional could also affect phenotypic differences between monogyne and polygyne colonies. The precise roles of this gene remain to be investigated.Does 5SiOBP12b′ is a particularly interesting candidate because it is an Sb‐specific paralog that has acquired high expression in the antennae. These genes, and especially SiOBP12b′, will be the subject of further behavioral genetic analyses.In summary, we have profiled gene expression of the fire ant worker antennae and found that supergene genotype and social environment equally affected antennal gene expression. We identified 81 DEGs including 13 putative metabolism and odorant perception genes that may be involved in queen discrimination. Of these, we found that None declared.V.D.D. and J.W. conceived and designed the experiments. V.D.D. conducted experiments. All authors contributed to bioinformatic analyses. V.D.D. and J.W. wrote the manuscript. All authors gave final approval for publication. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


EDG5), impacted the maintenance of pluripotency, actin cytoskeleton organization, colony integrity, and focal adhesions in human embryonic stem cells, which were associated with the alteration in the RhoA‐ROCK‐Cofilin‐PAXILLIN‐actin signaling pathway. Similarly, downregulation of EDG5 during the initiation stage of somatic cell‐induced reprogramming resulted in alteration of cytoskeleton, loss of human‐induced pluripotent stem cell colony integrity, and a significant reduction in partially and fully reprogrammed cells as well as the number of alkaline phosphatase positive colonies at the end of the reprogramming process. Together, these data point to an important role of EDG5 in the maintenance and acquisition of pluripotency. Stem Cells2019;37:318–331Direct reprogramming of human somatic cells toward induced pluripotent stem cells holds great promise for regenerative medicine and basic biology. We used a high‐throughput small interfering RNA screening assay in the initiation phase of reprogramming for 784 genes belonging to kinase and phosphatase families and identified 68 repressors and 22 effectors. Six new candidates belonging to the family of the G protein‐coupled receptors (GPCRs) were identified, suggesting an important role for this key signaling pathway during somatic cell‐induced reprogramming. Downregulation of one of the key GPCR effectors, endothelial differentiation GPCR5 ( Using a high‐throughput RNA interference screen, authors identified 22 effectors of somatic cell‐induced reprogramming, six of which belong to the G protein‐coupled receptor (GPCR) family. The present study describes a new role for the GPCR family member, endothelial differentiation GPCR5 , whose downregulation in human embryonic stem cells (hESCs) or during the initiation period of reprogramming leads to abrogation of the pluripotent stem cell colony formation because of the defects in cytoskeleton organization and focal adhesions. Together, the data provide for the first time substantive evidence for EDG5 as a critical GPCR for the maintenance of pluripotency in hESC and successful reprogramming of human fibroblasts to human‐induced pluripotent stem cells.OCT4, SOX2, KLF4, and c‐MYC (OSKM) transcription factors results in generation of human‐induced pluripotent stem cells (hiPSCs), which are similar to human embryonic stem cells (hESCs) in many of their properties Direct reprogramming of human somatic cells by ectopic expression of Pluripotency induction during reprogramming occurs in discrete stages  and is characterized by specific alterations in the cellular transcriptome, epigenome To identify novel regulators of reprogramming, we developed a high‐throughput RNA interference (RNAi) screening assay. This strategy allowed us to perform knockdown of 784 members of the different kinases and phosphatases at the initiation stage of reprogramming. We identified 90 reprogramming candidates: 68 repressors and 22 activators, among which 76 were novel. Importantly, our list included previously recognized candidates in human  and mouse cells  There are more than 800 GPCRs in the human genome, making it the largest receptor superfamily of cell‐surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric GTP‐binding (G) proteins. The human GPCR superfamily is divided in five distinct families: rhodopsin, secretin, glutamate, adhesion, and frizzled receptors. G‐proteins, composed of α, β, and γ subunits, are central components of the primary mechanisms used by cells to respond to diverse extracellular stimuli. Most GPCRs activate one or multiple G‐alpha (Gα) subunits, which can be subdivided into four major families: Gi, G12/13, Gs, and Gq. Once activated, G protein subunits modulate secondary messenger release and activate various downstream intracellular signaling pathways , and D, calcium mobilization, MAPK, extracellular signal‐regulated kinases‐1/2 ERK[1/2], PI3K, and the activation of small GTPases such as Rho and Rac), leading to regulation and adaptation of cellular functions to an external stimulus.Knockdown of some GPCRs and/or components of their signaling pathways  are embryonic lethal or associated with significant developmental anomalies in mice and humans EGD5 was identified as a novel gene required for stem cell pluripotency EDG5 during the initiation stage of reprogramming process resulted in loss of colony integrity, dysregulation of actin cytoskeleton, and a significant reduction in the number of pluripotent stem cell colonies. Together, our data provide for the first time, substantive evidence for EDG5 as a critical GPCR for the maintenance of pluripotency in hESC and successful reprogramming of human fibroblasts to hiPSCs.Two known positive regulators of pluripotency in hESCs, sphingosine‐1‐phosphate (S1P), and lysophosphatidic acid CytoTune‐iPS 2.0 Sendai reprogramming kit  was used for IPSC derivation as described recently on a feeder‐free culture on a plates covered with Matrigel  EDG5 was purchased from Dharmacon  was used.SMARTpool: siGENOME small interfering RNA (siRNA) for Protein extraction, Western blotting, and antibody/antigen complex detection were performed as published previously Primary antibodies were from Cell Signaling: for phospho‐cofilin (Ser3) , for total Cofilin , for GAPDH , for Phospho‐FAK , for total FAK (3285), for p‐MLC2 , for TESK1 (D4904), for NANOG , and for p‐ERK Thr202/Tyr204 (D13.14.4E). Primary antibodies from Abcam  were: for PAXILLIN , for total PAXILLIN (ab2264), for ROCK1 (ab58305), and for GAPDH (ab9485). Antibody for p‐LIMK2 (Thr505) was from Invitrogen (PA537630). Primary antibodies from Santa Cruz  were: for EDG5 , for vinculin , and for GAPDH (sc‐47724). Antibody for E‐cadherin was from ThermoFisher Scientific .http://nikon.com) using a CFl Plan Apochromat VC ×20/0.75 objective as described Immunocytochemistry was performed as before Cells were harvested, and the total RNA was extracted using TRIzol , according to manufacturer's instructions. All steps were performed as described before 6) were resuspended in a total volume of 200 μl PBS containing 1% bovine serum albumin and incubated with appropriate amounts of anti‐CD44‐BV421  and anti‐TRA‐1‐60‐FITC  monoclonal antibodies for 1 hour on a shaker, plate in the dark at room temperature. Finally, samples were washed using BD FACS Lyse Wash Assistant (BD Biosciences) and immediately analyzed on a flow cytometer. Fluorescence‐activated cell sorting (FACS) analysis was performed using BD FACS Canto II flow cytometer with FACSDiva software (BD Biosciences). A minimum of 20,000 events were recorded for each sample. Fluorescence minus one control (for each antibody) was used to gate the subpopulations.Cells were disassociated using Versene , washed with phosphate‐buffered saline (PBS), and fixed in paraformaldehyde  at 37°C for 10 minutes. After washing with PBS, the cells were permeabilized with prechilled methanol (−20°C) and incubated at 4°C for 30 minutes, followed by a washing step. Cells  according to manufacture instructions.t‐test analysis was used to assess differences between control and EDG5‐RNAi groups. The results were considered significant if p < .05. Significant differences indicated using asterisks .Student ). The average Z scores from three biological repeats were calculated for TRA‐1‐60+ cells versus total nuclei and a cutoff of ±1.65 was applied. This analysis resulted in identification of 68 candidate repressors and 22 candidate effectors, which included genes reported to play an important role in hiPSC generation, including MPP3, TGFBR2, ROCK1, ATM, BUB1, and TESK1 EDG5, GPR42, GPR20, GPR123, GPR116, and GPR37L1 as candidate effectors of the reprogramming process .Our examination of the expression of the named GPCR candidates in hESC and hiPSC compared to somatic cells using publicly available microarray expression data  and assessed the number of alkaline phosphatase positive colonies at day 28. This analysis indicated a significant reduction in the number of alkaline phosphatase of pluripotent stem cell colonies . Downregulation of EDG5 was confirmed using Western blotting  event N‐cadherin and SNAIL upregulation in EDG5 knockdown cells may mark the start of EMT transition instead of mesenchymal‐to‐epithelial transition (MET), which is critical for iPSCs generation. Further analysis of the genes involved in mesendodermal differentiation indicated overexpression of these markers  and filamentous actin under EDG5‐RNAi conditions in hESCs . Our results indicate that p‐PAXILLIN (Tyr118) is lost from FAs sites upon EDG5‐RNAi conditions : the same was found under ROCK inhibition (data not shown). FAK is a substrate for ROCK; in accordance, the expression of p‐FAK (Tyr397) and the total FAK were significantly reduced upon EDG5 knockdown in hESCs . In contrast to the control colonies, colocalization of p‐FAK (Tyr397) with p‐PAXILLIN (Tyr118) was not observed at the edges of the EDG5‐RNAi colonies at day 10 of reprogramming . This may be explained by the fact that localization of VINCULIN is regulated by TESK1 via integrin signaling cascade EDG5 as shown in Figure EDG5 knockdown, we did not detect alteration on E‐cadherin expression by immunofluorescence during reprogramming under EDG5‐RNAi conditions  and p‐PAXILLIN (Tyr118) and a “cornerstone” for the successful generation of hiPSCs colonies ies Fig. A, 6B, leies Fig. , akin to97 and p‐EDG5‐RNAi  and loss of colony integrity upon NAi Fig. C, we wenNAi Fig. A. Morpho and lossges Fig. , furtherSince the discovery that somatic cells could be reprogrammed to iPSCs in 2006 by Yamanaka and colleagues EDG5 resulted in loss of typical colony morphology, acquisition of filopodia, dysregulation of actin cytoskeleton, overexpression of mesendodermal and EMT markers, and a significant reduction in the number of alkaline‐positive colonies in hESCs. Similarly, downregulation of EDG5 during the initiation stages of human somatic cell‐induced reprogramming led to similar dysregulation of cytoskeleton, colony integrity, FAs, and reduced number of the pluripotent stem cell colonies at the end of the reprogramming process. Accordingly, these data imply that in hESCs, EDG5 functions cannot be substituted by its close EDG family member, EDG3, which has been suggested to compensate for Edg5 null mice, which are viable, fertile, and developed normally In our research, we used a 384‐well plate format for small interfering RNA (RNAi) screening of the library of 784 different kinases and phosphatases. This screening strategy allowed us to identify in addition to the known players in reprogramming new candidate effectors and repressors of this process. This screen revealed enrichment in GPCRs signaling and identified six new effectors  belonging to this family. We focused our attention on EDG5 and showed that downregulation of In hESCs, S1P receptors are linked to different G proteins, allowing this signaling pathway to elicit a variety of specific responses through the activation of Gi, Gq, G12/13, and Gs which control survival, proliferation, migration, and self‐renewal EDG5, in this process. As EDG5 signaling can be mediated via three different G‐α subunits resulting in the different cellular outcomes, we focused our study to the EDG5‐G12/13‐RoA‐ROCK signaling pathway. Our study demonstrated that EDG5 downregulation in hESCs abrogated Rho‐ROCK signaling and led to COFILIN/PAXILLIN/F‐actin cytoskeleton abnormalities. Earlier, the importance of actin filament organization for human somatic cell reprogramming was suggested by EDG5. This also occurred during the somatic cell‐induced reprogramming, thus highlighting for the first time that the importance of the EDG5‐G12/13 signaling is the maintenance and induction of pluripotency.Regardless the fact that G protein signaling is recognized as a key functional regulator of hiPSCs generation EDG5 knockdown, inhibition of ROCK signaling pathway led to reduced staining for filamentous actin in hESCs but not complete ablation (data not shown), suggesting that a direct comparison is not valid.It is known that actin filaments generate forces that drive changes in cell shape and mechanics through their interaction with myosin molecular motors. In fact, RhoA‐GTPase/Rho‐associated ROCK‐myosin‐II signaling can alter tension of the actin cytoskeleton and regulate survival of individual hESCs EDG5 expressing CHO cells exhibited inhibition of migration and chemotaxis toward Insulin–like growth factor 1(IGF1) in response to the S1P concentration gradient The assembly of filamentous actin was shown to play a direct role in controlling cytoskeletal and morphological aspects of the contact guidance response in hESCs EDG5 resulted in loss of PAXILLIN (Thr 118) which may be important for FA‐dependent regulation of cell morphology and hiPSCs development EDG5 knockdown. Furthermore, our data provide evidence that EDG5 plays an important role for the assembly of FAs as its downregulation resulted in significant decrease on the expression of two key components, namely p‐FAK and p‐PAXILLIN, in hESC and during reprogramming of human fibroblasts to hiPSC.Another aspect of our research is the link between actin cytoskeleton and FAs during reprogramming. FAs, the cell's mechano‐transducing units, were recently reported to be important for the generation of hiPSCs EDG5 (S1P2) downregulation in hESC and during reprogramming to hiPSC demonstrated alteration of the typical stem cell morphology, acquisition of filopodia, which was associated with downregulation of RhoA‐ROCK signaling, reduced expression of p‐LIMK2 (Thr505), ablation of the p‐COFILIN (Ser3) expression, and a significant reduction in filamentous F‐actin. Also, our data revealed alteration at the Rho‐ROCK‐p‐MLC2 signaling, the second axis important for actin cytoskeleton organization. Additionally, in agreement with that FAK is a substrate of ROCK, the expression of p‐FAK (Tyr 397) was reduced; consequently, p‐PAXILLIN (Tyr118), important for FAs organization at the edges of the colonies, was lost. Thus, we concluded that EDG5 signaling via G12/13‐RoA‐ROCK is important for (a) proper F‐actin cytoskeleton organization in hESCs and its remodeling during generation of hiPSCs and (b) the assembly of the PAXILLIN/F‐actin‐FAK FA complexes, which are important for pluripotency and colony integrity as summarized in the Graphical Abstract . Application of the specific FAK inhibitor (PF562271) at day 8 to day 10 of the reprogramming process significantly reduced the number of developing colonies, further supporting our data. Thus, EDG5 is a new indispensable gene for acquisition and maintenance of pluripotency.Using an RNAi screen at the initiation stage of reprogramming, we identified 90 new potential effectors and repressors of reprogramming. Of the 22 effectors, six new genes belonging to the GPCR family were identified. Detailed analysis of the I.N.: conception/design, collection and/or assembly of data, data analysis and interpretation, figure preparation, manuscript writing, final approval of manuscript; L.C., P.B., and K.G.: collection and/or assembly of data, data analysis and interpretation, final approval of manuscript; L.A. and G.L.: conception/design, financial support, final approval of manuscript; M.L.: conception/design, data analysis and interpretation, manuscript writing, financial support, final approval of manuscript; A.S.: statistical analysis and data interpretation.L.C. declared employment/leadership position with Fujifilm Diosynth Biotechnologies UK Ltd. The other authors indicated no potential conflicts of interest.Figure S1 Z‐score ranked distribution plot of all identified candidates from the RNAi screen. 20 genes with Z scores of <1.65 are considered potential candidate effectors of reprogramming. 68 genes, with Z scores of >1.65 are considered potential candidates that are repressors. The top 3 effectors  belonging to GPCRs family are shown in red bars. In addition, GPR123, GPR116 and GPR37L1 were identified as potential effectors of reprogramming (red bars). Genes, which were identified previously and recognized as primary hits in human cells  cells. Are shown in blue bars:  cells.Click here for additional data file.Figure S2 Densitometry analysis of the Western Blot data presented at Figure4A‐B and D. (A‐A’) Quantification of RhoA and ROCK protein expression; (B) Quantification of the p‐LIMK2 expression; (C) Quantification of the expression of p‐LIMK2, TESK1, p‐COFILIN and p‐MLC2. * p < .05.Click here for additional data file.Figure S3 Densitometry analysis of the Western Blot data represented at Figure . (A) Quantification of the expression of p‐PAXILIN, PAXILIN Total, VINCULIN and E‐cCadherin; (B) Quantification of the expression of p‐FAK and total FAK. * p < .05.Click here for additional data file.EDG5 downregulation during day 8–10 of reprogramming process. (A)Figure S4 Organization of VINCULIN cytoskeleton is not altered upon  Typical confocal immunofluorescence observation of the VINCULIN and E‐Cadherin, in Control and EDG5 siRNA treated groups during OSKM reprogramming at Day10. Scale bar 100 μm.Click here for additional data file.EDG5 downregulation alters the typical morphology of the emerging hiPSCs colonies. (A)Figure S5  Representative bright‐field images of the Control and EDG5 siRNA treated groups during OSKM reprogramming from day 8 till day 10. Black arrows point to the numerous projections in EDG5 siRNA treated RNAi cells. Note sparse distribution and absence of the define edges in EDG5 siRNA treated RNAi, n = 3. Scale bar 100um.Click here for additional data file.Supplement Table 1: XXX.Click here for additional data file.


Synovial sarcoma is a rare tumour, accounting for approximately 2.5–10% of all soft tissue sarcomas. In the thorax, it most often presents as a large, homogenous mass and, most commonly, is the result of extrathoracic tumour metastasis. Here, we report a case of a 73‐year‐old male who presented to the hospital after a motor vehicle collision. Chest computed tomography demonstrated a 2.0 × 2.4 cm left lower lobe pulmonary nodule with endobronchial extension and a 2.5 × 2.1 cm right‐sided kidney mass. He was eventually diagnosed with monophasic synovial sarcoma. To date, only seven other cases of primary pulmonary synovial sarcoma with endobronchial extension have been reported. A review of the cases and literature is discussed. Synovial sarcoma is a rare soft tissue sarcoma. It most commonly occurs in young adults in the extremities, especially in the close proximity of large joints. Pulmonary sarcomas are exceptionally rare, and metastases from extrapulmonary sarcomas are undoubtedly more common than primary pulmonary sarcomas. A diagnosis of synovial sarcoma is dependent on histological, immunohistochemical, and chromosomal translocation testing. Here, we report a case of an incidentally found primary pulmonary synovial sarcoma with endobronchial extension in a 73‐year‐old man and provide a review of the literature.A previously healthy 73‐year‐old man presented to the emergency department after a motor vehicle collision. Computed tomography (CT) of the chest, abdomen, and pelvis demonstrated a 2.0 × 2.4 cm left lower lobe pulmonary nodule with endobronchial extension and a 2.5 × 2.1 cm right‐sided kidney mass  with stable renal mass (2.0 × 2.0 cm). Our leading diagnosis was metastatic renal cell carcinoma (RCC) given the presence of a kidney mass on imaging. A CT‐guided biopsy of his left lower lobe lung mass was performed, and that demonstrated monotonous, densely cellular spindle cells in a vaguely fascicular pattern Fig. . PolymerOur patient did well post‐lobectomy. He subsequently underwent partial nephrectomy, with pathology demonstrating RCC. Follow‐up imaging five months after surgery showed no residual disease in the chest, abdomen, or pelvis.Incidental findings on CT are common. In a series, 44.5% of CTs performed for trauma showed unrelated findings Differential diagnosis also included other tumours such as primary tracheobronchial tumours and lymphoma and benign conditions such as amyloidosis, tuberculosis, and fungal infections that can involve airways and pulmonary parenchyma. Primary tracheobronchial tumours are rare, representing only 0.6% of all pulmonary tumours. Predominant primary malignant tracheobronchial tumours are squamous cell carcinoma, adenoid cystic carcinoma, and mucoepidermoid carcinoma Bronchoscopy is the usual approach to biopsy for pulmonary lesions with an endobronchial extension. However, bronchoscopy fails to provide diagnosis in 10–15% of endoscopically visible lesions, as seen in our case Synovial sarcoma is a rare tumour, accounting for approximately 2.5–10% of all soft tissue sarcomas Primary pleuropulmonary synovial sarcoma (PPSS) only accounts for 0.1% of all lung tumours Primary pulmonary synovial sarcomas are exceedingly rare to present with endobronchial extension. Seven such reported cases are summarized in Table Synovial sarcomas are of four histological subtypes: biphasic, monophasic spindle, monophasic epithelial, and poorly differentiated (round cell) Poor prognostic features of synovial sarcoma include size >5 cm, male gender, age >20 years, extensive tumour necrosis, large number of mitotic figures, neurovascular invasion, and SS18‐SSX1 variant Given the rarity of the disease, there are no treatment guidelines. However, patients are typically treated with multimodal therapy involving surgery, radiotherapy, and chemotherapy Appropriate written informed consent was obtained for publication of this case report and accompanying images.


Rice husks (RHs) as an agro-waste generated from rice production, while its application is limited. This study was designed to introduce a full utilization of rice husks, which extracted the phytochemical at first and then produced cellulose nanocrystals (CNCs) as the use of the residue. Furthermore, the phytochemicals extracted from rice husk was identified and its biological activity, including antioxidant activity, cellular antioxidant activity (CAA) and antiproliferative activity, had been detected as well. Results showed the bound fraction of rice husk had higher antioxidant than common fruit and grain. Free fraction of rice husk deserved to have further analysis in antiproliferative activity due to its low cytotoxicity. The CNCs produced by residue was using delignification process and acid hydrolysis treatments. The chemical composition of the residue obtained after phytochemical extraction was determined. CNCs morphological investigation was performed using an optical microscope and atomic force microscopy (AFM). Our strategy is to achieve a comprehensive utilization of rice husks with both economy and environment benefits. However, due to their tough structure and huge bulk, rice husk has limited applications and was treated by burying underground or open field burning. Thus, it is of great benefit to both the economy and environment to effectively convert rice husk into valuable products.The utilization of waste material and byproduct plays a vital part in solving economic issues at present. Rice husk is the byproduct of rice milling and the major agro-waste from rice production. World production of rice annually was 741 million tons, and approximately 148 million tons of husks were generated, corresponding to 20% of grain weight3. The strong link between antioxidant activity and phytochemical especially phenolics had been demonstrated by numerous reports, which indicate that phenolic compounds could be the dominant factor of antioxidant capacity4. Furthermore, phenolic compounds were found to inhibit the growth of tumor cell lines from human cancers5. Several literatures had been reported the in vitro antioxidant activity and phenolic compounds of rice husk6. However, none of these researches reported the cellular antioxidant activity of rice husk. Moreover, the antiproliferative activity of phytochemical extracted from rice husk was limited.Recently researches regarding obtaining higher values from RHs have been investigated, such as deriving bioactive phytochemicals due to its protection against oxidative damage8. Studies have shown that rice husk contains about 35–40% cellulose, 15–20% hemicellulose, and 20–25% lignin9. Hence, another way to take advantage of rice husk includes the utilization of the cellulose section. Johar et al.10 had successfully produced cellulose fibers and nanocrystals from rice husk using an acid hydrolysis treatment. CNCs has attracted a great deal of attention for its wide application, including reinforced nanocomposites, optically transparent functional materials12. Moreover, their use in films, membranes, catalyst support materials, and functionalized drug carriers has being explored recently13. In the study of Wijaya et al.14, CNC was extracted from passionfruit peels and used as tetracycline antibiotic drug carrier. Therefore, the use of rice husk as the primary source for producing cellulose nanocrystals is promising.In addition, agricultural solid residues with high cellulose content have been proposed to use in cellulose and nanocellulose productionThus, the objective of this study was to introduce a comprehensive approach to utilize rice husk by deriving phytochemicals primarily and gaining CNCs from residue. Moreover, phenolic compounds, antioxidant and antiproliferative activities were evaluated and morphological investigation of the CNCs was conducted. Through this method, phytochemical extracted from rice husk can be a potential ingredient in antioxidants or anticancer products. And the CNC made from the residue can be applied to other areas that generate economic value such as reinforcement materials and drug carriers.et al.2 claimed that bound phenolic contents of rice husk extracted by acetone accounted for 73.3 and 80.0% contributes to total phenolic and flavonoid contents, respectively. In addition, the content of phenolics had little difference compared to the content of flavonoids in free fraction. The total phenolic and flavonoid contents of rice husk were 14.90 ± 0.7 mg GAE/g and 3.08 ± 0.17 mg CE/g, which were consistent with results reported by Seungcheol et al.15. It is known that corn has a higher total phenolic content in whole grain and its total phenolic content was about 2.65 mg GAE/g16, which accounted for about 20% of that in rice husk. The same phenomenon was observed between some kinds of common fruit17, vegetable18 and rice husk, which strongly indicated the potential biological activity in rice husk extracts.The contents of phenolic and flavonoid of rice husk were presented in Table p-hydroxybenzoic acid, caffeic acid, p-coumaric acid and ferulic acid, were identified and quantified and their contents were summarized in Table P-coumaric acid and ferulic acid were the major phenolic acids in bound fraction and the contents were 265.4 ± 2.4 and 33.64 ± 1.01 mg/100 g, respectively. This data was significantly higher than those in brown rice, millet, and other cereals21. Previous reported showed the antiperoxidative potential of p-coumaric acid against adjuvant-induced arthritis in rats22. Janicke et al. also reported ferulic acid and p-coumaric acid could produce antiproliferative effects by acting on the cell cycle of Caco-2 cells23. Our data demonstrated the high content of p-coumaric acid and ferulic acid in rice husk extract, therefore, the antioxidant and antiproliferative activities of phytochemical extracted from rice husk were evaluated below.The HPLC chromatogram of free and bound phenolic acids of rice husk was shown in Fig. in vitro antioxidant and cellular antioxidant activity of rice husk extract, respectively. On the whole, the PSC value  and the median cytotoxicity dose (CC50). The IC50 value of free fraction was 46.90 ± 2.79 mg/mL, while bound fraction had a higher antiproliferative activity at IC50 37.58 ± 4.40 mg/mL and at CC50 67.86 ± 1.62 mg/mL. It was observed that concentration at IC50 of free fraction showed no cytotoxicity, which indicated that the inhibitory effect of free fraction was not due to cytotoxicity but to the antiproliferative effect. Previous researches indicated p-coumaric can inhibit MCF-7 cells proliferation with an EC50 value at 1856.90 ± 70.25 μM27, and ferulic acid at 10 μM concentration can inhibit 21.09% growth of HepG2 cells28. Results indicated that the antiproliferative activitiy of the free fraction of rice husk deserved to have further study due to its low cytotoxicity. The antiproliferative activity of free and bound fraction revealed less consistence to phenolic content and antioxidant activity, which was in accord with the report of Nzaramba et al.29, who found no significant correlations between antiproliferative activity and antioxidant activity, phenolics in both HT-29 and LNCaP prostate cancer cell lines. This may be because of different effects, such as additive, synergistic and/or antagonistic effects among component presented in rice husk extracts. Our result demonstrated that the antiproliferative activities could not be explained only by their phenolic compound contents; Besides, the interaction among components presented in extracts should be considered30.The antiproliferative activities and cytotoxicity were evaluated by HepG2 cells and shown in Fig. 31. Furthermore, more than a hundred particles were chosen randomly and the diameter and length of these were measured, which was shown in Fig. et al. produced CNC-gelatin hydrogels to act as drug carriers, which could be used in controlled drug delivery systems32. Tang et al. also found the potential of polyrhodanine coated cellulose nanocrystals to be antimicrobial agent33.The chemical composition of rice husk was given in Supplementary file. The content of cellulose, hemicellulose, lignin, and ash were detected after phytochemical extracted from rice husk, as shown in Fig. In conclusion, this work presented a fine example of the full application of rice husk, which is expected to promote effective utilization of ago-waste in the near future Fig. . Our resp-coumaric acid, caffeic acid, p–coumaric acid, ferulic acid were purchased from Sigma-Aldrich Ltd. . Chromatographic grade of acetonitrile used for HPLC analysis was obtained from Anpel Ltd. . Human liver cancer cell line HepG2 (ATCC HB-8065) were purchased from ATCC company . WME medium, fetal bovine serum (FBS), Hank’s balanced salt solution (HBSS), Trypsin-EDTA solution and other cell culture reagents were purchased from Gibco Life Technologies Co. .Rice husk was provided by the Rice Research Institute of Guangdong Academy of Agricultural Science, China. Folin-Ciocalteu reagent, fluorescein disodium salt, 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP), 2′,7′-Dichlorofluorescin diacetate (DCFH-DA), (+)-catechin, gallic acid, and chromatographic grade of 34. Briefly, the free phenolics in rice husk were extracted by chilled 80% acetone and then reconstituted using 70% methanol. The bound phenolics were digested and neutralized primarily, and then extracted five times with ethyl acetate. The ethyl acetate fractions reconstituted using 70% methanol. Both extractions and the remaining residue were stored at −40 °C for further use.Phenolics were extracted according to previously reported with a slight modification35 and gallic acid was used as the standard. The flavonoids were determined using the sodium borohydride/chloranil assay (SBC)36, using catechin as the standard. Data is expressed as milligram gallic acid equivalent per gram (mg GAE/g) and milligram catechin equivalent per gram (mg CE/g) of rice husk in triplicate, respectively.The phenolic content was measured by Folin-Ciocalteu method as reported previously37 with slight modifications. The samples were analyzed by an HPLC system , which was equipped with a 4.6 × 250 mm, 5 μm Sunfire C18 reversed phase column . The mobile phase consisted of purified water with 0.1% trifluoroacetic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 1.0 mL/ min using gradient elution as follows: 0–5 min 10% B, 5–20 min 25% B, 20–25 min 35% B, 25–40 min 90% B, 40–50 min 10% B, 50–60 min 10% B at 280 nm. Phenolic compounds in rice husk were identified by comparing the retention time and detected using an external standard method. Measured values were expressed as milligrams per 100 g of rice husk (mg/100 g).The identification of phytochemical was performed according to the method reported previouslyin vitro antioxidant activity was evaluated by the peroxyl radical scavenging capacity (PSC) assays as described previously25. Ascorbic acid (ASA) was used as the standard. In brief, diluted samples and standard solutions were mixed with equivalent volume DCFH–DA and followed by ABAP. The reaction was performed at 485 nm excitation and 538 nm emission by the multi-mode microplate reader . Results were calculated as micromoles of ascorbic acid equivalent per gram (μmol ASA equiv./g) and the data were reported as mean ± SD (n = 3).The 2.Human liver cancer cells HepG2 were cultured in WME medium supplemented with 2 mM L-glutamine, 10 mM Hepes, 5% fetal bovine serum, 5 μg/mL insulin, 50 units/mL penicillin, 0.05 μg/mL hydrocortisone, 50 μg/mL streptomycin and 100 μg/mL gentamycin at 37 °C in a humidified atmosphere of 5% CO26. Quercetin was used as the standard. In brief, HepG2 cells were incubated at a density of 6.0 × 104 cells/well for 24 h and followed by medium containing samples which were under the cytotoxicity dose plus 25 μM DCFH-DA. ABAP was added after incubated for 1 h and the fluorescence intensity was monitored at 485 nm excitation and 535 nm emission every 5 min for 1 h (Molecular Devices). The CAA value was presented as micromoles of quercetin equivalent per 100 g of rice husk (μmol QE/100 g).The CAA assay was conducted as described previously38. Briefly, cells were seeded at a concentration of 2.5 × 104 cells/well. For cytotoxicity test, cells were plated at the concentration of 4 × 104 cells/well. After incubated, a series concentration of diluted extracts in growth medium was added into each well and then stained for viable number counting. Absorbance was measured at 570 nm on Multi-Mode Microplate Reader (Molecular Devices). Each sample was measured at least three times. The cytotoxicity and antiproliferative activity of phytochemicals were assessed by half maximal cytotoxicity concentration (CC50) and median inhibition dose (IC50), respectively, which were expressed as mg/ml .The antiproliferative activity was measured in HepG2 cells by using a methylene blue assay that has been reported previously39 was used to measure the content of cellulose and hemicellulose and the lignin content was obtained according to T222 OS-8340. The ash content was determined after pyrolysis of the dry sample in a furnace at 525 °C for 6 h, following the standard procedure of T211 om-0241.The chemical composition of the residue was determined according to the Technical Association of Pulp and Paper Industry (TAPPI). T203 OS-7410 with slight modifications, which including delignification process and acid hydrolysis treatment. The residue was treated with the alkali solution  and cooked at 165 °C for 90 min. Then the solid was washed with distilled water several times. Then, hydrogen peroxide  and distilled water were added to ensure 12% concentration of pulp, followed by bleaching at 80 °C for 1 h. The mixture was filtered using excess distilled water. The acid hydrolysis treatment was conducted at 45 °C using concentrate sulphuric acid for 120 min under continuous stirring. The hydrolyzed material was washed by centrifugation at 10,000 rpm for 10 min. This step was repeated several times. And then the suspension was dialyzed using distilled water for several days until constant pH in the range of 5–6 was reached.The preparation of cellulose nanocrystals from residue was conducted according to previously reported42. Lengths and diameters were obtained using the section analysis tool of the NanoScope Analysis software  from height mode AFM image. More than a hundred CNCs were randomly measured in order to determine the average length and diameter.A drop of the cellulose fiber suspension after bleaching was placed on a glass slide for imaging with an optical microscope (OLYMPUS BX51). A drop of CNC suspension was placed on a piece of freshly cleaved mica, dried and then analyzed with Atomic Force Microscope P-values < 0.05 were regarded as significant.The data, expressed as the mean ± SD, included at least three replicates per sample. ANOVA and Tukey’s test were performed using Statistical Package for the Social Sciences . Graphical representations were performed using Sigma plot version 12.3 . supplement material


Here, leaf gas exchange, photosynthetic light–response and CO2–response curves, and total carbon (C) and nitrogen (N) accumulation in hot pepper leaves were investigated under three ECiw levels  and two LFs treatments (0.17 and 0.29).The modification effect of leaching fraction (LF) on the physiological responses of plants to irrigation water salinity  in higher ECiw, whereas the LF did not affect the intrinsic WUE. Carbon isotope discrimination was inhibited by ECiw, but was not affected by LF. ECiw reduced the carboxylation efficiency, photosynthetic capacity, photorespiration rate, apparent quantum yield of CO2 and irradiance–saturated rate of gross photosynthesis; however, LF did not influence any of these responses. Total C and N accumulation in plants leaves was markedly increased with either decreasing ECiw or increasing LF.Leaf stomatal conductance was more sensitive to ECiw depressed leaf gas exchange, photosynthesis capacity and total C and N accumulation in leaves, but enhanced intrinsic WUE. Somewhat surprisingly, higher LF did not affect the intrinsic WUE but enhanced the total C and N accumulation in leaves.The present study shows that higher EC Capsicum annuum L.), which is now one of the most widely grown crops in the world. In 2016, global pepper production (fresh and dry) from some 4 million ha was estimated at some 39 million tonnes, increasing by some 30% in the last decade .The PFD curve :6\documen–Ci curves were made at CO2 levels of 400, 250, 150, 100, 50, 500, 700, 1000 and 1500 μmol mol− 1 at a fixed PPFD of 1200 μmol m− 2 s− 1. The Pn were plotted against the respective Ci. A non–rectangular hyperbola curve was used to simulate Pn–Ci curve [ε is carboxylation efficiency (mol m− 2 s− 1); Pn sat is the photosynthetic capacity (μmol CO2 m− 2 s− 1); and Rp is the rate of photorespiration (μmol CO2 m− 2 s− 1).Measurements of PCi curve , 60:7\doiw and LF on gas exchange parameters, intrinsic WUE, Δ13C, δ15N, C content and total C and N accumulation, dry biomass of leaves and roots, Na+ content, the parameters obtained from the Pn–PPFD and Pn–Ci curves. All analyses were conducted in the SPSS software package . Correlations between the measured parameters were determined with regression analyses. The slopes of the relationships between Pn and gs under different ECiw levels and LFs were tested by a standard analysis of covariance (ANCOVA). Pn was analyzed through a General Linear Model (GLM) of the natural logarithm of gs. The ECiw (or LF) and the interaction with the linear predictor were included to test for differences in slope. If there was no significant interaction between ECiw (or LF) and linear predictor, the slopes were assumed to be the same.Two-way analysis of variation using the general linear model-univariate procedure was performed to assess the effects of the EC


Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. J Orthop Res 36:3188–3195, 2018.Blood vessel and neurite ingrowth into the degenerating intervertebral disc (IVD) are related to pain. In reported studies, notochordal cell (NC)‐conditioned medium (NCCM) induced a regenerative response of nucleus pulposus (NP) cells, but also inhibition of neurite and vessel formation. NC matrix (NCM) derived from NC‐rich NP tissue, induced even stronger anabolic effects than NCCM. Thus, the aim was to investigate whether NCM has similar anti‐neurogenic and ‐angiogenic properties as NCCM. NCM and NCCM where produced from porcine NC‐rich NP tissue. Human umbilical vein endothelial cells (HUVECs) were cultured in base medium , NCCM (produced at 300 and 400 mOsm), NCM, or with chondroitin sulfate  in angiogenesis‐inducing medium, after which vessel length was measured. Although CS alone inhibited vessel growth, NCCM (both osmolarities) stimulated vessel formation by HUVECs. NCM did not affect vessel growth relative to BM. SH‐SY5Y cells were cultured in BM, NCCM, and NCM on poly‐D‐lysine coated and polystyrene surfaces, and analyzed for neurite length and percentage of neurite expressing cells. On coated surfaces, neither NCCM nor NCM affected neurite growth. On a polystyrene surface, NCCM and NCM induced a higher number of neurite‐expressing cells. NCCM's previously reported anti‐angiogenic and ‐neurogenic effects were not observed in this study. Although addition of CS inhibited HUVEC vessel formation, other factors may be present in NCCM and NCM that affect neurite and vessel growth. Therefore, future studies testing an NC‐based regenerative strategy should carefully assess the risk of such adverse effects in an in vivo setting. © 2018 The Authors.  Intervertebral discs (IVDs) are the structures in between the vertebrae that transmit loads and provide flexibility to the spine. The IVD consists of a hydrated core, the nucleus pulposus (NP), which is circumferentially confined by the annulus fibrosus (AF). Negatively charged glycosaminoglycans (GAGs), bound within proteoglycans (PGs), indirectly attract water into the NP. Thereby the swelling pressure inside the NP rises, which is crucial for sustaining high compressive loads. During IVD degeneration, the swelling pressure decreases due to a loss of PGs, and compressive loads are increasingly exerted on the AF, possibly leading to annular tears and further degenerative changes.Annular tears, which are more frequently found with increasing age,Previously, nerve growth factor (NGF) was suggested as the driving factor for nerve ingrowth in the IVD.Notochordal cells (NCs), and more specifically NC‐conditioned medium (NCCM), has received considerable attention due to its IVD regenerative potential. NCCM has been produced by incubating either isolated NCs or NC‐rich NP tissue in culture medium for several days. The factors secreted by NCs induced an anabolic response in vitro of nucleus pulposus cells (NPCs) and bone marrow‐derived mesenchymal stem cells (BMSCs) in various animal models.Recently, we proposed an alternative to NCCM, that is, natural NC‐derived matrix (NCM), which can be produced by lyophilizing and pulverizing porcine NP tissue that was mostly populated by NCs. By using NCM, the NCCM incubation period, in which bioactive factors may be subject to degradation and inflammatory factors may be produced as a result of free swelling conditions+, BD Biosciences, Breda, the Netherlands) and 50 μg/ml ascorbic acid‐2‐phosphate  at 30 ml medium per gram of tissue as described previously.2 and 5% O2 after which the NP tissue was removed from the medium by filtration with a 70 μm pore size cell strainer. Subsequently, the medium was filtered through a 3 kDa cut‐off filter tube , and the strained material was resuspended in the same amount of fresh culture medium. For HUVEC culture, this material was resuspended in Medium 200 . For SH‐SY5Y cell culture, the strained material was resuspended in a 1:1 mixture of Eagle's minimum essential medium (EMEM) and F‐12 K medium (both from Life Technologies). To produce NCM, the other half of the NP tissue of each spine was lyophilized overnight and pulverized to a fine powder using a microdismembrator . NCCM and NCM (n = 5 donors) were aliquoted and stored at −80°C until further use.NC‐rich NP tissue was harvested within 6 h after slaughtering from the cervical, thoracic, and lumbar IVDs of five young (approximately 3‐month‐old) pigs, obtained from the local abattoir. The IVDs were opened aseptically, and the NP was taken out, careful not to include AF tissue. To produce NCCM, half of the NP tissue from each spine was incubated in high glucose Dulbecco's modified eagle's medium  supplemented with 1% penicillin/streptomycin , 1× insulin transferrin selenium . A dimethyl methylene blue (DMMB) assay was performed,NCCM contained 902 ± 112 μg GAG/ml, whereas NCM contained 472 ± 22 μg GAG/mg NP tissue (average and standard deviation from five different donors). Therefore, for NCM cultures approximately 2 mg NCM was dissolved in 1 ml BM, to apply a similar GAG concentration between NCCM and NCM. The protein concentration in NCCM was 614 ± 69 μg/ml and that for NCM was 290 ± 13 μg/mg (average and standard deviation from five different donors). Hence, normalization for GAG content, resulted in an approximately similar protein concentration between NCCM and NCM employed in the culture experiments.2 in three different medium conditions: BM, NCCM with the same supplements as BM or NCM added to BM . The NCM concentration in medium was adjusted to obtain similar GAG content to NCCM. After 24 h, cells were incubated for 15 min with 10 μM Calcein AM and 10 μM propidium iodide. Per well, three pictures were taken at 4× magnification using a fluorescence microscope . Per sample, the images were taken at approximately the same locations, covering the largest part of the well without overlap. To assess whether effects of NCCM or NCM may be dependent on HUVEC donor variation, the same experiment was repeated with HUVECs (used at passage 2) from two separate additional donors .HUVECs  were expanded in basal medium  consisting of Medium 200 supplemented with low serum growth factors  and 1% P/S until 80% confluency. A 24‐well plates were coated with 120 μl/well Geltrex LDEV‐free , after which HUVECs at passage two were seeded at 80,000 cells per well. Cells were incubated for 24 h at 37°C and 5% COn = 5 biological repeats for each medium condition). Physiological osmolarity NCCM was produced similar to basal NCCM, but with addition of a 1% salt solution (5 M NaCl/0.4 M KCl) to the medium during the 4 day incubation period, increasing medium osmolarity by 108 mOsm as previously described.Since osmolarity changes are known to affect NC phenotype,2 in expansion medium to allow them to attach. After 24 h, medium was aspirated and the wells were washed twice with phosphate buffered saline  to remove serum. Thereafter, wells were divided into 4 different medium conditions: Basal medium (BM: EMEM:F‐12 K (1:1) supplemented with 1% P/S), positive control medium  to induce neurite growth, NCCM + PDGF and NCM + PDGF . NCCM and NCM contained the same supplements as BM + PDGF. The NCM concentration in medium was adjusted to obtain similar GAG content between NCCM and NCM. Cells were incubated for 48 h at 37°C and 5% CO2. After 48 h, 10 phase contrast images per well were taken at 10× magnification using a bright‐field microscope . The images were taken at random location, ensuring no overlap. To determine the effect of the culture surface on which SH‐SY5Y cells were incubated, this experiment was repeated  with polystyrene six‐well plates.SH‐SY5Y cells (Sigma) were cultured up to 70% confluency in EMEM:F‐12 K (1:1) supplemented with 10% fetal calf serum  and 1% P/S. Subsequently, cells were seeded at a density of 50,000 cells per well in a poly‐D‐lysine coated six‐well plate that had been employed before for similar experiments exploring the anti‐neurogenic effects of NCCM.HUVEC vessel length as well as SH‐SY5Y cell neurite length were measured using ImageJ Simple Neurite Tracer. For HUVECs, only the vessels themselves were measured, not taking into account the branch points. To ensure that actual vessels were measured, only processes larger than 50 μm were taken into account. The total vessel length from three pictures per well was expressed per surface area. For SH‐SY5Y cells, only cells and neurites that were in the field of view in their entirety were counted and measured. For branching neurites, only the longest branch was measured, and only neurites longer than 20 μm were taken into account. From this, the percentage of neurite‐expressing cells and average neurite length were calculated.t‐test post hoc testing with Bonferroni corrections. For non‐normally distributed data, a Kruskal–Wallis test was used, followed by Mann–Whitney U tests with post‐hoc Bonferroni corrections. Statistical significance was accepted for P values <0.05.Statistical analysis was performed with Statistical Package for Social Sciences . Normality was tested using the Shapiro–Wilk test. Normally distributed data were analyzed using a one‐way analysis of variance (ANOVA), followed by independent After 24 h of incubation, only limited cell death was observed, with little difference between experimental groups. Vessel formation was observed in all groups Fig. . Howevern = 2 biological repeats per NCCM/NCM donor). Although there were donor differences in total vessel length, the overall pattern between culture groups was similar to the initial experiment; vessel length increased with NCCM  compared to BM , whereas it was unaffected by NCM , similar as observed with the pooled HUVECs of the initial experiment.To assess whether this finding could be due to HUVEC donor variation in response to NCCM and NCM, HUVECs from two additional donors were subjected to the same experiment  have been detected in porcine NCCM.The question still remains what causes the different results between the current study and that by Cornejo et al. HUVEC donor variation is not likely, as a stimulatory effect on vessel formation was observed for all three independent HUVEC donors tested in the current study. Furthermore, addition of CS to the HUVEC cultures had comparable inhibitory effects on vessel formation, indicating that the HUVECs do respond to proper anti‐angiogenic stimuli. The difference between studies is more likely NCCM‐related. NCCM was generated from NP tissue from 12‐week‐old pigs in our study, whereas NP tissue was harvested from 6‐ to 8‐week‐old pigs in the study by Cornejo et al. The small age difference may have contributed to the contradicting results. Additionally, it cannot be excluded that NCCM produced from NP tissue of different pig breeds has differential effects on HUVEC vessel formation. Furthermore, in previous studies that investigated NCCM's anti‐angiogenic and ‐neurogenic potential, NCCM was produced at 1% oxygen, whereas it was produced at 5% oxygen in the current study. Previous studies comparing NC behavior in normoxia (21% oxygen) and hypoxia (1–3.5% oxygen) found oxygen‐related differences in NC morphology, gene expression and lactate production,While NCCM has been reported to inhibit neurite growth by SH‐SY5Y cells,In conclusion, application of a NC‐based regenerative treatment strategy should, in the first place, provide an anabolic response to the resident NPCs to restore the IVD to a healthy state. Ideally, concomitant inhibition of neurite and vessel ingrowth may contribute to pain management. Such an effect was however not established in the current study. In fact, although CS indeed inhibited vessel formation, other factors are present in NCCM and/or NCM that may affect blood vessel formation and neurite growth. Such adverse effects pose a potential risk of an NC‐based regenerative therapy and should be carefully evaluated in more physiologically relevant models, for example, using a chorioallantoic assay in the developing chick embryo for angiogenesis and dorsal root ganglia rather than the SH‐SY5Y cell line for evaluation of neurite growth.SdV, MvD, and KI designed the HUVEC and SH‐SY5Y cell culture experiments. SdV prepared NCCM and NCM. SdV and MvD set up experiments and took neurite and vessel pictures. SdV analyzed vessel length, neurite length, and number of neurite‐expressing cells. SdV, MvD, MT, and KI interpreted these data. SdV prepared the manuscript, and all authors contributed and provided critical feedback. KI oversaw the study, and gave the final approval for the submitted version of the manuscript.


RUNX2 located on the short arm of chromosome 6p21. In general, there are intrafamilial variations in height among CCD patients. Few studies have reported data on recombinant human growth hormone (rhGH) treatment for patients with CCD; thus, it remains to be elucidated whether rhGH treatment can improve short stature. Here, we report a case of a 6-year-old girl with CCD who has growth hormone deficiency (GHD) and a novel mutation of RUNX2.Cleidocranial dysplasia (CCD) is a rare skeletal disorder with autosomal dominant inheritance that is characterized by hypoplastic clavicles, delayed closure of the cranial sutures, dental abnormalities, and short stature, among other features. The responsible gene for CCD is At 5 years of age, this patient was diagnosed with GHD and rhGH treatment was initiated. Thereafter, she was diagnosed with CCD due to the presence of hypoplastic clavicles and an open fontanelle, which was also observed in her mother and brother. She responded well to rhGH treatment; her height improved from − 3.2 SD to − 2.4 SD after 13 months.A detailed patient history and physical examination are necessary for the early diagnosis of CCD. Similarly, to ascertain the effect of rhGH treatment, careful evaluation of the patient’s final height post-therapy is needed. RUNX2), also known as core-binding factor alpha-1 (CBFA1), located on 6p21, can cause CCD [Cleidocranial dysplasia (CCD) is a rare skeletal disorder with autosomal dominant inheritance, which is characterized by abnormalities in systemic membranous ossification. The characteristic symptoms are hypoplastic clavicles, delayed closure of the fontanelles, delayed eruption of primary and permanent dentition, and short stature. Considerable phenotypic variation has been reported, even within the families . The freause CCD . Herein,The patient was born uneventfully at a gestational age of 40 weeks and 3 days. Her weight, length, and head circumference at birth were 3112 g (− 0.15 SD), 48.5 cm (− 0.89 SD), and 33 cm (− 0.62 SD), respectively. There was no abnormality in screening for inborn errors of metabolism. Her father’s height was 171 cm, while her mother’s was 157 cm. There was no family history of endocrine disorders or metabolic bone diseases. When she was 3 months old, she was referred to our hospital because of a large anterior fontanelle. Her head circumference was within normal range; thus, no further testing was done.When she was 3 years and 9 months old, she was presented to our hospital with short stature. Her height and weight were 11.5 kg (− 2.24 SD) and 88.3 cm (− 2.69 SD), respectively. Blood test revealed a low level of serum insulin-like growth factor-1 (IGF-1) (24 ng/mL). She had poor growth velocity, with her height SD score decreasing to − 3.2 SD at 5 years and 5 months of age. GH provocation tests were performed to evaluate for GHD. The maximal GH responses to arginine and GHRP-2 were 3.26 ng/mL and 2.97 ng/mL, respectively. Based on these results, she was diagnosed with GHD, and recombinant human GH treatment (0.175 mg/kg/week) was started. Her wrist X-ray showed her supposed bone age at 5 years and 8 months, according to the Japanese TW2-RUS method, was only 3 years and 10 months. Brain MRI showed no morphological abnormalities of the pituitary gland.RUNX2 gene was analyzed by Sanger sequencing. Genomic DNA was extracted from peripheral blood leukocytes, and polymerase chain reaction (PCR) was performed to amplify each exon using the primer sets shown in Additional file RUNX2 gene analysis revealed a heterozygous mutation ‘p. Q282X’ (c. 844C > T)  Fig. . The patRUNX2 located on the short arm of chromosome 6p21. Genetic analysis reveals a heterozygous mutation of RUNX2 in almost 70% of patients [RUNX2 is a transcription factor belonging to the Runx family , and is expressed in osteoblasts and chondrocytes. RUNX2 is essential for the differentiation of multipotent mesenchymal cells to osteoblasts. It cooperates with Sp7 and canonical Wnt pathway [RUNX1 and RUNX3 can compensate for the RUNX2 function to some extent, even if the expression of RUNX2 is halved due to mutation. However, for intramembranous ossification, RUNX2 is crucial. Thus, open fontanelles and sutures, together with hypoplastic clavicles, are characteristic findings in CCD since the formation of these bones is related to intramembranous ossification [RUNX2 could lead to various phenotypic features even in the same family, as previously reported [CCD is a rare skeletal disorder characterized by hypoplastic clavicles, delayed closure of fontanelles, delayed eruption of primary and permanent dentition, and short stature. It is diagnosed clinically. The prevalence is 1 in 1,000,000. The gene responsible for pathogenesis is patients . RUNX2 ireported .RUNX2 mutation in the present case is a novel nonsense mutation with intact Runt domain. Her short stature could be attributable to a nonsense mutation. However, her mother with the same mutation has a normal height. This is compatible with a previous paper reporting probable intrafamilial variabilities in height [RUNX2 for the differentiation of osteoblast [RUNX2 decreases due to gene mutation, GH signaling does not work properly and leads to growth hormone deficiency. Additionally, the effect of rhGH might vary depending on the activity of RUNX2 based on the genotype. Randomized controlled studies are needed to evaluate the effects of rhGH therapy inpatients with CCD.The present case has a short stature (− 3.2 SD), one of the characteristic features of CCD. The final height of patients with CCD is reported to be − 1.47 SD for men and − 1.89 SD for women [n height , while on height . Howevern height . The effteoblast . Thus, iRUNX2 and GHD. The family history should be evaluated in detail, and characteristic features of CCD, such as clavicle hypoplasia and open fontanelles, should be checked for when a patient with short stature is encountered. There is a risk of misdiagnosis because of mild symptoms due to variation in phenotypes. Although rhGH treatment was effective in our case, further studies, such as randomized controlled trials, are warranted to reveal the effectiveness and safety of rhGH treatment in CCD patients.In the present study, we have reported a case of CCD with a novel mutation in Additional file 1: Table S1. Primer sets used for the RUNX2 mutation analysis.