Petromyzon marinus) to complete their life cycle. The synthesis and release of a releaser/primer pheromone 3-keto petromyzonol sulfate (3kPZS) by spermiating males have been well characterized. 3kPZS evokes sexual behaviors in ovulatory females, induces immediate 3kPZS release in spermiating males, and elicits neuroendocrine responses in prespawning adults. Another primer pheromone released by spermiating males, 3-keto allocholic acid (3kACA), antagonizes the neuroendocrine effects of 3kPZS in prespermiating males. However, the effects of 3kACA and 3kPZS on pheromone production in prespawning adults is unclear. To understand the foundation of pheromone production, we examined sea lamprey bile salt levels at different life stages. To investigate the priming effects of 3kACA and 3kPZS, we exposed prespawning adults to vehicle or synthetic 3kACA or 3kPZS. We hypothesized that endogenous bile salt levels are life-stage and sex dependent, and differentially affected by 3kACA and 3kPZS in prespawning adults. Using ultra-performance liquid chromatography tandem mass spectrometry, we found that sea lampreys have distinct mixtures of bile salts in the liver and plasma at different life stages. Males usually had higher amounts of bile salts than females. Petromyzonamine disulfate was the most abundant C27 bile salt and petromyzonol sulfate was the most abundant C24 bile salt. Waterborne 3kACA and 3kPZS exerted differential effects on bile salt production in the liver and gill, their circulation and clearance in the plasma, and their release into water. We conclude that bile salt levels are life-stage and sex dependent and differentially affected by primer pheromones.Pheromonal bile salts are important for sea lampreys ( Summary: Sea lamprey bile salts are pheromones produced in a sex- and life-stage-dependent manner. Primer pheromones differentially affect bile salt production, circulation, clearance and release. Petromyzon marinus) is a vertebrate residing at an important evolutionary juncture where jawed vertebrates (Gnathostomata) branch out from jawless vertebrates (Agnatha). Chemical communication is important for sea lampreys to complete their life cycle, including the prespawning migration aided by migratory pheromones released from larvae  or 24-carbon (C24) backbone  and petromyzonsterol disulfate (PSDS); (2) C24 bile salts with multiple double bonds: 3,12-diketo-4,6-petromyzonene-24-sulfate (dkPES) and several bile salt-like dienones; and (3) a group of structurally related C24 bile salts: 3kPZS and its presumptive precursors petromyzonol sulfate (PZS), petromyzonol (PZ), 3-keto petromyzonol (3kPZ), allocholic acid (ACA) and 3kACA  were collected from Saint Marys River in 2014 and 2015 by agents of the US Fish and Wildlife Service  and Department of Fisheries and Oceans Canada Sea Lamprey Control Centre . Larval sea lampreys  were collected by the survey crew of US Fish and Wildlife Service, Ludington Biological Station  in 2016. They were sent to US Geological Survey, Hammond Bay Biological Station, Great Lakes Science Center . Prespermiating males were visually identified and separated from preovulatory females by carefully applying pressure to the lower abdomen to feel for eggs. Ovulatory females and spermiating males were obtained by holding prespawning animals in cages (0.25–1.0 m3) in the lower Ocqueoc River  to induce maturation (https://www.ncbi.nlm.nih.gov/books/NBK54050/pdf/Bookshelf_NBK54050.pdf).Prespawning migratory turation . They we2H5]3kPZS were custom-synthesized from Bridge Organic Inc. .Acetonitrile (ACN), ethanol, methanol and water were HPLC grade . Ammonium acetate (HPLC grade) and ethyl 3-aminobenzoate (MS222) were purchased from Sigma-Aldrich Co. . PZ and 3kPZ were purchased from Cayman Chemical . ACA, 3kACA, dkPES, PZS, 3kPZS, PADS, PSDS and deuterated 3kPZS; 10 µl per sample). The final volume of the liver homogenate was brought up to 1.5 ml by adding 1100 μl of 75% ethanol. To dissolve and extract bile salts, liver homogenate (in 75% ethanol) was incubated at room temperature overnight, and centrifuged twice at 15,800 g, 4°C for 20 min. The supernatant was transferred to a new tube and freeze-dried using a CentriVap Cold Trap Concentrator . Plasma samples  were processed similarly but without homogenization. Water samples were freeze-dried directly. Samples were then reconstituted with 1 ml 50% methanol (in HPLC water) and transferred to autosampler vials before UPLC-MS/MS analyses.Tissue samples were weighed  and homogenized with 400 μl of 75% ethanol (in HPLC water) with 1 ng internal standards  Xevo TQ-S mass spectrometer coupled with a Waters ACQUITY H-Class UPLC system was used. A Waters BEH C18 column  was used with the column oven temperature set at 30°C. Mobile phase A was 10 mmol l–1 ammonium acetate in water, and mobile phase B was ACN. Separation was achieved using the following gradient program at a flow rate of 200 l min−1 for 15 min: 3% B for 0.5 min, increased to 40% B from 0.5 to 10 min, increased to 95% B from 10 to 12 min, maintained at 95% B from 12 to 13 min, and returned to 3% B from 13 to 15 min. The injection volume was 10 µl. Mass spectra were acquired using electrospray ionization in negative ion mode with multiple reaction monitoring. The capillary voltage, extractor voltage and rf lens settings were 3.17 kV, 4 V and 0.3, respectively. The flow rates of cone gas and desolvation gas were 20 and 400 l h−1, respectively. The source temperature and desolvation temperature were set at 110 and 350°C, respectively. Collision-induced dissociation employed argon as the collision gas at a manifold pressure of 2×10−3 mbar (200 Pa), and collision energy and source cone potentials were optimized for each transition using Waters QuanOptimize software. Data were acquired with MassLynx 4.1, and calibrated and quantified by QuanLynx software.Solutions for the calibration standard curve and the internal standard were prepared freshly from 1 mg ml−1 to 0.02 ng ml−1 in plasma and tissues, and 0.4 pg ml−1 to 0.05 ng ml−1 in water (Extraction efficiency (recovery) for sea lamprey bile salts ranged from 76.7±6.2% to 85.9±7.0% in gill homogenates; 76.3±5.2% to 98.7±8.6% in liver homogenates; 70.5±5.5% to 89.1±6.7% in plasma; and 84.1±3.2% to 90.3±2.2% in water , 2015. Tin water , 2015.post hoc tests (Dunn multiple comparison post hoc tests with Bonferroni correction) were performed if the ANOVA test was significant (P<0.05). Some prespermiating males began spermiating at the time of sample collection, and these data were separated as groups of spermiating males. Liver sample size: n=6 per group except prespermiating males in the control (n=11), 3kACA-treated (n=12) or 3kPZS-treated (n=9) groups, and spermiating males in the 3kACA-treated group (n=3). Plasma sample size: preovulatory females in the control group n=8, 3kACA-treated group n=8 and 3kPZS-treated group n=10; prespermiating males in the control group n=13, 3kACA-treated group n=12 and 3kPZS-treated group n=7; spermiating males in the control group n=23, 3kACA-treated group n=16 and 3kPZS-treated group n=26. Gill sample size: n=6 per group except prespermiating males in the 3kPZS-treated group (n=3), and spermiating males in the control (n=11), 3kACA-treated (n=9) and 3kPZS-treated (n=12) groups. Water sample size: n=2 (repeated measures). Sample size varied according to the availability of animals at the time. Correlation analyses were performed using the life-stage data to examine the relationships between hepatic and plasma bile salt concentrations from the same animals.One-way analysis of variance (ANOVA) was used to compare the bile salt concentration in tissues and plasma  among different life stages or different pheromone treatment groups. Bonferroni/Dunn P<0.0001), except PZ , likely as a result of the accumulation of bile salts in the gall bladder  and PADS  showed significant sex differences. Spermiating males had higher amounts of hepatic PZS  and PADS  than preovulatory females . Prespermiating males had the highest amounts of plasma PZS , significantly different from those in preovulatory females  and spermiating males . Prespermiating males also had the highest amounts of plasma 3kPZS , significantly different from those in preovulatory females  and spermiating males . Spermiating males had the highest amounts of plasma PADS , significantly different from those in preovulatory females  and prespermiating males . None of the adults had detectable amounts of plasma 3kACA and 3kPZ, and ovulatory females did not have detectable amounts of plasma ACA. Plasma PSDS was only detected in ovulatory females, plasma PZ was only detected in prespermiating males, and plasma dkPES was only detected in spermiating males . Interestingly, plasma dkPES concentration was also positively correlated with hepatic PZS and PADS concentrations (P<0.05).To determine the relationships between hepatic and plasma bile salt concentrations, correlation analyses were performed. As expected, hepatic bile salt concentrations were positively correlated with their plasma concentrations, and bile salts in the same branch of the synthetic pathway were positively correlated with each other , P<0.05.trations , P<0.05.P=0.0167; P=0.0332; 24 bile salts (PZ and/or 3kACA) in the liver and gill but facilitate the production of C27 bile salt PADS in the gill.Exposure to waterborne 3kACA led to a reduction of hepatic 3kACA A and plaExposure to waterborne 3kACA increased hepatic 3kPZ A, whereaIn spermiating males, pheromone exposure had no significant effects on hepatic bile salt levels A. Exposu5-C27-steroid dehydrogenase/isomerase (HSD3B7) activity (We found that sea lampreys produced distinct mixtures of bile salts at different life stages. It is likely that different bile acid synthetic enzymes were upregulated or downregulated at specific life stages. For example, sea lamprey larvae did not produce detectable amounts of dkPES, probably because of the lack of 3β-hydroxy-Δactivity . In contactivity , these bWe found life-stage and sex differences in bile salt levels in sea lampreys. This phenomenon persists in the spawning stage where only spermiating males release a large quantity of bile salts as sex pheromones, whereas ovulatory females barely release any detectable amounts of bile salts , 2017b. We found that pheromonal bile salts exerted differential effects on bile salt biosynthesis, circulation and clearance. In addition, 3kPZS only induced bile salt release in spermiating males. PZS was the first bile salt identified in sea lamprey larvae , and it CYP7A1 gene expression. Therefore, the JNK/c-Jun signaling pathway inhibits bile acid synthesis and protects hepatocytes against the toxic effect of inflammatory agents (Bile acid synthesis is regulated by multiple feedback inhibition mechanisms in mammals . One mecy agents . As 3kACy agents ,b, it isOne interesting finding in this study is that not all bile salts were produced continuously in the liver, and not all bile salts were released into the circulation and water. In prespawning sea lampreys, 3kACA and 3kPZ are more likely metabolic intermediates produced in and confined to the liver, whereas other bile salts are more likely produced in the liver and exert remote functions via the circulation. In spermiating males, 3kPZS was the major pheromonal bile salt released into the water accompanied by lesser amounts of PZS, ACA and 3kACA, and the release could be influenced by exogenous 3kPZS. Interestingly, sea lampreys contain other organs that can produce, modify or release bile salts, such as the gill and intestine . TherefoIn summary, bile salt levels are life-stage and sex dependent in sea lampreys. Pheromonal bile salts exert differential effects on bile salt production, transportation and clearance in prespawning adults, whereas only 3kPZS induces the water release of bile salt in spermiating males.


Second-generation antipsychotic (SGA) drugs have been associated with the development of type 2 diabetes and the metabolic syndrome in patients with schizophrenia. In this study, we aimed to investigate the effects of two different SGA drugs, olanzapine and aripiprazole, on metabolic state and islet function and plasticity.−1 day−1) for 6 months. Glucose and insulin tolerance tests, in vivo glucose-stimulated insulin secretion and indirect calorimetry were performed at the end of the study. The effects of SGAs on beta cell plasticity and islet serotonin levels were assessed by transcriptomic analysis and immunofluorescence. Insulin secretion was assessed by static incubations and Ca2+ fluxes by imaging techniques.We analysed the functional adaptation of beta cells in 12-week-old B6;129 female mice fed an olanzapine- or aripiprazole-supplemented diet , glucose intolerance (p<0.01) and impaired insulin secretion (p<0.05) vs mice fed a control chow diet. Aripiprazole, but not olanzapine, induced serotonin production in beta cells vs controls, likely by increasing tryptophan hydroxylase 1 (TPH1) expression, and inhibited Ca2+ flux. Of note, aripiprazole increased beta cell size (p<0.05) and mass (p<0.01) vs mice fed a control chow diet, along with activation of mechanistic target of rapamycin complex 1 (mTORC1)/S6 signalling, without preventing beta cell dysfunction.Treatment of female mice with olanzapine or aripiprazole for 6 months induced weight gain (Both SGAs induced weight gain and beta cell dysfunction, leading to glucose intolerance; however, aripiprazole had a more potent effect in terms of metabolic alterations, which was likely a result of its ability to modulate the serotonergic system. The deleterious metabolic effects of SGAs on islet function should be considered while treating patients as these drugs may increase the risk for development of the metabolic syndrome and diabetes.The online version contains peer-reviewed but unedited supplementary material available at 10.1007/s00125-021-05630-0. In recent years, an increased incidence of type 2 diabetes in patients taking chronic pharmacological treatment has been reported . In pati1A, 5-HT2A, 5-HT2C, 5-HT3, 5-HT6 and 5-HT7), histamine H1 receptor (H1R) or muscarinic M1–M5 receptors (M1R–M5R) )M1R–M5R) ; howeverM1R–M5R) . Beta ceM1R–M5R) . As reviM1R–M5R) , olanzapHerein, we used the chemically unrelated SGAs, olanzapine (a commonly prescribed SGA that is highly diabetogenic) and aripiprazole (the metabolic side effects of which are less well-known) to study the effects of prolonged treatment with SGAs on blood glucose levels, islet morphometry and beta cell function in female mice.Animal experiments were approved by the Animal Ethics Committees of the Spanish National Research Council and Comunidad de Madrid in accordance with Spanish (RD 53/2013) and European Union (63/2010/EU) legislation (PROEX 037/17).−1 day−1) was calculated considering daily food intake. After 6 months on the diet, mice were euthanised by cervical dislocation and pancreatic islets, whole pancreases, white adipose tissue (WAT) depots  and blood were collected and processed for analysis. As a positive control for serotonin expression in islets we used 12-day pregnant B6 female mice, aged 16 weeks, bred in-house with B6 male mice.Details of the B6;129 mice used in this study have been previously reported . Mice weA simple and sensitive LC-MS/MS method  was used for simultaneous determination of aripiprazole and olanzapine levels in plasma, as reported previously  and detaFood intake was measured manually using KERN PCB2500-2 scales  during the first month of treatment in mice housed in group cages and the mean food intake per mouse and per day was calculated.2 release were measured, and respiratory exchange ratio (RER) was determined as 2. Energy expenditure (EE) was calculated as EE = (3.185 + 1.232 × RER) × After 6 months on the diets, metabolic assays were performed, including i.p. GTT, i.p. ITT, glucose-stimulated insulin secretion (GSIS) and indirect calorimetry see ESM . In brieeviously . Analysi2 in complete RPMI-1640 medium  containing 5.6 mmol/l glucose. The next day, islets were pooled, randomised and incubated with 6 μmol/l olanzapine or aripiprazole , 1–500 μmol/l serotonin  (1–24 h incubation) or 10 μmol/l 4-Chloro-dl-phenylalanine . Control islets were treated with 0.01% (vol./vol.) DMSO.Islets were isolated by collagenase P  digestion (13.5 U/ml in cold Hank’s buffer), as described previously . For ex 2 in KRB containing 2.8 mmol/l glucose, 115 mmol/l NaCl, 5 mmol/l KCl, 1.2 mmol/l NaHCO3, 1.1 mmol/l MgCl2, 1.2 mmol/l NaH2CO4, 2.5 mmol/l CaCl2, 25 mmol/l HEPES and 0.25% (wt/vol.) BSA. Incubations were then performed using 2.8 mmol/l or 16.7 mmol/l glucose at 37°C, 5% CO2 for 1 h. Insulin levels were determined by ELISA (Mercodia) and values were normalised to islet number.For each individual mouse, 3–6 groups of three islets matched by size were placed in each well of a 96-well plate. Islets were pre-incubated for 1 h at 37°C and 5% COInsulin was extracted from 20 islets/mice using glycine/NP-40 lysis buffer  and measured by ELISA.Islets treated ex vivo with SGAs were pre-incubated with Fura-2 AM  and perfused with KRB containing glucose (2.8 mmol/l or 16.7 mmol/l). Fluorescence measurements were obtained at excitation wavelengths of 340 nm and 380 nm with an Axiovert 200 inverted microscope  with appropriate filters. Data acquisition was performed with the Aquacosmos 2.6 software . Recordings were expressed as the ratio of fluorescence at 340 nm and 380 nm (F340/380).Pancreases were fixed in Bouin’s solution  overnight at 4°C. Paraffin embedding and tissue sectioning were performed as described previously . LongituPancreatic sections were processed as described above using antibodies against insulin, glucagon, serotonin, p-S6 and Ki67, and secondary Alexa-Fluor conjugated antibodies (see ESM n = 10 beta cells from three independent mice/group) were assessed using ImageJ software (NIH). Insulin granules from beta cells were classified into four categories: mature (with an electron-dense core); immature (with a less electron-dense core); empty (lacking the core); and atypical (insulin granules with an irregular shape).For transmission electron microscopy (TEM) analysis, pools of 300 pancreatic islets from three mice per condition were processed as described in ESM Supernatants collected from static incubation experiments were used for the measurement of serotonin levels using the ELISA Fast Track kit . Values were normalised to islet number.www.bioinformatics.babraham.ac.uk/projects/fastqc, access date 27 May 2019), the reads were aligned to reference transcripts with the Kallisto algorithm [p value (p-adj) of <0.1 when performing a Wald test between two conditions and a Benjamini–Hochberg adjustment. Over-representation analyses (ORAs) of the DEGs were completed with the WEB-based GEne SeT AnaLysis Toolkit  [Islets were isolated in TRIzol  and total RNA was extracted using the PureLink RNA Mini Kit . Total RNA expression was analysed using Illumina TruSeq Stranded RNASeq technology . The libraries were sequenced (2 × 100 bp) with a mean output of 40 million reads in a NovaSeq 6000 sequencer (Illumina). After a quality control check with FastQC  .Protein levels were assessed in pancreatic islets using antibodies against IRS-2, mechanistic target of rapamycin (mTOR), p-mTOR (Ser2448), S6K1, p-S6K1 (Thr389), p-S6 ribosomal protein, tryptophan hydroxylase 1 (TPH1) and vinculin (ESM Table e PCR (RT-qPCR) using Power SYBR Green PCR Master Mix  and 7900HT Fast Real-Time PCR System (ThermoFisher Scientific), as described in ESM Gene expression was determined by quantitative real-timt test was used to compare mean differences between two groups, and for three or more groups, one-way ANOVA with Bonferroni post hoc test was used. For data with non-parametric distributions, differences between groups were examined with Mann–Whitney U test for two groups, or Kruskal–Wallis test for three or more groups. Two-way ANOVA was employed to compare two different categorical, independent variables. Where other statistical analyses have been used, this has been indicated in the figure legends. Data are expressed as mean ± SEM. Tests were two-sided and p<0.05 was considered statistically significant. Mice and islets were randomly and blindly distributed for the treatments by experimenters. Experimenters were not blind in outcome assessment.Statistical analysis was performed using Prism 8 . Datasets were first analysed for normal distribution. For data with parametric distributions, unpaired Student’s p<0.01). Aripiprazole-treated mice also gained more weight than the controls over the treatment period (p<0.05) but, as body weight stabilised in the last month of the treatment in this group, there was less body weight gain compared with olanzapine-treated mice (p>0.05)  and aripiprazole-treated (p<0.001) mice had a significant increase in visceral adiposity and showed a slight, but not significant, increase in iWAT/body weight ratio vs controls  for 6 months. Figure 05) Fig. . Both olols Fig. .Fig. 1Ep<0.05; Fig. p<0.05; Fig. We further studied the effects of the two SGAs on food intake. Consistent with a previous report , food cop<0.01) or aripiprazole-supplemented diet  and aripiprazole-treated animals (p<0.05), as compared with islets from chow-diet-fed mice  or aripiprazole (p<0.001) vs controls , but this effect was not observed in olanzapine-treated mice  and more empty granules (p=0.09) in beta cells from olanzapine-treated mice vs controls , but not from mice receiving olanzapine , basal fluorescence  or response time to high glucose (time islets take to respond to change in glucose concentration by opening voltage-gated Ca2+ channels) vs controls . PCA showed differential gene expression in islets from mice under SGA treatment Fig. . DEGs wep value <0.05, as assessed by the Wald test between two conditions, and relevant findings were further validated by RT-qPCR. A total of 289 genes dysregulated by aripiprazole and 136 by olanzapine were included in the ORA (data not shown). We found that the top-five upregulated pathways in the aripiprazole arm appeared to be related to serotonin biosynthesis  shows that genes encoding the serotonin-synthetising enzymes Tph1 and Tph2 were upregulated in islets from aripiprazole-treated mice, whereas the gene encoding the Htr3a receptor was downregulated in islets of olanzapine-treated mice  is a small number of genes for ORA, the analysis was performed with all genes with a fold change ≤ −1.5 or ≥1.5 and a sis Fig. . The heaice Fig. . NotablyTph1 and Tph2 expression vs controls  and that there was an apparent increase in THP1 protein levels, along with increased serotonin secretion (p<0.05) vs controls  for 1 h increased mTORC1 activity, as reflected by apparent increases in mTOR, S6K1 and S6 phosphorylation vs controls Fig. . MoreoveThis study provides novel findings on the effect of the SGAs olanzapine and aripiprazole in inducing glucose intolerance and reducing insulin secretion. We demonstrate that aripiprazole modulates the serotonergic system in islets, increasing mTOR/S6 phosphorylation, as well as elevating TPH1 expression and serotonin production in beta cells. By contrast, the effects of olanzapine on insulin secretion seem to be independent of the serotonergic system. Since type 2 diabetes develops gradually through life, and chronic medication is needed to tackle schizophrenia, we analysed the metabolic disturbances in female mice treated with these two chemically unrelated SGAs via supplementation in the diet over 6 months. To our knowledge, this is the first preclinical study in rodents to report the metabolic outcomes of long-term administration of olanzapine and aripiprazole that focuses on islet function.2C [1R receptors in the hypothalamus [Olanzapine treatment induced weight gain by increasing food intake; this finding is in agreement with previous studies that also demonstrated that this effect was mediated by the 5-HT2C  and H1R thalamus . Conversthalamus . Of clinthalamus . Yet, rethalamus . Remarkathalamus , and alsthalamus  weight gthalamus , the metthalamus , the cur2+ signalling, whereas olanzapine likely inhibits insulin secretion through a mechanism distal to Ca2+ entry into the beta cell. Of interest, while the GSIS test addresses insulin secretion exclusively, we cannot exclude alterations in hepatic insulin clearance or the beta cell insulin-degrading enzyme, both of which impair insulin secretion [Female mice treated with olanzapine or aripiprazole developed glucose intolerance that was associated with insulin resistance in aripiprazole-treated mice. However, we cannot exclude that longer treatments or more sensitive assays to assess insulin sensitivity, such as the euglycaemic–hyperinsulinaemic clamp, would reveal greater effects on blood glucose levels and insulin sensitivity. A step further, this is the first study to unravel a unique effect of aripiprazole in interfering with glucose-regulated Caecretion .Notably, mice treated with the SGAs had larger islets, particularly the aripiprazole-treated group, in which beta cell mass was twofold higher than that of the control group. In obesity and pregnancy, beta cell expansion is associated with enhanced insulin secretion, which compensates for insulin resistance. On the contrary, aripiprazole impairs insulin secretion despite beta cell expansion, indicating that increased beta cell mass, per se, is not sufficient to overcome beta cell dysfunction. The apparent paradoxical effects on mass and function were more prominent in aripiprazole-treated mice in which doubling of beta cell mass was associated with blunted insulin response.p=0.09), which deserves further investigation. Additionally, olanzapine-treated mice had higher alpha cell mass, pointing to potential pancreatic alterations beyond beta cells.Treatment with SGAs did not affect beta cell proliferation, which remained low, as previously reported in middle-aged mice . HoweverTph1 and Tph2 genes, and the induction of Tph1 mRNA and TPH1 protein levels (the rate-limiting isoform for serotonin biosynthesis) was associated with increased serotonin content and release in islets from aripiprazole-treated mice. These results were supported by: (1) the ex vivo treatment of islets with aripiprazole, which similarly resulted in increased TPH1 expression; (2) the decrease in insulin secretion in islets treated with serotonin that, like aripiprazole, activated mTORC1/S6 signalling; (3) and the recovery of GSIS in islets treated ex vivo with aripiprazole together with a TPH1 inhibitor, pointing to serotonin-mediated inhibition of insulin secretion by this SGA. Our findings are in agreement with a recent study showing that Sirtuin 3 deficiency in beta cells increased Tph1 expression, along with impairment of GSIS in obese mice [The complexity of the dopaminergic and serotonergic systems in pancreatic islets, which regulate insulin secretion , 37, togese mice .Htr3a, which encodes a serotonin receptor, in islets, potentially playing a role in the impairment of insulin secretion by this SGA, as previously reported [db/db mice, which exhibited increased expression of Htr2c [Transcriptomic analysis also showed that olanzapine downregulated the expression of reported , 40. Notof Htr2c . So far,of Htr2c , 23 and of Htr2c . Recent of Htr2c . In the of Htr2c , reinforof Htr2c , additioIn summary, we have identified alterations in islet plasticity and insulin secretion in female mice treated with the SGAs olanzapine and aripiprazole, with important translational implications. In the case of aripiprazole, in which the serotonergic system was activated, specific TPH1 inhibitors that do not cross the blood–brain barrier could be used to prevent intra-islet and peripheral serotonin dysregulation without affecting serotonin levels in the brain .ESM(PDF 902 kb)


Purpose: To evaluate and compare the efficacy and safety of interferon alpha-2a (IFN-α2a) and cyclosporine-A (CsA) in patients with refractory Behçet’s uveitis (BU).Methods: In this 12-month randomized, controlled, prospective trial, 26 participants (44 eyes) completed the study. Patients were randomly allocated to the IFN-α2a or CsA groups. All patients in both groups received a standardized prednisone burst and tapering schedule as per protocol. The primary outcome measures were response rate, complete remission rate, and tolerance rate. The secondary outcome measures included time to achieve complete remission, the logarithm of the minimum angle of resolution (logMAR) of best-corrected visual acuity (BCVA), and Behçet’s disease ocular attack score 24 (BOS24). T-tests and non-parametric tests were used to compare quantitative variables, and chi-square tests were performed to compare qualitative variables.Results: The response and complete remission rates were 85.7% (12/14 patients) and 50.0% (7/14 patients) in the IFN-α2a group, compared with 66.7% (8/12 patients) and 25.0% (3/12 patients) in the CsA group, respectively (p > 0.05). Complete remission was achieved at 3.3 and 7.0 months after initiation of IFN-α2a and CsA (p = 0.023). LogMAR BCVA significantly improved 1 month after IFN-α2a initiation (23 eyes) (p = 0.002), and this beneficial effect remained statistically significant during the entire follow-up period (p < 0.05); however, this improvement was not observed in the CsA group (21 eyes). At the endpoint, LogMAR BCVA in the IFN-α2a group was significantly better  with a higher improvement rate . Moreover, compared to the CsA group, more eyes in the IFN-α2a group had a lower BOS24 score . None of the patients had any side effects that influenced the medication adherence.Conclusion: Compared to CsA plus corticosteroid, IFN-α2a plus corticosteroid appears to induce a better treatment response, a significantly greater improvement in visual acuity, and more stable remission of intraocular inflammation in a 12-month study period. Clinical Trial Registration: Interferon α2a Versus cyclosporine for refractory Behçet’s disease uveitis, NCT03209219.Behçet’s disease (BD) is a multisystemic chronic inflammatory disease of unknown cause characterized by recurrent oral aphthous ulcers, ocular lesions, genital ulcers, gastrointestinal, and central nervous system manifestations . UveitisCurrent treatments for BU mainly include glucocorticoids, conventional immunosuppressants such as cyclosporine-A (CsA) and azathioprine (AZA), and biological agents such as interferon-alpha (IFN-α) and anti-tumor necrosis factor-alpha (anti-TNF-α) agents . While hIFN-α2a has long been reported to be effective in BU patients with different genetic backgrounds . IFN-α2aClinicalTrials.gov (NCT03209219).This 12-month randomized controlled prospective study was conducted at the Department of Ophthalmology at Peking Union Medical College Hospital between June 2017 and August 2020. All recruited patients with refractory BU were randomly assigned (1:1) to the IFN-α2a or CsA groups using a random number table. The study protocol was approved by the Institutional Review Board of Peking Union Medical College Hospital  and conducted according to the tenets of the Declaration of Helsinki. Informed consent was obtained from all participants. This study was registered in The study population was adult (18 ≤ age ≤ 65) refractory BU patients with acute uveitis attack. BD was diagnosed according to the International Criteria for Behçet’s Disease (ICBD) . Uveitis3, platelet count < 100,000/mm3, Hgb < 8.5 g/dl), urine tests, liver and kidney function  or aspartate transaminase (AST) 2× above the normal) were not eligible.Patients with any of the following conditions were excluded: 1) patients who had previously received any biological agent , had used CsA but did not tolerate, or had any systematic contraindication  that prevent using glucocorticoids; 2) patients with malignancy, pregnant, breast-feeding, mental illness, depression, cognitive impairment, poorly controlled hypertension or diabetes mellitus, alcohol abuse or drug abuse, history of acute or chronic inflammatory joint or autoimmune disease, systemic infectious diseases, including hepatitis B virus, hepatitis C virus, HIV, syphilis, or tuberculosis (TB) infection were also excluded; 3) patients with severe extra-ocular involvement; 4) patients who showed a presence of severe pupillary adhesion, cataract and posterior capsular opacification that obscured the fundus observation, and/or had other ocular diseases, and intraocular surgery in the previous 3 months; and 5) patients with significant laboratory abnormalities in complete blood counts , intraocular pressure, slit-lamp biomicroscopy, and fundoscopy, were performed at baseline (before the initiation of 60 mg/day prednisolone) and at each follow-up visit. BCVA was examined using standard logarithmic visual acuity charts and then converted to the logarithm of the minimum angle of resolution (LogMAR) for statistical analysis.An ocular inflammatory attack was defined as a new-onset of intraocular inflammation and/or worsening of preexisting uveitis, necessitating treatment intensification. The severity of ocular inflammation at baseline and each follow-up visit was evaluated using the BOS24 scoring system, which is a novel and more definite tool for scientific analysis . The BOSBone mineral density and infection screening tests were performed at baseline. Blood pressure was measured at baseline and monthly during the study period. Laboratory tests, including complete blood counts, urine tests, and biochemical tests, were performed monthly or bimonthly.The endpoints of this study were relapse of posterior or pan-uveitis, drug  withdrawal due to intolerance, and completion of the 12-month follow-up since initiation of 60 mg/day prednisolone.The primary efficacy outcome measures were the response and complete remission rates. Specifically, treatment response was categorized into complete remission, partial remission, and treatment failure. Complete remission was defined as a decrease in vitreous haze to no more than grade 0.5+ and complete disappearance of signs of active fundus inflammation including retinal infiltrates, hemorrhage, and vascular sheathing , withoutThe primary safety outcome measure was the tolerance rate to IFN-α2a or CsA treatment. The secondary safety outcome measures included the incidence of adverse effects, significant abnormal changes in vital signs or laboratory test results, and the adverse effects profile.p value of <0.05.Statistical analysis was conducted using the Macintosh software . The Kolmogorov–Smirnov test was used for normality testing. Normal variables are presented as the mean and standard deviation (SD), and non-normal variables as the median and interquartile range (IQR). T-tests were used to compare the means of normally distributed quantitative variables; otherwise, the Mann-Whitney U test was used. The non-parametric Wilcoxon test was used to compare continuous variables. Chi-square tests were used to compare the qualitative data. Statistical significance was set at Sample size analysis was conducted using PASS 11.0 software . This randomized controlled prospective study was designed to have a statistical power of 80% and a significance level of 5%. Based on our clinical experience and previous studies, we estimated that the primary endpoint of participants, namely, complete remission rate of IFN-α2a and CsA therapy, was 80 and 30% , respectA total of 28 eligible patients were included in the study from June 2017 (enrollment of the first patient) to August 2020 (the date of the last follow-up visit). Two patients who did not follow the treatment protocol were excluded. Therefore, 26 patients with refractory BU (44 eyes) completed the trial and were included in the analysis. As shown in Prior to enrollment, all patients were treated with corticosteroids in combination with a median of 1 immunosuppressant . The median dose of prednisolone was 20.0 mg/day . The baseline immunosuppressive agents taken by patients included CsA , AZA , CTX , THD , MMF , TAC , and MTX . The IFN-α2a and CsA groups were not significantly different in basic demographic data, baseline clinical features, and treatments.p = 0.365). Notably, complete remission (no relapse within the 12-month follow-up period) was achieved in 7 (50.0%) patients in the IFN-α2a group, compared to only 3 (25.0%) patients in the CsA group (p = 0.248). Of those patients who completely responded to the therapy, the duration between the therapy initiation to a complete absence of ocular inflammation was 3.3 and 7.0 months in IFN-α2a and CsA group, respectively (p = 0.023). On the other hand, for incomplete responders and nonresponders who suffered further uveitis attacks during the study period, the relapses occurred on average 4.7 ± 3.7 and 4.8 ± 2.2 months after IFN-α2a and CsA initiation, respectively (p = 0.966).Of the 26 patients, 20 (76.9%) responded  to IFN or CsA treatment . SpecifiThe analysis included 23 eyes in the IFN-α2a group and 21 eyes in the CsA group with refractory BU.p = 0.147). BCVA equal or below 20/50 and 20/200 were found in 16 eyes (69.6%) and 9 eyes (39.1%) in the IFN-α2a group, compared to 17 eyes (81.0%) and 13 eyes (61.9%) in the CsA group (p > 0.05), respectively.The baseline LogMAR BCVA was 0.52 (0.10–1.00) in the IFN-α2a group and 1.00 (0.61–1.52) in the CsA group (p < 0.001), and this beneficial effect sustained to the endpoint visit (p = 0.026) (p > 0.05). Notably, at the end of the study, the median LogMAR BCVA increased to 0.22 (0.00–0.92) and 0.92 (0.31–1.70) in the IFN-α2a group and CsA group, respectively (p = 0.031).Of the 23 enrolled eyes in the IFN-α2a group, the improvement in LogMAR BCVA started at the first month’s visit after treatment initiation (= 0.026) . In contFurthermore, of all eyes in the IFN-α2a group, BCVA improved ≥0.2 LogMAR from study initiation in 8 eyes (34.8%), improved but less than 0.2 LogMAR in 6 eyes (26.1%), remained stable in 3 eyes (13.0%), and worsened in 6 eyes (26.1%). On the other hand, in the CsA group, BCVA improved by ≥0.2 logMAR in only 4 eyes (19.0%), but it stabilized and deteriorated in 6 eyes (28.6%) and 7 eyes (33.3%), respectively.p = 0.001), remained statistically significant at every follow-up visit, and eventually improved to 0.31 (0.00–0.98) at study endpoint (p = 0.020). Meanwhile, in the complete remission subgroup, a total of 13 eyes showed similar VA progression (p > 0.05).Among 20 eyes that responded  to the IFN-α2a therapy, LogMAR BCVA was 0.60 (0.17–1.30) at baseline, and significantly increased 1 month after treatment initiation (gression . Howeverp = 0.803). Of all eyes in the IFN-α2a group, the BOS24 score showed a significant decrease 1 month after treatment initiation (p < 0.001) and remained low during the entire study period (p = 0.001) (p = 0.124), respectively.The median baseline BOS24 scores were 5 (3–7) and 5 (3.5–6.5) in the IFN-α2a and CsA groups, respectively (= 0.001) . Howeverp = 0.042). In contrast, only 2 eyes (8.7%) showed a higher BOS24 score when relapse occurred during the IFN-α2a treatment period, while 5 eyes (23.8%) in the CsA group had an elevated score at the endpoint, indicating a more severe ocular inflammation status (p = 0.232).Moreover, at the endpoint visit, a decreased BOS24 score was obtained in 20 out of 23 eyes (87.0%) and 12 out of 21 eyes (57.1%) in the IFN-α2a and CsA groups, respectively (p < 0.05).Among eyes with complete and partial remission, the BOS24 score decreased over time in both the IFN-α2a and CsA groups . Comparep = 0.695). The median corticosteroid dosage before enrollment was 20.0 mg (16.9–24.4) and 20.0 mg (20.0–30.0) per day in the IFN-α2a and CsA groups, respectively (p = 0.207). At the endpoint, the average dosage of corticosteroid was significantly decreased to 15.0 mg per day in complete remission patients treated with IFN-α2a (p = 0.024). Nevertheless, no obvious corticosteroid-sparing effects were observed in patients in the other subgroups, including partial remission and treatment failure patients in the IFN-α2a group, and all CsA subgroups (p > 0.05).After IFN-α2a or CsA treatment, the prednisolone dose was reduced in 8 (57.1%) and 5 (41.7%) patients at the end of the study, respectively (p = 0.665). Compliance with IFN-α2a was satisfactory. IFN-associated side effects, which were mild and reversible, included flu-like syndrome associated with fever, myalgia, and headache (at the initiation phase of the treatment) , mild elevation of serum liver enzymes , hair loss , skin disorders , minor leukopenia , dryness of mouth , and mild depression . The side effects related to the CsA treatment were as follows: increased ALT/AST , increased uric acid , hyperlipidemia , hypertension , hematuria , and increased bilirubin . Hirsutism was observed in one female patient in the CsA group.The tolerance rate of both the IFN-α2a and CsA groups was 100% in this study. No treatment discontinuation was required because of the side effects. No serious adverse drug effects were observed. The incidence of adverse events in patients treated with IFN-α2a and CsA was 78.6% (11/14) and 66.7% (8/12), respectively  response rates of IFN-α2a for treatment of BU . The ratThe advantage of IFN-α2a over CsA was also reflected by the time to reach complete remission, and the improvements in visual function and disease severity, as indicated by LogMAR BCVA and BOS24 score, respectively. Our current study showed that the use of IFN-α2a treatment led to a significantly earlier complete remission in refractory BU patients than CsA treatment. Additionally, during the entire 12-month period, treatment with IFN-α2a can effectively achieve sustained disease control by markedly increasing visual acuity and reducing BOS24 score, regardless of whether the patients achieved complete remission. Consequently, at the endpoint of the study, more patients in the IFN-α2a group achieved a prominent visual acuity improvement with amelioration of intraocular inflammation, as compared to the CsA group. Therefore, this randomized prospective comparative clinical trial provides multiple lines of evidence suggesting that in the treatment of refractory BU, IFN-α2a treatment can not only reduce the dosage of glucocorticoids but also display superiority in inducing rapid disease remission and maintaining disease quiescence in 12 months.Our study also revealed generally favorable safety profiles for both IFN-α2a and CsA regimens. Although adverse effects were recorded in 78.6 and 66.7% of the patients in the IFN-α2a and CsA groups, respectively, they were all reversible and well tolerated. The most frequent side effects of IFN-α2a and CsA were flu-like symptoms (71.4%) and renal toxicity (33.3%), respectively, which are in accordance with previous studies . We alsoThis study has some limitations. First and most importantly, the sample size was relatively small and inadequate for more detailed analyses and comparisons. The approval of adalimumab for refractory non-infectious uveitis in March 2020 in China and the COVID-19 pandemic have made it difficult to recruit participants further. Second, we noticed that there was a difference in the baseline BCVA between the IFN-α2a and CsA groups, although this disparity was not statistically significant. Third, the current study period was not long enough to evaluate the long-term efficacy of IFN-α2a and CsA treatment. It would be of higher clinical qualifications to conduct the study over a longer time span.In conclusion, this randomized, controlled, prospective clinical trial provides multiple lines of evidence suggesting that IFN-α2a is superior to CsA when combined with glucocorticoid for refractory BU during a study period of 12 months. Compared to CsA, IFN-α2a induces a higher rate of treatment response, a significantly better improvement in visual acuity, and a more stable disease remission in 12 months for refractory BU.


Shock waves altered the molecular pattern of proliferation, activation, differentiation, and apoptosis, as well as proliferation/ differentiation regulators, including Bax, Bcl2, ASK1, p21, p27, and Notch1. In summary, we show that extracorporeal shock wave therapy regulates the proliferation and differentiation of keratinocytes derived from hypertrophic scar to maintain normal epidermal integrity. Hypertrophic scars represent a common complication in burn patients. In addition to cosmetic defects, they may cause serious sensory abnormalities such as pain and itching, severe dysfunction depending on the site, and emotional disorders such as anxiety and depression. The present study aimed to identify the molecular mechanisms underlying the use of extracorporeal shock wave therapy in keratinocytes. Keratinocytes derived from hypertrophic scar tissue were cultured and expression of proliferation markers (keratin 5 and 14), activation markers (keratin 6 and 17), differentiation markers , apoptosis factors , and proliferation/differentiation regulators (p21 and p27) was investigated to compared with that of those in keratinocytes derived from normal skin tissue. Scar-derived keratinocytes were treated with extracorporeal shock waves under 1000 impulses at 0.1, 0.2, and 0.3 mJ/mm It is accompanied by excessive deposition of extracellular matrix, which not only increases scar thickness and decreases elasticity of the scar skin, but also causes continuous scar pain and itching in burn patients2. In addition, thicker scars in the joint area decrease the range of motion of the joint and become an important impediment to daily life movements and walking during the rehabilitation process1.Hypertrophic scar hyperplasia is one of the most common complications after burn injury. Whereas the exact mechanism remains unknown, the phenomenon is characterized by the pathological proliferation of scar tissue caused by abnormal wound healing and scar maturation processes3. Triamcinolone is used for intralesional injection treatment, effectively reducing scar thickness and inhibiting proliferation3. However, as it affects also continuous progression of scar growth3, it is used only for localized scars. Therefore, a new, non-invasive, and effective treatment that can be applied continuously on a wide scar area is required.Various treatments, such as compression clothing, silicone gel, scar massage, laser treatment, intralesional injection, and surgical removal, are combined in clinical practice to treat hypertrophic scars in burn patients4. A single treatment of ESWT upregulated with prolonged period time, 25–30 key pro-angiogenic gene expression that are decreased in both diabetic and wound healing models. ESWT inducing pro-angiogenic factors included for platelet–cell adhesion molecule‐1 (PECAM‐1), vascular endothelial growth factor (VEGF) and its receptor (VEGFR), endothelial nitric-oxide synthase (eNOS), and hypoxia-inducible factor 1α (HIF-1 α)5. ESWT not only induced endothelial cell proliferation, but also enhanced proliferation of other cell types8. Several previous studies both in vitro and vivo have shown an anti-inflammatory mechanism of ESWT, which treatment inhibited NF-κB activation and decreased oxygen radical production, leukocyte infiltration, chemokine expression, and pro-inflammatory cytokine expression11. In recent our study, shown ESWT down-regulated transforming growth factor beta 1 (TGF-β1) expression and reversed epithelial mesenchymal transition (EMT) in fibroblast derived from HTS12. The signal transduction associated with above events by ESWT is not clear. However, the activation of ERK1/2 or p38 MAPK may be responsible for the biological effects of ESWT14. Moreover, it has been reported to improve perfusion, ameliorate lymph circulation in patients with lymphedema, stimulate osteoblasts and union in fractures showing nonunion for more than 6 months, and promote regeneration of damaged peripheral nerves15. However, so far, the application of extracorporeal shock waves to treat hypertrophic scars resulting from burns has been limited to animal models16.Extracorporeal shock wave therapy (ESWT) is used to reduce inflammation, diminish pain, and regenerate soft tissues in musculoskeletal diseases1. Although the normal epidermal keratinocytes actively interact with dermal fibroblasts17, little is known about whether keratinocytes seen in hypertrophic scars play a particular role in scar proliferation. A study using a tissue-engineered human skin model showed that pathological keratinocytes in the epidermis induced fibrotic endothelial matrix formation and inhibited extracellular matrix degrading factors, resulting in hypertrophic scar formation18. Therefore, examining the characteristics of epidermal tissue-derived keratinocytes, which many studies have focused on, will provide basic data for developing new treatments against thickening burn scars.Although normal epithelial cells actively interact with interstitial fibroblasts, they come from most studies on the etiology of hypertrophic scars have revealed pathological proliferation of the dermis20, and showed that treatment changed the levels of hypertrophic scar-related molecules in fibroblasts derived from hypertrophic scars12. Examining the effect of extracorporeal shock waves on keratinocytes derived from hypertrophic scar tissue at the molecular level will provide important evidence about the effectiveness of this therapy for hypertrophic scar treatment.Previously, we reported the therapeutic effects of ESWT on scar pain and pruritus in burn patients with hypertrophic scar21. The hypertrophic scar tissue and normal skin tissue are paired from four patients that were obtained during surgical procedure, washed with 70% ethanol and cold phosphate-buffered saline containing antibiotics and antimycotics . The tissue was cut into 2–4-mm-wide pieces, which were placed in 50-mL conical tubes containing 30 mL dispase II  solution and agitated for 16 h at 4 °C. After digestion, the epidermis was obtained from the sample tissues by sterile forceps, and then digested with TrypLE™ Express Enzyme (Gibco) at 37 °C for 45 min. The solutions were added with KGM-Gold™ keratinocyte growth medium containing supplements  to the enzyme, and then filtered and centrifuged at 1500 rpm for 5 min. The pellet was suspended in the medium and cultivated at 37 °C in a 5% CO2 atmosphere. Human normal keratinocytes (HNKs) and human hypertrophic scar keratinocytes (HTSKs) were used for future experiments at passage 1 or 2.All experimental protocols of this study were approved by Institutional Review Board of Hallym University Hangang Sacred Heart Hospital, and were carried out in compliance with the guidelines . Written informed consent was obtained from all subjects. After that, the procedure of primary cell culture was as published previously® cell detachment solution  and suspended in complete medium in a 4.5-cm-long conical tubes at 2.0 × 105/mL. The cells treated a focused shock wave generated from an electromagnetic cylindrical coil source using Duolith SD-1® device . Cells were exposure to 1000 impulses/cm2 under an energy flux density  and a frequency (4 Hz). After ESWT, that cells collected, centrifuged and further cultivated in complete medium in T75 culture plates. Cultivation for 24 h or 72 h, the cells collected, and used to analysis of mRNA and protein expression.Keratinocytes were collected from cell culture dish by dissociated with Accutase21. The primer sequences are listed in Table −△△Ct method.Total RNA was extracted using a ReliaPrepTM RNA Miniprep system  according to the manufacturer’s instructions. RNA concentration was measured, cDNA was synthesized, and quantitative real-time PCR were performed as previously describedg and 4 °C, and the protein concentration was measured with a BCA kit (Thermo Fisher Scientific). Electrophoresis, primary and secondary antibody attachment, development (protein detection), and quantification were performed as previously described21. The primary antibodies are listed in Table HNKs or HTSKs were harvested and lysed in ice-cold RIPA buffer  pre-added a complete phosphatase inhibitor cocktail  and protease inhibitor cocktail , and maintained for 1 h at 4 °C with constant agitation. The cell lysates were centrifuged for 15 min at 13,000×P < 0.05 was considered statistically significant.All experimental results are presented with the mean ± standard deviation (SD). Comparisons between two groups were performed using the Mann–Whitney U test. Statistical analyses were performed using PASW statistics 24 , and 22. The expression of keratin 5 and 14 at mRNA and protein levels was no significant difference between HTSKs and HNKs    Figure A–D. The P < 0.05)       Figure E,F. In cP < 0.05)  [Figure 2     Figure . AnotherP < 0.05)      Figure A. Howeve) Figure B. The ke) Figure C. These P < 0.05)      Figure  D and E.P < 0.05)        2 in mesenchymal cell line14, 1000 impulses at 0.14 mJ/ mm2 in human tenocytes25, and 0.06–0.50 mJ/mm2 in human osteoblasts26. Recent we applied ESWT with 4 Hz frequency, 0.05–0.30 mJ/mm2 energy flux density, and 1000 to 2000 impulses/cm2 per treatment according to patient's tolerance to pain. This treatment improved scar thickness, vascularity, and functionality, when treat to HTS of hand without obvious side effects24. Moreover, in our previous study, that ESWT with 1000 impulses/cm2 at 0.03, 0.1, and 0.3 mJ/mm2 not only altered the expression of fibrosis-related molecules in fibroblast derived from HTS, but did not affected the cells viability. Therefore, in this study, we selected the parameter with 1000 impulses/cm2 at 0.1, 0.2, and 0.3 mJ/mm2, applied to keratinocytes, and determined the molecular changes of cells.In the literature on the ESWT, no agreement on how to achieve maximum potential. There are several in vitro studies has been used with 100–300 pulses at 0.03–0.13 mJ/mm27. Moreover, differentiation marker, filaggrin and involucrin were abnormally detected in HTS28. The increased expression of involucrin also found in other hyperproliferative disease, such as psoriasis29. The authors considered that abnormal expression of involucrin possibly affect stratum corneum formation28. In our results, the characteristic of HTSKs is similar to those of above previous study that is activated keratinocytes highly expressed keratin 6 and 17, and involucrin expression was also increased. Activated keratinocytes may play an important role in the development of HTS through the epidermal-mesenchymal interaction17. In HTSKs, the anti-apoptotic factor Bcl2 expression at mRNA and protein levels increased, while pro-apoptotic factor Bax expression was unchanged, compared to HNKs that suggest up-regulated anti-apoptosis in HTSKs. This results also reflect the low level of apoptosis is a cause of HTS formation30. The differentiation marker, keratin 1 and involucrin expression were increased in HTSKs, suggest differentiation of HTSKs was increased than HNKs. Moreover, the cell cycle regulator, p21 and p27 expression were also up-regulated in HTSKs. Study revelated the keratinocyte proliferation and differentiation were tightly regulated by the p21 and p27 expression. Moreover, over-expression of p21 and p27 induced keratinocyte differentiation and inhibited proliferation31. On the contrary, down-regulation of p21 and p27 expression resulted inhibition of differentiation with decrease of keratin-1 expression32. Therefore, increased expression of keratin 1 and involucrin may be related to up-regulated p21 and p27 expression in HTSKs.At present, the fundamental epidermal pathology of HTS is still unclear. In previous studies, determined the expression of keratin 5 and 14, both are proliferation marker, and keratin 6, 16, and 17, they are activation marker by immunohistochemistry in HTS tissues that provided evidence that HTSKs were activated, but there was no difference in proliferation compared with site matched normal skin tissues33, including Bax, Bcl2, ASK1, and Caspase 14 etc. In normal epidermal development, Bcl-2 is highly expressed in basal proliferating keratinocytes and absent from suprabasal keratinocytes34. Bax expression has been reported to be stronger in suprabasal than in basal keratinocytes35. Over-expression of ASK1 in keratinocytes doses not induce apoptosis, but induce cell differentiation along with increasing the expression of involucrin36. Caspase-14 is uniquely expressed in skin tissues unlike other caspases type expressed ubiquitously37 and its accumulation is associated with keratinocyte differentiation and stratum corneum formation38. In our results, Bcl2 expression was correlated with Bax expression increased at 24 h decreased at 72 h after ESWT. Moreover, ASK1 expression was increased at 24 h and 72 h after ESWT. Caspase 14 expression was increased just at 72 h after treatment. These results are further supported the effect of shock wave therapy regulate HTSKs proliferation and differentiation. To sum up the above, that over- or down-expression of apoptosis-related factors reflects ESWT treatment induces the proliferation or differentiation phenotype keratinocyte.We found the proliferation marker, keratin 14 expression was decreased at 24 h and increased at 72 h, while activation marker keratin 6 expression was increased at 24 h and decreased at 72 h. Moreover, early time differentiation marker keratin 1 expression was decreased at 24 h and increased at 72 h, and later differentiation marker, involucrin was increased at 24 h and decreased at 72 h. These results indicate that ESWT coordinately regulate HTSKs proliferation, activation, and differentiation, rather than blindly increase or decrease. Whereas, expression of keratin 6, and keratin 1 and involucrin in HNKs at 24 h after ESWT was increased and decreased at 72 h. These results suggest that ESWT exhibit different effects depending on physiological phenotypes. To maintain normal epidermal integrity, the proliferation-differentiation-death cycle must operate seamlessly. The several apoptosis-related factors physiologically involved in keratinocyte proliferation and differentiationOverall, the shock wave therapy altered the expression of proliferation and differentiation—related molecular in HTSKs. The increased or decreased expression of molecular in HTSKs 24 h or 72 h after treatment was close to that of those in the normal cell; for instance, keratin 1 expression at 24 h, involucrin, bcl2, p21, and p27 expression at 72 h vs. that of those in HNKs. The consequence its dynamic regulation may be partially involved in pathological phenotype restore to normal cell physiological state contribute to maintain normal epidermal homeostasis.42. Specifically, activation of Notch1 upregulation of p21 to induce differentiation associated cell arrest, whereas p27 activation promotes the differentiation of keratinocytes42. In our results, expression of Notch1, p21, and p27 was increased at 24 h and decreased at 72 h after ESWT. Their expression pattern was consistent with that expression of keratin 14 and involucrin. Therefore, the effect of ESWT on the proliferation and differentiation of HTSKs possibly through regulate expression of Notch1, p21, and p27.Notch1, p21, and p27 are also involved in regulating the proliferation and differentiation of keratinocytesIn summary, present study demonstrated that extracorporeal shock wave dynamically regulates proliferation, activation, and differentiation of keratinocytes originated from hypertrophic scar tissue Table . This evSupplementary Figures.


Modiolus kurilensis bivalve mollusk widespread in the Northwest Pacific was purified. The isolation procedure included ammonium sulfate precipitation followed by affinity chromatography on pectin-Sepharose. The full-length MkC1qDC sequence was assembled using de novo mass-spectrometry peptide sequencing complemented with N-terminal Edman’s degradation, and included 176 amino acid residues with molecular mass of 19 kDa displaying high homology to bivalve C1qDC proteins. MkC1qDC demonstrated antibacterial properties against Gram-negative and Gram-positive strains. MkC1qDC binds to a number of saccharides in Ca2+-dependent manner which characterized by structural meta-similarity in acidic group enrichment of galactose and mannose derivatives incorporated in diversified molecular species of glycans. Alginate, κ-carrageenan, fucoidan, and pectin were found to be highly effective inhibitors of MkC1qDC activity. Yeast mannan, lipopolysaccharide (LPS), peptidoglycan (PGN) and mucin showed an inhibitory effect at concentrations three orders of magnitude greater than for the most effective saccharides. MkC1qDC localized to the mussel hemal system and interstitial compartment. Intriguingly, MkC1qDC was found to suppress proliferation of human adenocarcinoma HeLa cells in a dose-dependent manner, indicating to the biomedical potential of MkC1qDC protein.C1q domain-containing (C1qDC) proteins are a group of biopolymers involved in immune response as pattern recognition receptors (PRRs) in a lectin-like manner. A new protein MkC1qDC from the hemolymph plasma of  The specific interaction of proteins with carbohydrate underlies a variety of cell-to-cell communications and cooperation with extracellular matrix (ECM). Cell surface forms unique glycosylation patterns, which play a significant role in the immune defense providing the self and non-self-discrimination abilities. Meanwhile, the most known group of carbohydrate binding proteins are lectins, which demonstrated diversified affinity to a variety of carbohydrate determinants. Initially all carbohydrate binding proteins were recognized as lectins. However, extensive genomic research distinguished separate groups of proteins recognizing glycans of pathogen-associated molecular patterns (PAMPs) . Among tCrassostrea gigas [Pinctada fucata [Modiolus philippinarum [Ruditapes philippinarum [Saccostrea glomerata [Crassostrea virginica [In the vertebrates, the C1q protein is a key component of the complement system, triggering the classical pathway of its activation. In invertebrates, C1q domains and corresponding proteins show a wide range of ligands, including a variety of PAMPs. Interestingly, the largest number of genes encoding C1qDC proteins was found in bivalve species genomes: 337 in ea gigas , 296 in ppinarum , 1182 inppinarum , 554 in irginica ,11,12, birginica ,13, shelirginica ,15,16 anirginica . Until rirginica ,18,19 orirginica .Currently, lectins as biomedical and biotechnological tools are a very active area of research ,23,24,25M. kurilensis (MkC1qDC) was identified and an effective protocol for its isolation was developed. Immunohistochemical detection showed intracellular localization of target protein only in hemocytes and MkC1qDC presence to the hemal system, ECM and interstitial components. The physicochemical and functional properties of MkC1qDC was characterized, including the carbohydrate specificity and antimicrobial activity. Furthermore, MkC1qDC demonstrated the inhibition of HeLa proliferation in a dose-dependent manner, suggesting biomedical potential of this protein.In this work, a novel C1qDC bivalve protein from M. kurilensis is characterized by the highest hemagglutination (HA) activity towards human erythrocytes equally for all groups of the AB0 system displaying 1/64–1/256 titers against 6 × 107 cells per mL. Analysis of the carbohydrate specificity of M. kurilensis plasma agglutinins by hemagglutination inhibition (HAI) assay showed that mucins type II and type III, mannan, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, sialic acid, D-(–)-ribose, as well as D-glucuronic and D-galacturonic acids, were inhibitory. Fraction 0–15% of ammonium sulfate precipitation of plasma proteins had no agglutinating activity. Fractions 15−30%, 30−45%, 45−60%, 60−75% and 75−85% had agglutinating activity, but only 60−75% and 75−85% was inhibited by uronic acids and citrus pectin containing a polygalacturonic acid structure. The inhibition activity was Ca2+-dependent as 30 mM Na2EDTA disposed of it. Thus, in the presented final scheme for the isolation of the target protein (2+-binding eluate (M. kurilensis hemolymph plasma.Cell-free hemolymph (plasma) of  protein a, ammoni protein b, Lane 1 protein b, Lane 2 protein b, Lane 3 protein c the ban protein b, Lane 4g eluate b, Lane 5g eluate c. On avehttps://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 26 May 2021).We initially sequenced the protein by Edman sequencing. MkC1qDC samples were purified independently at five different time points, and we were able to sequence 13 to 41 residues a. HoweveTo complement the data, we applied de novo mass spectrometry peptide sequencing to determine the amino acid sequence of the protein. The purified protein was digested with trypsin and several peptides of up to 50 residues with an Average Local Confidence >80% were identified. Analysis of these fragments allowed us to assemble a sequence that matched to data obtained by Edman sequencing b,c. In aTo further explore the properties of MkC1qDC we used 2D electrophoresis. Purified MkC1qDC was found to be a single polypeptide with a molecular mass of 19 kDa mass a and a pPurified MkC1qDC had high agglutinating activity after incubation for 1 h at temperatures of 0–40 °C, which slightly decreased at 50 °C, and were completely lost at 60 °C or higher b. At theThe highest MkC1qDC agglutination activity was against human erythrocytes with no prevalence with respect to blood group. Therefore, all succeeding experiments was performed only with human group 0 erythrocytes.Saccharomyces cerevisiae, LPS from Escherichia coli, PDG from Staphylococcus aureus and mucin type II from porcine stomach were moderately inhibitory at concentrations three orders of magnitude greater than for the most effective saccharides (0.10–0.49 mg/mL). Alginate, κ-carrageenans, fucoidan and citrus pectin were highly effective inhibitors characterized by the lowest IC50 (less than 0.002 mg/mL).Among 23 monosaccharides tested by the HAI assay, only six were inhibitory . L-gulosBacillus subtilis, S. aureus) and Gram-negative  bacteria . The only exception was Vibrio sp. The decrease in the density of the Vibrio sp. cultures in the presence of MkC1qDC occurred only after 26 h of cultivation (p < 0.05) when the control was in a stationary phase  or 16 h (1 and 2 μg/mL) . FurtherM. kurilensis plasma, we observed a strong inhibitory effect on D-glucuronic acids. Earlier, in a closely related species Modiolus modiolus, the same analysis did not reveal the same specificity [M. modiolus had a different molecular weight [Chlamys farreri (CfC1qDC) and Argopecten irradians (AiC1qDC-1) [Using a HAI technique to study the carbohydrate specificity of the cificity . Lectinsr weight ,29 from r weight . MoreoveC1qDC-1) ,9.Pseudoalteromonas sp. and B. subtilis (C. farreri (CfC1qDC) and A. irradians  scallops [M. kurilensis plasma [C1qDC proteins have been found to be immune factors in many cases. Various pathogens and their components induce an increase in C1qDC proteins expression, as well as C1qDC proteins demonstrating the ability to bind PDG, LPS, poly I:C, mannan, β-1,3-glucan, yeast glucan and live bacteria, generally indicating their role as PRRs ,36,37,38subtilis . Agglutiscallops ,11. Intes plasma , which pIt is currently known that C1qDC proteins in the bivalve can be expressed in in all organs ,17,20,37M. kurilensis and Coccomyxa parasitica [The role of C1qDC proteins as PRRs implies their specificity for various PAMPs; this study also showed the high sensitivity of MkC1qDC towards mannan, LPS and PDG . Howeverrasitica . At the rasitica . Regardirasitica ,18,30, wrasitica ,44. It wrasitica  or galacrasitica  as well rasitica .Haliotis discus discus, which is also a representative of the C1qDC proteins [The range of carbohydrate specificity determines the potential in the use of lectins and lectin-like proteins in biotechnology and biomedical science. During tumor cells transformation, a change in the carbohydrates types of their surface occurs. Therefore, the lectins and lectin-like proteins that are able to recognize particular carbohydrate patterns also show antitumor activity. Impotence of cell surface sialylation in tumorigenesis and metastasis was known for a long time ,49. The proteins , possessproteins ,51,52. Oproteins . Hence, proteins ,55. Addiproteins ,57. In tM. kurilensis. The PRR properties such as antibacterial activity and interactions with PAMPs  were observed. At the same time, agglutination activity and growth inhibition of bacteria had various effects on different strains, although MkC1qDC properties were not associated with their Gram classification. The investigated range of carbohydrate affinity of C1qDC protein has a unique character that, together with its ability to inhibit the growth of the HeLa cells, indicates possible implementation of MkC1qDC protein in biotechnology and biomedicine. Taking into account that the MkC1qDC protein is capable of recognizing acidic galactans and mannans, it may be of interest for creating systems for targeted delivery of therapeutic agents to cancer cells, and can also be included in diagnostic kits for phenotyping the cell surface carbohydrate profile. This is of interest for further research and development involving both native and recombinant MkC1qDC.Thus, this study first described a novel C1qDC protein, which has immune functions and is associated with the hemal system and interstitial compartment of the marine mollusk M. kurilensis. Healthy adult mollusks with shells over 70 mm in length were selected from Peter the Great Bay, Sea of Japan . Hemolymph was isolated immediately from the hemal sinus of the dorsal adductor muscle. Cells were removed by centrifugation for 15 min at 4 °C and 300× g. Plasma from different individuals was mixed, clarified by centrifugation for 30 min at 4000× g and 4 °C and stored at −80 °C until the next stage. The MkC1qDC was purified from cell-free hemolymph (plasma) of M. kurilensis plasma proteins with 0−15%, 15−30%, 30−45%, 45−60%, 60−75% and 75−85% salt saturation at 0 °C was produced. Protein coagulates were centrifuged for 30 min at 4000× g and 0 °C to precipitate. The obtained precipitates were dissolved in Tris buffer saline , dialyzed against a large volume of TBS three times at 4 °C, clarified by centrifugation  and added CaCl2 up to 15 mM just before using. The target activity was determined by hemagglutination assays described below, and included inhibition by uronic  acids and adding chelants  for Ca2+-dependence determination. The isolation of MkC1qDC was carried out in a few steps. Firstly, ammonium sulfate precipitation  of M. ku2. The elution was carried out with a large buffer solution containing a chelant . All stages were performed with the flow 0.2 mL/min. The eluate was dialyzed against TBS at 4 °C and divided into aliquots containing 100 μg of protein in cryovials, which were frozen and stored in liquid nitrogen at −196 °C.Citrus pectin (Copenhagen Pectin A/S) covalently linked to Sepharose CL-4B  by divinyl sulfone  was applied for affinity chromatography on the final purification step. Loading of prepared fractions with target activity and column washing from unbound components was performed in TBS with 15 mM CaClProtein concentration was determined by the Bradford method , using BTo analyze the quality of protein isolation at each stage, SDS-PAGE based on 12% polyacrylamide gel stained with Coomassie Brilliant Blue G-250 solution was produced .The isoelectric point (pI) and composition of the purified MkC1qDC were determined by 2D electrophoresis using ReadyStrip™ IPG strips pH of 4−7  in the first dimension and a gradient of 4−20% polyacrylamide gel in the second dimension.N-terminal sequencing was performed according to Edman’s degradation method  by a Prog for 10 min and desalted on homemade C18 stage tips as described previously [http://web.expasy.org/compute_pi/, accessed on 26 May 2021).For mass spectrometry, MkC1qDC was dissolved in 6 M guanidinium-HCl, 100 mM Tris pH 8, 10 mM chloroacetamide, 5 mM Tris (2-carboxyethyl) phosphine hydrochloride, heated at 95 °C for 5 min. The sample was diluted with water 10-fold and 1/100 trypsin/substrate was added. Digestion was performed at 37 °C for 4 h. The digest was acidified to 1% trifluoroacetic acid final concentration, spun at 15,000× eviously . Peptideeviously . This reeviously . The theHemagglutination (HA) was performed as described previously , using fTo investigate the carbohydrate specificity of MkC1qDC, a hemagglutination inhibition (HAI) test with the presence of one of 41 different saccharides was performed, 12 of which were polymers, 6 were oligomers and others were monomers . Each ca2+ with high buffering capacity .Isotonic (150 mM NaCl) buffer solutions were used to determine the pH dependence: glycine-HCl (pH 3), sodium acetate-acetic acid , sodium cacodylate-HCl , Tris-HCl (pH 8), glycine-NaOH (pH 9) and carbonate-bicarbonate (pH 10) at a concentration of 20 mM. HA activity was checked after 1 h of incubation at room temperature in TBS/CaTo study the thermal lability, MkC1qDC samples were incubated for 1 h at temperatures of 0, 10, 20, 30, 40, 50, 60 and 70 °C and then were brought to room temperature before HA reaction.Vibrio sp., Ruegeria sp., E. coli, Pseudoalteromonas sp.) and Gram-positive  bacteria from marine organisms were used for analysis. Strains grew for 2-4 days to log-phase at room temperature in the liquid medium based on sterile-filtered sea water [Gram-negative , washed three times with HEPES buffer saline  and fixed by 4% PFA solution in HBS. Next, bacterial cells were stained with a fluorescent dye Fluorescein isothiocyanate  with a final concentration of 0.1 mg/mL, vortexed at 800 rpm for 1 h, and washed 3 times in HBS. The stained suspensions with a final optical density (A600) of 1 were mixed in HBS with 50 mM CaCl2 and 0.1 mg/mL MkC1qDC, or without it as control (3 replicates for each condition). The mixtures were vortexed at 100 rpm for 1 h at room temperature. Visualization was performed using laser scanning microscope FV1200MPE-FV12M-5XX-3XX (Olympus). Agglutination assay was performed as described previously  with som600 = 0.05 and 50 μL placed into each well of the 96-well flat-bottom microplate. Then 50 μL of 0.2 mg/mL MkC1qDC solution in PBM was added in experimental wells, or 50 μL PBM as a positive growth control, or 50 μL antibiotic solution (penicillin 2000 units/mL and streptomycin 2 mg/mL) in PBM as negative control (6 replicates for each condition). The results of bacterial growth inhibition were estimated at 600 nm using the Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). The inhibition efficiency was evaluated by the delay time in the bacteria growth in the presence of lectin vs the positive control.For the evaluation of MkC1qDC antimicrobial activity, bacteria were diluted in poor broth medium  to AThe commonly used immunization protocols  with somThe immunoglobulin fraction was purified by standard protocol  using amPrimary antibody activity was measured by the commonly used protocol of indirect ELISA  with horM. kurilensis plasma and purified MkC1qDC as samples.In addition, antibody validation using Western blotting was performed by Mini Trans-Blot® Module for Mini-PROTEAN® Tetra Cell . After electrophoresis, samples were transferred onto the PVDF membrane and placed for 2 h in a 3% BSA solution in TBS with 0.05% Tween-20 (TBST). The purified primary antibody was used at 1/2500, 1/5000 and 1/10000 dilutions  in 0.5% BSA prepared on TBST, followed by incubation at 23 °C for 2 h. Then it was incubated with mouse antirabbit secondary antibody conjugated with horseradish peroxidase  at 1/20000 dilution in TBST, and incubated for 1 h at 23 °C. After that, a substrate for chemiluminescent staining Pierce ECL Plus Western Blotting Substrate  was applied to the membrane according to the industrial protocol. The result was recorded on a ChemiDoc Touch Imaging System . Analysis was performed using native 2HPO4-KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4), washed 10 times in PBS and dehydrated through a progressive series of ethanol, infiltrated by xylene and embedded in paraffin. Tissue sections with a thickness of 10 μm were obtained using an HM-360 rotary microtome . The paraffin sections were dewaxed and washed 3 times in PBS with 0.05% Tween-20 (PBST) with further staining without drying.Tissue fragments of the intestine, mantle, muscle, pericardium, gills, gonads, digestive gland and kidney were excised and immediately fixed for 2 h in paraformaldehyde (PFA) solution in phosphate buffered saline  directly to the culture medium to a final concentration of 1.5%. Then the liquid was taken from the wells, 300 μL of cold methanol was added to them and left at 4 °C for 10 min, after which the chamber was left for longer storage at –20 °C. Immediately before staining, hemocyte preparations were washed 3 times with PBST solution.Suspension of live hemocytes was obtained as previously described , dilutedFurther, in order to permeabilize the membranes, PBS with 0.5% Triton X-100 was applied to the sections and hemocytes for 10 min at 23 °C. The primary antibodies in PBS with 0.5% BSA at a dilution of 1/500 were incubated with preparations for 2 h at 23 °C; secondary goat antibodies conjugated with Alexa Fluor-488  with the same dilution were incubated for 1 h at 23 °C; nuclei were stained by DAPI  with a concentration of 1 μg/mL in PBS for 5 min at 23 °C. Between all steps, samples were washed 3 times for 10 min in PBST and, after the last step of staining, embedded in the water-soluble medium Mowiol 4-88 . The described procedure with pre-immune rabbit serum as primary antibody was used as a control. The samples were analyzed using FluoView FV1200MPE-FV12M-5XX-3XX laser scanning microscope (Olympus).2. The cells were cultured at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle’s medium  supplemented with 10% FBS. The cells were cultured for their adhesion and growth stabilization for 18 h, then treated with 1, 2 and 4 μg/mL of MkC1qDC and monitored for 90 h applying time-lapse phase-contrast imaging. Untreated wells were utilized as a control sample group. All series were cultivated and analyzed in six replicates. Cell proliferation data are presented as growth curves indicating total cell count produced automatically based on machine learning. Cell proliferation assay was performed using a high-content imaging system Cell-iQ MLF (CM Technologies). Human adenocarcinoma cell line HeLa (ATCC) was plated in tissue-culture grade 24-well plates at a density of 15,000 cells/cmp-value less than 0.05 was considered to be statistically significant. Experimental results are presented as a mean ±95% confidence interval.Proteomics experiments were conducted on one purified protein sample. For the analysis of MkC1qDC effect on bacteria and HeLa growing, the Mann–Whitney U test and Kruskal–Wallis H test were used. A


Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries and are currently used around the world via Asian catering. The MPs biosynthetic pathway has been well-illustrated; however, the functions of a few genes including mrpigH in the MPs gene cluster of M. ruber M7 are still unclear. In the current study, mrpigH was disrupted in Δmrlig4ΔmrpyrG, a highly efficient gene modification system, using mrpyrG as a selection marker, and ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG have been obtained. Subsequently, their morphologies, biomasses, MPs and citrinin (CIT) production were analyzed, respectively. These results have revealed that the deletion of mrpigH has significant effects on the morphology and growth of M. ruber M7. Moreover, compared with M. ruber M7, the yields of MPs and CIT were drastically increased and decreased in mrpigH mutants, respectively. Monascus species are famous medicinal and edible filamentous fungi used in traditional fermentation in Asian countries, such as China, Japan, and the Korean Peninsula for nearly 2000 years [Hongqu, also called red fermented rice, red yeast rice and red mold rice, are widely used as food additives and nutraceutical supplements worldwide owing to their production of abundant beneficial secondary metabolites (SMs), such as Monascus pigments (MPs), monacolin K (MK) and γ-amino butyric acid (GABA) [Monascus spp., restricts the application of Monascus fermented products [00 years ,2. At prd (GABA) ,3. Howevproducts .M. ruber M7, which can produce MPs and CIT, without MK, was subjected to whole-genome sequencing analysis [M. ruber M7 have been investigated by gene manipulation [M. ruber M7, such as mrpigH and mrpigI, which have not been investigated [mppE in M. purpureus KACC (highly homologous to mrpigH in M. ruber M7) encoded a reductase which can decrease orange pigments (OPs) and red pigments (RPs) in the biosynthesis of MPs [analysis . And thepulation . Howeverstigated ,7. In 20s of MPs . In 2019mrpigH from M. ruber M7. Subsequently, mrpigH was disrupted in the highly efficient system Δmrlig4ΔmrpyrG [mrpyrG selection marker, and two mrpigH deletion strains ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG have been constructed. Finally, the morphologies, biomasses, MPs and CIT production of these mrpigH mutants were assessed. The results revealed that the deletion of mrpigH led to a dramatic reduction in biomass accumulation. Crucially, the inactivation of MrPigH resulted in an increase of MPs and a decrease of CIT.In this study, we firstly cloned 4ΔmrpyrG  using thM. ruber M7 , which can produce MPs and CIT, but no MK, is an original strain, which was isolated from Hongqu and preserved in our laboratory [mrlig4ΔmrpyrG, the highly efficient gene modification system for M. ruber M7, also named as the markerless disruption strain [mrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG. All strains used in this study are described in 4Cl, 1 g (NH4)2SO4, 0.5 g KCl, 0.5 g NaCl, 1.0 g KH2PO4, 0.5 g MgSO4·7H2O, 0.02 g FeSO4·7H2O, 20.0 g glucose, distilled water to 1 L, pH 5.5). When required, 10 mM uridine and/or 0.75 mg/mL 5-fluoroorotic acid (5-FOA) were added. For observation of colonial and microscopic morphologies, four different types of media, PDA, malt extract agar (MA), Czapek yeast extract agar (CYA) and 25% glycerol nitrate agar (G25N) were utilized [boratory . Δmrlig4n strain  was usedutilized . PDA wasmrpigH using Oligo 6 software . PCR was carried out to amplify mrpigH from the genome of M. ruber M7. Amino acid sequence encoded by mrpigH was predicted using Softberry’s FGENESH program , and the MrPigH functional regions were analyzed using the Pfam 33.1 program . Homology of the deduced amino acid sequence was analyzed using the BLASTP program on the NCBI website .A pair of primers, pigH F1–pigH R1 , was desM. ruber M7 used for PCR was isolated as described previously [mrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG were constructed using site-directed homologous recombination. The mrpigH gene markerless deletion cassette (5′UTR-mrpyrG-5′-1UTR-3′UTR) was constructed by seamless cloning, and shown schematically in The genomic DNA of eviously . The mutmrpigH were amplified with the primers pigHpyrG 5F–pigHpyrG 5R, pigHpyrG 3F–pigHpyrG 3R and pigHpyrG 5F-1–pigHpyrG 5R-1, respectively. The 1.28-kb mrpyrG marker cassette was amplified from M. ruber M7 genomic DNA with the primer pair pigHpyrG pyrGeF–pigHpyrG pyrGeR. Then the four amplicons  were mixed at a 1:1:1:1 molar ratio and cloned into vector pBLUE-T using the seamless cloning and assembly kit (Beijing Zoman Biotechnology). Subsequently, both the cloned DNA fragment and the pCAMBIA3300 plasmid were digested with HindIII and XbaI, and ligated by T4 DNA ligase to generate plasmid pCPGPIGH for the mrpigH knock-out harbouring mrpyrG selection marker.The 5′ and 3′ flanking regions  and the 5′-1 flanking region (531 bp) of Agrobacterium tumefaciens EHA105 using a freeze-thaw method [mrlig4ΔmrpyrG, a markerless disruption strain, was used as a host strain to delete mrpigH with the mrpyrG recyclable marker [A. tumefaciens clones containing pCPGPIGH were incubated for transformation with Δmrlig4ΔmrpyrG to generate a mrpigH gene deletion mutant (ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG) by minimal medium without uridine/uracil. The conidia of ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG were collected and spread onto PDA with 0.75 mg/mL 5-FOA and 10 mM uridine. After incubated at 28 °C for 6 days, the surviving colonies were transferred to a new PDA under the same conditions for 4 days. The final surviving colonies were selected and verified by PCR. Selected transformants were designated as ΔmrpigHΔmrlig4ΔmrpyrG.The plasmid pCPGPIGH was transformed into w method . Δmrlig4e marker . The A. M. ruber M7 can produce MPs and CIT, but no MK. Previous researches have shown that MPs mainly accumulate in the mycelia, while CIT exists in the media [5 cfu/mL) of each strain were inoculated on PDA plate coated with cellophane membranes and incubated at 28 °C for 11 days. 20 mg freeze-dried mycelia or media powder were suspended in 1 mL 80% (v/v) methanol solution, and subjected to 30 min ultrasonication treatment . Then, the extraction solutions were separated by centrifugation at 10,000× g for 15 min and filtered with a 0.22 μm filter membrane for further analysis.he media . TherefoThe pigments groups concentration was measured using a UV−vis UV-1700 spectrophotometer  at 380, 470 and 520 nm which are the maximal absorption of yellow, orange, and red pigments, respectively. The results were expressed as optical density (OD) units per gram of dried mycelia multiplied by a dilution factor .The CIT was detected on Waters ACQUITY UPLC BEH C18 column  by fluorescence detector  in accordance with a previously described method .mrpigH deletion on gene expression in MPs and CIT gene cluster, ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and the wild-type strain (M. ruber M7) were selected for quantitative real-time PCR (RT-qPCR) detection. The ΔmrpigHΔmrlig4ΔmrpyrG was lacked of mrpyrG and had to supply uridine, which might have had an effect on the yields of MPs and CIT.To analyze the influence of 5 cfu/mL) of each strain were inoculated on PDA plate and incubated at 28 °C and samples were taken every other day from the third day to the ninth day. RT-qPCR was performed according to the method described by Liu et al. [One milliliter freshly harvested spores  which consists of one exon and encodes 369-amino acids. A database search with Pfam 33.1 program has shown that MrPigH pertains to the alcohol dehydrogenase GroES-like domain. Besides, a database searched with NCBI-BLAST has been demonstrated that the deduced 369-amino acid sequences encoded by mrpigH share 65.31% similarity with the enoyl reductase (GenBank: PCH03974.1), 57.84% similarity with oxidoreductase of Glonium stellatum (GenBank: OCL03635.1), and 56.64% similarity with dehydrogenase of Hyphodiscus hymeniophilus (GenBank: KAG0646231.1). The specific function of mrpigH is still unclear.A 1.24-kb fragment containing the putative mrlig4ΔmrpyrG strain is a promising host for efficient gene targeting in M. ruber M7 and analysis of biosynthesis of SMs. The deletion of mrpigH was executed in Δmrlig4ΔmrpyrG with the mrpyrG marker  and Δmrlig4ΔmrpyrG(2.85 kb) differed in size when primers pigHpyrG 5F- pigHpyrG 3R annealing to homologous arms were used.The ΔG marker a. After mrpigHΔmrlig4ΔmrpyrG::mrpyrG strain, the mrpigH deletion cassette contains two completely homologous sequences (5′UTR and 5′-1UTR) between the mrpyrG expression fragments. If ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG was incubated on PDA plates containing 5-FOA, the strains that lost the mrpyrG expression fragments by homologous recombination should be selected. As predicted, ΔmrpigHΔmrlig4ΔmrpyrG strains without the mrpyrG fragment could be isolated from ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG after growth on PDA containing 0.75 mg/mL 5-FOA and 10 mM uridine. Total 12 putative ΔmrpigHΔmrlig4ΔmrpyrG strains with 5-FOA resistance were obtained and analyzed, and one of them was shown as follows. In PCR analysis as shown in mrpigH gene and a 0.5-kb product of the mrpyrG gene could be amplified from M. ruber M7 using primers pigH F-pigH R and pyrG F2-pyrG R2, while nothing was obtained from ΔmrpigHΔmrlig4ΔmrpyrG, respectively. Meanwhile, amplicons of ΔmrpigHΔmrlig4ΔmrpyrG (1.73 kb) and M. ruber M7 (2.85 kb) differed in size when primers pigHpyrG 5F- pigHpyrG 3R annealing to homologous arms were used. Those results indicated that there was mrpigH markerless deletion was constructed in Δmrlig4ΔmrpyrG.In the ΔmrpigH was markerlessly deleted by mrpyrG in Δmrlig4ΔmrpyrG, M. ruber M7 and its mrpigH markerless mutants were cultivated on PDA supplemented the appropriate additive (10 mM uridine for the uridine/uracil auxotrophy).The results  to observe their colonial and microscopic characteristics. The results showed that the colonial morphologies of mrpigH mutants  than 2 mrpigH mutants (ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG), whereas the ability of producing MPs among 2 mrpigH mutants showed no obvious difference. After 11 days of cultivation, the YPs, OPs and RPs production in ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG were 2.04–2.22, 8.76–10.06, 4.29–4.69 times those of M. ruber M7, respectively.In order to evaluate the effect of detecting M. ruber M7 and all mrpigH mutants showed an obvious difference. At the end of the 11 days of fermentation, CIT production in ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG decreased dramatically, and was two to three orders of magnitude less than that of M.ruber M7. Among 2 mrpigH mutants, CIT production in ΔmrpigHΔmrlig4ΔmrpyrG was higher than that of ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG. The possible cause was that the ΔmrpigHΔmrlig4ΔmrpyrG lacked mrpyrG and had to supply uridine, which might have an effect on the yields of CIT. As to CIT, the UPLC has been performed to detect the production during fermentation. As shown in mrpigHΔmrlig4ΔmrpyrG::mrpyrG and M. ruber M7, were analyzed by RT-qPCR. As shown in mrpigA, mrpigB, mrpigC, mrpigD, mrpigE, mrpigF, mrpigG, mrpigJ, mrpigK, mrpigM, mrpigN, mrpigO and mrpigP in ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG were obviously higher than that of M. ruber M7 on the fifth to ninth days. Therefore, the deletion of mrpigH increased the majority of MPs gene expression level, which might correspond with the enhanced MPs production.The gene expression in MPs and CIT gene clusters in ΔM. aurantiacus including 16 genes. He and Cox [mrl7), dehydrogenase (mrl6), glyoxylase-like domain (mrl5), NAD(P)+ dependent aldehyde dehydrogenase (mrl4), transcriptional regulator (mrl3), Fe(II)-dependent oxygenase (mrl2), serine hydrolase (mrl1), non-reducing polyketide synthase (mrpks),and major facilitator superfamily (MFS) protein (mrr1). As shown in mrl6, mrl5, mrl4, mrl2, mrl1, mrpks and mrr1 were dramatically lower than those of M. ruber M7 at 3rd and 5th day. Therefore, the deletion of mrpigH decreased the expression level of core CIT genes, which might correspond with the reduced CIT production. In a previous study, Li et al.  consider and Cox  demonstrMonascus spp. have been widely used in food fermentation for nearly 20 centuries in East and Southeast Asian countries due to their ability of producing vivid MPs [Monascus spp. and Penicillium spp. [mrpigA-mrpigP) in M.ruber M7 [ivid MPs ,19, whicium spp. ,20,21. Mruber M7 ,7.mrpigH, we used the Δmrlig4ΔmrpyrG as the starting strain to generate two kinds of mrpigH disruptants, namely ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG. The colonial and microbiological phenotypes and biomasses of the mrpigH mutants showed an obviously difference from those of M. ruber M7 (mrpigH mutants accumulated more slowly than that of M ruber M7. Our laboratory previously constructed MPs gene knockout strains with the resistance selection markers (hph/neo) in M7, and found that the morphologies and biomasses of mrpigC, mrpigE, mrpigF, mrpigM and mrpigO knockouts were comparable to those of M. ruber M7, whereas the growth rates of mrpigA, mrpigJ, and mrpigK knockouts were increased, and the deletion of mrpigN resulted in a reduction in biomass accumulation compared with M. ruber M7 [In the current study, in order to explore the function of ruber M7 b, d. In ruber M7 .mrpigH in M. ruber M7 can enhance YPs, OPs and RPs  and Liu et al. (2014) separately identified that the overexpression of mrpigB and mrpigE in MPs gene cluster of M. ruber M7 resulted in a reduction of CIT production [orf6) in CIT gene cluster of M. purpureus, and found that the deletion of orf6 could improve the MPs and CIT yields at the same time [M. ruber M7), yielding the typical YPs monascin and ankaflavin. The other branch of the pathway produces the typical OPs rubropunctatin and monascorubrin by a FAD-dependent oxidoreductase (MrPigF in M. ruber M7). Furthermore, rubropunctatin and monascorubrin were converted into RPs rubropunctamine and monascorubramine through an amination reaction [mrpigH, the relative expression of mrpigF dramatically increased . When mrpigH was deleted, the majority of the genes in the cluster  were obviously down-regulated  increased dramatically. However, the CIT production of the mrpigH mutants decreased drastically. This work will make some contribution to the regulation of MPs and CIT production in M. ruber M7.In conclusion, the


Human T-lymphotropic virus type-1 (HTLV-1) is a neglected infection most often associated with an indolent process. However, a subset of HTLV-1 seropositive patients face the risk to develop life-threatening T-cell lymphoma/leukemia, or the highly disabling and incurable HTLV1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Over the years, other complications to HTLV-1 have been proposed and debated intensely. One of these, although rare, associations include cerebellar ataxia occurring most often in Japanese patients with manifest HAM/TSP. Here we present a HTLV-1 seropositive patient from the Middle East featuring a slowly progressive cerebellar syndrome with cerebellar atrophy but not evidence of spastic paraparesis. In addition, this patient suffered from autoimmune conditions such as Sjögren’s syndrome and vitiligo which are putatively associated with HTLV-1. Infection with human T cell lymphotropic virus type 1 (HTLV-1) is in most cases indolent; however, some patients develop adult T cell leukemia, associated with poor prognosis, or the highly disabling and incurable HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) , normal Achilles reflexes, and absence of Babinski’s sign. In addition, her muscle tone, strength, and sensation to touch and pin prick were normal. Vibration sense in her malleoli and proprioception were impaired with subsequent abnormal Romberg’s test; the retropulsion test was normal. Her altering hand movements were irregular; bradykinesia was absent. The patient also displayed hypermetric saccades. Her Scale for the Assessment and Rating of Ataxia score rose from 7.5 to 9.5 and Instituto de Pesquisa Clinica Evandro Chagas HAM disability scale, from 8 to 10, over 5 years.Neuroimaging demonstrated cerebellar atrophy Fig.  but not The most common genetic ataxias, syphilis, Lyme’s and Whipple’s disease, and autoimmune ataxias were ruled out (Supplementary document). Thus, onconeuronal antibodies, antibodies for celiac disease, and against GAD were absent. PVL in blood was initially 0.54% and 1.4% when repeated whereas serum β2M increased from 2.5 to 4.4 mg/L (ref < 2.0) over 4 years. In the CSF, white cell count, albumin concentration, and neurofilament light protein (NfL) concentration were normal, with no malignant cells, protein 14-3-3 or oligoclonal bands. However, CSF PVL (4 HTLV DNA copies/15 cells), neopterin 3.7 pg/mL (reference 2–3 pg/mL), and CXCL10 249.1 pg/mL (reference 115–160.8 pg/mL) were high.Computed tomography of her chest and abdomen showed an enlarged thyroid gland but the patient was euthyroid; a biopsy aspirate demonstrated colloid cells only. Mammography was initially normal but at age 68, she was diagnosed with a breast tumor treated with surgery and chemotherapy. Oral steroids were given with chemotherapy, but this did not alleviate the patient’s motor symptoms.So far, 20 HTLV-1 seropositive patients displaying ataxia have been reported, mostly in Japan. All but two had manifest HAM/TSP, which suggests that pyramidal signs may overshadow subtle cerebellar signs and nystagmus . In this case, β2M and PVL increased during follow-up and PVL in the CSF was high. Of note, determining PVL in the CSF is challenging due to the low number of leukocytes in CSF.Importantly, neurological complications are associated with high PVL . β2M is a component of the major histocompatibility complex class 1 and a key element in immediate immune response. As a consequence of the inflammatory reaction, chronic inflammation of the nervous system may occur in predisposed individuals, leading to permanent neurological dysfunction. The pattern of interleukin elevation in the CSF has been reported in HAM/TSP patients with very slow progression  Supplementary file2 (DOC 59 KB)Below is the link to the electronic supplementary material.