HTLV-1 infection is a neglected disease, despite infecting 10–15 million people worldwide and severe illnesses develop in 10% of carriers lifelong. Acknowledging a greater risk for developing HTLV-1 associated illnesses due to immunosuppression, screening is being widely considered in the transplantation setting. Herein, we report the experience with universal HTLV testing of donors and recipients of solid organ transplants in a survey conducted in Spain.All hospitals belonging to the Spanish HTLV network were invited to participate in the study. Briefly, HTLV antibody screening was performed retrospectively in all specimens collected from solid organ donors and recipients attended since the year 2008.A total of 5751 individuals were tested for HTLV antibodies at 8 sites. Donors represented 2312 (42.2%), of whom 17 (0.3%) were living kidney donors. The remaining 3439 (59.8%) were recipients. Spaniards represented nearly 80%.Overall, 9 individuals (0.16%) were initially reactive for HTLV antibodies. Six were donors and 3 were recipients. Using confirmatory tests, HTLV-1 could be confirmed in only two donors, one Spaniard and another from Colombia. Both kidneys of the Spaniard were inadvertently transplanted. Subacute myelopathy developed within 1 year in one recipient. The second recipient seroconverted for HTLV-1 but the kidney had to be removed soon due to rejection. Immunosuppression was stopped and 3 years later the patient remains in dialysis but otherwise asymptomatic.The rate of HTLV-1 is low but not negligible in donors/recipients of solid organ transplants in Spain. Universal HTLV screening should be recommended in all donor and recipients of solid organ transplantation in Spain. Evidence is overwhelming for very high virus transmission and increased risk along with the rapid development of subacute myelopathy. HTLV-1 infection is a neglected disease. As with other human retroviruses, i.e. HIV, acute infection is universally followed by long-lasting persistence of the virus. Clinical manifestations develop in roughly 10% of carriers lifelong, typically as subacute myelopathy which is known as tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM) . ScreeniSpain is leading solid organ transplantation worldwide. With 47 million people, 5261 transplants were performed in 2017 (kidney 3269), liver (1247), lung (363) and heart (304). Overall it represents 20% of transplants within the European Union. Until recently, the Spanish law only recommended HTLV testing for persons with high epidemiological risk of infection [A nationwide network of hospitals/clinics/labs was set up in Spain in 1989 and a national register of HTLV-1 cases works since then. Currently, more than 50 centres distributed across the Spanish geography belong to this group. Further information on this network has been reported elsewhere . All hosT-test or Mann-Whitney U tests. All analyses were 2-tailed and only p values below 0.05 were considered as significant. All statistical analyses were performed using SPSS software version 16.0 .All numerical variables are reported as absolute values and percentages. Categorical variables were compared using χ2 or Fisher exact tests whereas non-categorical variables were compared using Student A total of 5751 individuals were tested for HTLV antibodies at 8 sites. Table Overall, 9 individuals were initially reactive for HTLV antibodies. Table The two HTLV-1 positive cases were donors. Thus, the overall rate of HTLV-1 among solid transplant donors in our study was 0.09%. The first individual was a 49-year old female, born in Colombia, diagnosed in 2015, at the time she was assessed to be a kidney living donor. Before moving to the surgical procedure, she was discharged as potential donor following knowledge of her HTLV-1 seropositive status.The second HTLV-1 case was a 38-year old male, born in Spain, that died in 2015. Both kidneys were inadvertently transplanted. TSP/HAM developed within 1 year in one recipient, despite antiretroviral prophylaxis attempted within the first weeks. The second recipient seroconverted for HTLV-1 but the kidney had to be removed soon due to rejection. Immunosuppression was stopped and to date, 3 years later, the patient remains in dialysis but otherwise asymptomatic.The three HTLV-2 cases were two donors and one recipient. All had been born in Spain. The last patient died before transplantation. The two donors were finally discharged from transplantation because they were also positive for HIV and hepatitis C.not to donate blood, semen, body organs or other tissues” [More than 35 years after the discovery of HTLV-1 , donor/rtissues” . Neverthtissues” . AlongsiHigh rates of HTLV-1 have been found in specific groups in non-endemic regions of North America  and EuroSince the year 1988 there is a national register of HTLV-1 cases in Spain. A total of 369 cases had been reported until December 2018. Most cases are concentrated around the largest urban areas (Madrid and Barcelona) where the greatest immigrant populations are living. However, HTLV-1-infected persons have been identified across the whole Spanish geography. Although many were immigrants from endemic areas, mostly in Latin America, up to 18% were native Spaniards [The rapid development of subacute myelopathy was seen in one of our recipients from a kidney from the HTLV-1 positive cadaveric donor. Similar cases of subacute myelopathy shortly after transplantation have already been reported in the literature –14, inclIn response to the worldwide evolving foci of HTLV-1 infections and the very poor prognosis of post-transplant virus-associated illnesses, there are urgent calls for a broader HTLV-1 screening of live and -more difficult- deceased organ donors. Organ procurement organizations and transplant programs should determine local prevalence to guide HTLV-1 screening efforts . Targetepoint-of-care (PIC) assays) and improve the specificity of HTLV-1 screening tests to minimize unwanted organ discharge.The screening costs for HTLV-1 are small in comparison with the cost of post-transplant illnesses and/or death associated with TSP or ATL following HTLV-1 infection . For allOur findings call into question the current view that anti-HTLV screening of donated organs is not needed or just recommended when there is suspicion, as it is recommended in Spain . This opWe report a low but not negligible rate of HTLV-1 infection among donors and recipients of solid organ transplants in Spain. Of note, unaware HTLV-1-infected donors were not exclusively foreigners from highly endemic areas but native Spaniards that most likely have been exposed to HTLV-1 by sexual contact. Therefore, universal HTLV screening should be recommended in all donor and recipients of solid organ transplantation in Spain. Evidence is overwhelming for very high -if not uniform- virus transmission and increased risk as well as rapid disease progression, mostly subacute myelopathy.


HTRA1 and NOTCH3. The protease HTRA1 cleaves the NOTCH3 ligand JAG1 implying a mechanistic link between HTRA1 and Notch signaling. Here we report that HTRA1 is essential for VSMC differentiation into the contractile phenotype. Mechanistically, loss of HTRA1 increased JAG1 protein levels and NOTCH3 signaling activity in VSMC. In addition, the loss of HTRA1 enhanced TGFβ-SMAD2/3 signaling activity. Activation of either NOTCH3 or TGFβ signaling resulted in increased transcription of the HES and HEY transcriptional repressors and promoted the contractile VSMC phenotype. However, their combined over-activation led to an additive accumulation of HES and HEY proteins, which repressed the expression of contractile VSMC marker genes. As a result, VSMC adopted an immature phenotype with impaired arterial vasoconstriction in Htra1-deficient mice. These data demonstrate an essential role of HTRA1 in vascular maturation and homeostasis by controlling Notch and TGFβ signaling.Vascular smooth muscle cell (VSMC) dysfunction is a hallmark of small vessel disease, a common cause of stroke and dementia. Two of the most frequently mutated genes in familial small vessel disease are  NOTCH3 and HTRA1 being two of the most frequently mutated genes1. In addition, several loss-of-function mutations in HTRA1 are causative for cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL syndrome)2, which is similar to dominantly inherited CADASIL syndrome caused by neomorphic NOTCH3 mutations3. A common feature of these diseases is vascular smooth muscle cell (VSMC) dysfunction on small arterial blood vessels leading to episodes of impaired blood perfusion in certain brain regions.Familial small vessel disease is a major cause of stroke and dementia under the age of 60 with 4.Since VSMC are critical regulators to maintain vascular homeostasis they show high phenotypic plasticity, where contractile and synthetic VSMC represent the two ends of a spectrum with intermediate phenotypes, which have different morphologies and functions. While naïve VSMC display a synthetic phenotype and are unable to contract but important for maintenance, contractile VSMC control blood flow and pressure. During development, vascular remodeling and injury, synthetic VSMC secrete extracellular matrix proteins and exhibit higher growth rates and migratory activity than contractile VSMCHES and HEY genes. These encode basic helix-loop-helix (bHLH) transcription factors, which repress gene expression through either binding other bHLH factors or through interacting directly with DNA at promoter regions5. In muscle stem cells, HeyL interacts with Hes1 to bind DNA sites with high affinity causing anti-myogenic effects6. In VSMC, HES and HEY proteins can inhibit transcription of contractile VSMC marker proteins8. As such, the effect of Notch signaling on promoting the contractile VSMC phenotype can be counteracted by HES and HEY bHLH factors. This indicates that the outcome of Notch signaling activity is strictly dose-dependent.Notch signaling is a juxtacrine signaling mode, which controls numerous cell differentiation processes. The signal sending cell expresses Notch ligands of the Delta-like (DLL) and Jagged (JAG) families which activate Notch receptors on adjacent signal receiving cells. The interaction induces receptor cleavage and translocation of the Notch intracellular domain (ICD) to the nucleus, where it interacts with RBP-Jκ and promotes cell type-specific gene expression and induction of the 9. Interestingly, TGFβ signaling can also activate HEY and HES gene expression in certain cell types11. Provided that this also occurs in VSMC, HTRA1 might function through controlling expression levels of the HES and HEY transcriptional repressors via Notch and TGFβ signaling.Similar to the Notch pathway, TGFβ signaling has also been shown to promote VSCM differentiation13 and is known to cleave several intracellular17 and extracellular substrates18. Loss of HTRA1 leads to increased levels of TGFβ1 availability and TGFβ1 signaling, potentially caused by the ability of HTRA1 to cleave either pro-TGFβ1 or GFD621. Recently, we have shown that the Notch ligand JAG1 is a substrate for HTRA1. After cleavage of JAG1 by HTRA1 in the cytosol the remaining JAG1 protein was rapidly degraded22. NOTCH3 and JAG1 are both abundantly expressed on VSMC8. In arterial blood vessels, JAG1/NOTCH3 signaling is required for differentiation, maintenance and contractility of VSMC27, which is crucial for vasoconstriction and proper organ perfusion. Such blood vessel functions are impaired in familial small vessel disease. Thus, we hypothesized that HTRA1 functions not only to control TGFβ signaling but also to fine-tune NOTCH3 activity in VSMC by regulating the abundance of its ligand JAG1. As both signaling pathways are critically involved in controlling VSMC differentiation29, loss of HTRA1 may lead to impaired VSMC function and vessel contraction capacity.Here we aimed at better understanding how the serine protease HTRA1 controls Notch and TGFβ signaling in VSMC and how this affects the VSCM phenotype. HTRA1 is strongly expressed in VSMC and endothelial cells3, as well as our recent finding that HTRA1 cleaves the Notch ligand JAG122, prompted us to investigate the potential interplay between HTRA1 and NOTCH3 signaling. Therefore, HTRA1 was silenced in primary human umbilical artery SMC (HUASMC) using established siRNAs22 . We observed that silencing HTRA1 increased mRNA levels of the Notch target genes HES1 and HEYL   Fig. .Figure 4−/− mice. As expected these had lower α-SMA mRNA expression levels compared to aortic VSMC isolated from their wildtype littermate controls  with a resulting flow of ~0.07 mL/min. Arteries which showed no myogenic response were excluded from the analyses. Pressure-induced changes in vessel diameter were measured using the VediView software .40. Transfection of cells was done as previously described22. Generation of lentivirus and transduction of cells with lentivirus was done as shown elsewhere41. To inhibit Notch signaling, cells were incubated with 25 µM DAPT (Merck) overnight. TGFβ signaling was inhibited by adding 10 µM ALK5 inhibitor  for two hours to basal cell medium.VSMC were freshly isolated and cultured as previously describedFor the co-culture experiment, the same numbers of HUASMC were seeded with 3T3 cells expressing a TGFβ reporter (Luciferase cassette under the control of 12 SMAD binding sites (5′-CAGA-3′) and cultured overnight in basal medium. Luciferase activity was determined using the Luciferase Assay System  and a multiwell plate reader .Adenovirus was obtained from abm Inc. : HES1 (096900 A), HEYL (096954 A). HUASMC were infected with MOI = 50 and cell lysate were prepared 24 hours later.Mice were sacrificed by cervical dislocation. After exposing the heart and the descending aorta, fat and adventitia were dissected. The aorta was removed and rinsed in a 60 mm dish with cold PBS containing penicilin and streptomycin and cut into small rings, approximately 1 mm long. Rings were transferred in new 35 mm dishes with 2 ml DMEM with 15% FBS and penicilin/streptomycin containing collagenase at a 10 mg/ml concentration. Rings were incubated overnight. On the next day aortic rings and cells in suspension were transferred to a 15 ml tube, centrifuged  and suspended in DMEM with 15% FBS. Cells were incubated until confluence was reached.Total RNA was isolated using the innuPREP RNA Mini Kit . cDNA was synthesized from 1 µg of DNA-free total RNA using M-MLV Reverse Transcriptase (Life Technologies) and random hexamer primers (Life Technologies). Gene-specific transcription levels were determined using the POWER SYBR-Green Mastermix (Life Technologies) and an ABI StepOnePlus real-time cycler (Applied Biosystems). Ct-values were determined with LinReg software and normalization was done with the housekeeping genes OAZ1 (human) and Gapdh (mouse). Primers used were as follows: hOAZ (fw): 5′-gagccgaccatgtcttcatt-3′, hOAZ (rev): 5′-ctcctcctctcccgaagact-3′; mGapdh (fw): 5′-aactttggcattgtggaagg-3′, mGapdh (rev): 5′-acacattgggggtaggaaca-3′; hHES1 (fw): 5′-tcaacacgacaccggataaa-3′, hHES1 (rev): 5′-ccgcgagctatctttcttca-3′; mHes1 (fw): 5′-gtcacctcgttcatgcactc-3′, mHes1 (rev): 5′-tctggaaatgactgtgaagca-3′; hHES5 (fw): 5′-gcatggcccccagca-3′, hHES5 (rev): 5′-acgaaggctttgctgtgctt-3′; mHes5 (fw): 5′-caaggagaaaaaccgactgc-3′, mHes5 (rev): 5′-ggctttgctgtgtttcaggt-3′; hHEYL (fw): 5′-gcaagccaggaagaaacaca-3′, hHEYL (rev): 5′-agaatcctgtcccaccagtg-3′; mHeyL (fw): 5′-tatgatccctctgcgcttct-3′, mHeyL (rev): 5′-catcgatgtgggtcaagaga-3′; hα-SMA (fw): 5′-ctgttccagccatccttcat-3′, hα-SMA (rev): 5′-ccgtgatctccttctgcatt-3′; hSMTN (fw): 5′-cctggtgcacaacttcttcc-3′, hSMTN (rev): 5′-aagacacacttggggtcagg-3′; hCNN1 (fw): 5′-ggagctgagagagtggatcg-3′, hCNN1 (rev): 5′-gtgccaattttgggttgact-3′; hMYH11 (fw): 5′-gtgaacgcactcaagagcaa-3′, hMYH11 (rev): 5′-tattcactggccttggttcc-3′Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Whatman). The membranes were blocked with 5% skim milk or 3% bovine serum albumin in 0.1% Tween-20 in TBS and incubated with primary antibodies at 4 °C over-night. The membranes were incubated with peroxidase-conjugated secondary antibody for 2 hours at room temperature followed by ECL detection of the antibody. Band intensities were analyzed by the Image lab software (Biorad).Anti-JAG1 , anti-HTRA1 , anti-GAPDH , anti-α-SMA , anti-SM22α , anti-NOTCH3 , anti-myosin , pSMAD2/3  were used as primary antibodies. F-actin was stained with Alexa Fluor 568-conjugated phalloidin (Invitrogen).HUASMC were seeded on glass slides coated with 0.5% gelantine. Cells were washed twice with PBS, fixed with 4% PFA for 10 min, washed three times with PBS, permeabilized with PBS-T (containing 0.1% TritonX) and washed again three times with PBS. After blocking with 3% BSA in PBS, cells were incubated with the primary antibodies over night at 4 °C and secondary antibodies  for 1 hour at room temperature. Sections were counterstained with DAPI, washed three times with PBS and mounted with Fluoromount . Confocal images were obtained using an LSM 700 microscope (Carl Zeiss) and analyzed using Fiji software.Statistical analysis was performed with the Prism 6.0 software (GraphPad Software). Statistical significance was determined by an unpaired student t-test for two samples or by 1-way ANOVA followed by Dunnett post-hoc test when more than 2 groups were compared. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001.Human umbilical smooth muscle cells were freshly isolated from umbilical cords according to the declaration of Helsinki and with approval of the Heidelberg University ethics review board. Written informed consent from the donor was obtained prior to inclusion in the study. The Animal Care and Use Committee of the Bezirksregierung Baden-Württemberg, Germany, approved all procedures performed on mice.Supplementary figure


During the last 100 years, cell signaling has evolved into a common mechanism for most physiological processes across systems. Although the majority of cell signaling principles were initially derived from hormonal studies, its exponential growth has been supported by interdisciplinary inputs, e.g., from physics, chemistry, mathematics, statistics, and computational fields. As a result, cell signaling has grown out of scope for any general review. Here, we review how the messages are transferred from the first messenger (the ligand) to the receptor, and then decoded with the help of cascades of second messengers . The message is thus relayed from the membrane to the nucleus where gene expression ns, subsequent translations, and protein targeting to the cell membrane and other organelles are triggered. Although there are limited numbers of intracellular messengers, the specificity of the response profiles to the ligands is generated by the involvement of a combination of selected intracellular signaling intermediates. Other crucial parameters in cell signaling are its directionality and distribution of signaling strengths in different pathways that may crosstalk to adjust the amplitude and quality of the final effector output. Finally, we have reflected upon its possible developments during the coming years. Gr., arousing or excite) to explain, “The chemical messengers which spread from cell to cell along the bloodstream, may coordinate the activities and growth of different parts of the body” . . 146]. T neurons . Another neurons . In the  neurons , while i neurons   pathway where a loop exists from mTOR complex-1 to its downstream ribosomal S6 kinase to insulin/IGF signaling and mTOR complex-2. These proteins regulate many cellular processes by forming two biochemically and functionally distinct complexes mTORC1 and mTORC2 . Under hThe interactions between signaling pathways are dependent on the molecular diversity of the signaling components. The spatio-temporal sorting of signals can occur in highly organized networks with a multitude of interacting partners . Just asPTMs are reversible or irreversible chemical modifications in proteins following the process of translation. Proteolytic cleavage is one of the fundamental modifications, which adds or removes additional amino acids or a certain portion of protein. Nonetheless, in eukaryotes, the spectrum of PTMs extends to acetylation, amidation, biotinylation, farnesylation, formylation, geranylgeranylation, glycation, glycosylation, hydroxylation, methylation, mono/poly-ADP-ribosylation, myristoylation, oxidation, palmitoylation, SUMOylation, and phosphorylation. Ubiquitination is the most crucial PTM in eukaryotes since it governs the protein turnover in the cell by acting as a proteasomal degradation signal. More so, specific signals (degrons) can link a range of protein PTMs directly to the ubiquitin-proteasome system (UPS)  and therCyanobacteria, that exert ‘pervasive control’ over cellular processes including global gene expression  and status of methylation and acetylation can also affect the engineered signaling outcomes. Synthetically imposing regulation on kinome may provide a viable strategy to enhance signaling. For example, the behavior of Ste5 in yeast gives us information on how multisite phosphorylation can be read out via bulk electrostatic effects to yield a powerful biochemical switch [The successful engineering of signaling pathways would be greatly influenced by how scrutinized and robust the engineered models are in mimicking the in vivo behavior of cells. The minimization of context dependence should also be taken care of . Finally, manipulation of kinome for phosphorylation may greatly influence the outcome of the system and uncovering new phosphorylation targets may bring the novelty and stratify behavior of the modeled system . The wavl switch .Studies on cell signaling explain how cells sense and respond to the environmental cues, adapt, and coordinate with other components within the system to maintain homeostasis. While the genomic studies identified the organization and expression of the genes that influence cell signaling, the complex functional relationships between the signaling intermediates or their configurations into multi-strata signaling networks remain an enigma ,258. Str


We took a diblock copolymer of methoxy poly (ethylene glycol)-poly(L-lactic acid) (mPEG-PLA) as carrier material to prepare UA-PMs by the thin-film dispersion method. MTT assay and wound-healing assay were investigated to assess the inhibition effect of UA-PMs against HepG2 cells on cell-growth and cell-migration. Further, we chose KM mice for the acute toxicity experiment and assessed the antitumor effect of UA-PMs on the H22 tumor xenograft. UA-PMs could markedly inhibit the proliferation and migration of HepG2 cells. In vivo study showed that UA-PMs could significantly inhibit the growth of H22 xenograft and prolong the survival time of tumor-bearing mice. It demonstrated that UA-PMs possess great potential in liver cancer therapy and may enlarge the application of UA in clinical therapy.Ursolic acid (UA) is widely found in many dietary plants, which has been proved to be effective in cancer therapy. But unfortunately its hydrophobic property limits its clinical application. Polymer micelles (PMs) are constructed from amphiphilic block copolymers that tend to self-assemble and form the unique core-shell structure consisting of a hydrophilic corona outside and a hydrophobic inner core. PMs could entrap the hydrophobic substance into its hydrophobic inner core for solubilizing these poorly water-soluble drugs and it is widely applied as a novel nano-sized drug delivery system. This study aimed to develop the drug delivery system of UA-loaded polymer micelles (UA-PMs) to overcome the disadvantages of UA in clinical application thus enhancing antitumor effect on hepatocellular carcinoma. UA-PMs was prepared and characterized for the physicochemical properties. It was investigated the cell-growth inhibition effect of UA-PMs against the human hepatocellular carcinoma cell line HepG2 and human normal liver cell line L-02. UA-PMs was evaluated about the  Unfortunately, some current therapeutic methods like resection, transplantation and ablation for HCC are often ineffective due to tumor metastasis and poor patient conditions  in vivo. What's more, PMs possess the small and uniform particle size (10-100 nm) and can passively accumulate at tumor tissues as a result of an enhanced permeation and retention (EPR) effect, thus enhancing the antitumor activity and decreasing the side effects. In addition, as the carrier material of PMs, methoxy poly (ethylene glycol)-poly(L-lactic acid) (mPEG-PLA), has the advantages of good biodegradability and biocompatibility. PLA chains as hydrophobic segments are popularly utilized on account of its biodegradability In recent years, various formulations of UA such as carrier-based and carrier-free nanoparticles Furthermore, there are several kinds of methods to prepare polymer micelles such as dialysis method, self-assembled solvent evaporation method, thin film dispersion method and so forth. In the preparation of polymer micelles, the solubility of polymers as the primary factor should be taken into consideration in vitro and in vivo.Therefore, based on those findings mentioned above we hypothesize that mPEG-PLA as a drug carrier could be used to prepare UA-loaded polymeric micelles (UA-PMs) thus overcoming the limitations of free UA and improving the efficacy of UA in the treatment of liver cancers in vivo toxicity of UA-PMs in normal Kunming mice and the antitumor effect of UA-PMs in H22 tumor-bearing mice.In this study, we prepared UA-PMs by the thin-film dispersion method and investigated the cell-growth inhibition effect of UA-PMs against the human hepatocellular carcinoma cell line HepG2 and human normal liver cell line L-02 as shown in the Graphical abstract. We also assessed the 2000-PLA2000were from Jinan Daigang Biomaterial corporation . Pyrene was purchased from Macklin .Both ursolic acid and standard ursolic acid were obtained from Nanjing Zelang pharmaceutical technology corporation . mPEGDulbecco's Modified Eagle's Medium (DMEM) with high glucose and Rosewell Park Memorial Institute (RPMI) 1640 medium were purchased from HyClone . Fetal bovine serum (FBS) was obtained from Sijiqing . Paraformaldehyde was purchased from Jinshan Chemical Company . Trypsin, penicillin G, streptomycin and phosphate-buffered saline were obtained from Gibco Invitrogen . 3--2,5-diphenyltetrazolium bromide (MTT) was from Amresco . All other reagents and chemicals were of analytical grade and purchased from Keyang Biotechnology Corporation .Male Kunming mice with an average weight of 20 ± 2 g were purchased from the Animal Experiment Research Center of the Southwest Medical University, Luzhou, China. Mice were allowed to acclimate for a week before the experiment. All animals were bred with standard pellet diet in a standard animal-grade room with five animals in each cage on a 12-h light/12-h dark cycle. The temperature was set as room temperature. All the animal experimental procedures were conducted according to with the guidelines and protocols (Permit No. 20160142) approved by the Committee on the Use and Care of Animals of Southwest Medical University.2 and saturated humidity.Human hepatocellular carcinoma cell line HepG2, human normal liver cell line L-02 and mouse hepatocellular carcinoma cells line (H22) were got from Shanghai Cell Institute, China Academy of Sciences and preserved in our laboratory. HepG2 cells were cultured in DMEM supplemented with 10% FBS, both H22 cells and L-02 cells in RPMI 1640 with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C under an atmosphere containing 5% CO2000-PLA2000 (40 mg) with a mass ratio of 1:10 were dissolved by acetonitrile (4 mL) in a round-bottomed flask. Then the acetonitrile was completely removed through rotary evaporation in a 60 °C water bath to obtain a thin polymer film containing UA. The resultant thin film was hydrated with 2 mL of NaCl solution  to get a crude dispersion of the micelles. The micellar solution was mixed by water-bath ultrasound for 15 min followed by stirring for one hour at room temperature and filtered through a 0.22 µm film to remove the un-encapsulated UA. The blank polymeric micelles were prepared by the same way except addition of UA. The resulting micellar solution was lyophilized and stored at 4 °C for later use UA-PMs were prepared by the thin-film dispersion method. Briefly, both UA (4 mg) and mPEGThe size distribution, polydispersity index and zeta potential of micelles were determined by photon correlation spectroscopy (PCS) . Before measurement, the samples were diluted at a concentration of 1 mg/mL. The measurements were performed at 25 °C, at a fixed angle of 90. The zeta potential was derived from mobility of particles in electric field by applying the Smoluchowsky relationship. The measurement was set for 2 min in each turn (n = 3).The morphology of micelles was detected by the scanning electron microscopy (SEM) detection. Before SEM measurement, the samples were diluted at a concentration of 1.2 mg/mL. Sample was put on stubs with carbon adhesive disks and silver DAG, sputter coated, and viewed in a S-4800F (HITACHI) scanning electron microscope under the parameter setting of HV 5.0 kv, mag 20.0k ×, WD 8.6 mm and mode SE, respectively./min. It was monitored at 210 nm, and the injection volume was 10 μL. Samples were diluted with methanol and filtered through a 0.22-μm membrane before injection. The method was fully proved in terms of selectivity, linearity, precision, recovery and so on.A method based on high-performance liquid chromatography (HPLC) was developed for determining the content of ursolic acid total-Wfree) /Wtotal × 100%; DLR (%) =  / WUA-PMs × 100%.EE (%) =  and drug loading rate  of UA-PMs, the micellar solution loaded with UA was dissolved in methanol and analyzed by HPLC method. EE and DLR of UA-PMs were obtained according to the following formula as: in vitro release profile of UA-PMs was evaluated by a dialysis method. In detail, at least three batches of newly prepared UA-PMs were separately placed into dialysis bags (MWCO = 3500) and submerged in 100 mL PBS medium (pH 7.4 and pH 5.5) with gentle continuous-stirring at 37 °C. release medium. At predesigned time points of 0.5, 1, 2, 4, 6, 8, 12, 14, 24, 48, 72, 96 h, 2 mL of the solution was taken out from the release medium and supplemented with fresh PBS. After filtration through a 0.22-μm membrane (Millipore), all samples were measured using HPLC method mentioned above.The 4 cells/well and incubated for 24 h until reaching confluence. Then, they were treated with free UA and UA-PMs at different concentration of 2.5, 5, 10, 20, 30, 40, 50, 60, 80, 100 μmol/L using DMEM medium as negative control and 5-FU as positive control. After incubation for 24 h, 20 μL of MTT solution (5 mg/mL) was added and incubated for another 4 h in the dark. The supernatant medium was removed and the left MTT-formazan crystals were dissolved in 150 mL DMSO. Then the plate was incubated for 10 min at 37 °C with gentle shaking before determining the absorbance at 490 nm using Varioskan Flash instrument . The inhibitory rate of cell proliferation was obtained through the following formula as: sample / Acontrol) × 100%.Inhibition Rate (%) =  with 5-fluorouracil (5-FU) as a positive control and DMEM containing 2% FBS as the negative control.When the HepG2 cells came to the logarithmic growth phase, they were inoculated into the marked plates at the concentration of 5 × 100 h - SW24 h or 48 h) / SW0 hWound healing rates (%) =  at 24, 48 h to the wound width at 0 h and the wound healing rate was estimated by the following equation: in vivo study 8 cells/mL with 0.9% saline. Under sterile conditions, 0.5 mL of cells suspension was injected into the peritoneal cavities of mice. After one week, ascites was extracted from the tumor-bearing mice for collecting the H22 cells by centrifugation and the cells were suspended by saline. Then, 0.2 mL of the cell suspension (1.0× 107 cells/mL) was inoculated subcutaneously into the right flank of each mouse. Five days post tumor transplantation, when the tumors grew to about 100 mm3, the H22 tumor-bearing mice were divided randomly into seven groups (n=10). The treatments were separately initiated by intraperitoneal injection every two days for six times with normal saline as control, blank PMs, UA (50 mg/kg), 5-FU (25 mg/kg), UA-PMs (25 mg/kg), UA-PMs (50 mg/kg), UA-PMs (100 mg/kg), in which the formulation of free UA was prepared by dissolving UA into PEG400 (0.2%) and further being diluted with saline before injection. At the same time, every treatment group was randomly divided into part A and part B (n=5). In part A, the mice were treated every two days for 6 times and were sacrificed by cervical dislocation on day 17. The tumors were weighed and subjected to pathological analysis. In part B, the mice were also given the same treatment but they were kept feeding to record the death dates of mice for survival rate analysis. All the mice were monitored every day and the two axes of tumor  were measured using a vernier micrometer for measurement of tumor size after treatment. Tumor volume (mm3) was calculated as ½ (L x W2). Meanwhile, Antitumor activity was assessed by tumor growth inhibition (TGI), which is the mean tumor weight (MTW) of the treated group (TG) relative to the saline-treated control group (CG) on day 17. It was obtained according to the formula as: TGI (%) = (CGMTW -TGMTW) / CGMTW✕ 100 %. Further, to assess tumor necrosis areas, the tumor tissues were fixed in 4% paraformaldehyde and then embedded by paraffin. Paraffin sections were stained with hematoxylin and eosin (H&E) and analyzed by ImageJ software. The average necrosis rate was estimated by the following equation: Average necrosis rate (%) = Tumor necrosis areas / Total tumor areas × 100%.Murine H22 cells were inoculated in the Kunming mice for the P < 0.05 was considered to be statistically significant, and P < 0.01 was considered as highly significant in all cases.All experiments were performed at least three times with a minimum of three independent experiments. All values were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) or Student't-test was performed for statistical analysis between different groups. The Kaplan-Meier method was used in survival analysis. The result showed that the preformed blank PMs and UA-PMs prepared by thin film dispersion method were clear and transparent solution. If the micellar solution was stored at 4 °C condition, no visible precipitate appeared within 24 h. As shown in Figure in vitro release behavior of UA-PMs was evaluated at 37 °C using PBS as releasing medium at pH 7.4 and pH 5.5 conditions . However, the scratches in the other treatment groups recovered more slowly the cell migrations were reduced compared with the saline negative control and the blank PMs group at both 24 h and 48 h after treatments (**P< 0.01). Compared with the 5-FU group, all treatment groups except the high-dose group of UA-PMs (##P< 0.01) showed significant differences, in which the high-dose UA-PMs (80 μmol/L) showed same inhibition effect as the positive control of 5-FU (P > 0.05) at both 24 h and 48 h. Furthermore, we found the wound healing rate in UA-PMs groups was lower than that in the UA groups at the same concentrations. These results revealed that the migratory potential of HepG2 cells treated with UA-PMs was significantly reduced in a concentration-dependent manner compared to UA groups (★★P< 0.01).The wound-healing assay was performed to determine the inhibition effect of UA-PMs on the cell metastasis of HepG2. It can be seen from Figure in vitro cell proliferation and cell migration studies about HepG2 cells and in vivo acute toxicity study of UA-PMs as shown in in vivo. On day 5 after H22 tumor cells were implanted, different formulations taking saline as negative control and 5-FU 25 mg/kg as positive control were injected intraperitoneally into mice every other day for six times.Following the As shown in Figure P> 0.05). Treatments of UA-PMs with different concentration of 100, 50 and 25 mg/kg led to a tumor inhibition rate of 61.43%, 41.04% and 25.40%, respectively, in which the obvious differences were shown between them with ##P< 0.01 and #P< 0.05, respectively. Meanwhile, UA-PMs produced stronger tumor inhibition than free UA did at the same concentration of 50 mg/kg.Moreover, the tumor inhibition rate of 5-FU 25 mg/kg and UA-PMs 100 mg/kg is 58.46% and 61.43%, respectively, showing no significantly statistical difference between them (##P< 0.01). The mice treated with UA-PMs 50 mg/kg showed a longer survival time than that treated with UA-PMs 25 mg/kg (★P< 0.05). Moreover, at the same concentration of 50 mg/kg, UA-PMs obviously prolonged the mean survival times compared to free UA (★P< 0.05). The weight of mice in each group reported everyday  showed high degree of differentiation, which may be the evidence for the higher antitumor activity of UA-PMs. This result is consistent with that of antitumor effect mentioned above, suggesting that UA-PMs could cause cell necrosis of H22 and lead to the tumor growth inhibition.in vitro and the normal Kunming mice in vivo. Further, we found UA-PMs could suppress the tumor growth of H22 xenograft and prolong the survival time of the tumor-bearing mice. These results are consistent with the previous report In present study, we successfully developed the novel drug delivery system of UA-loaded polymeric micelles (UA-PMs) and revealed that UA-PMs significantly enhanced cell-growth and cell-migration inhibition effects against the human hepatocellular carcinoma cells HepG2 while showing no toxicity to both human normal liver cell line L-02 -3, 2.3 × 10-3 mg/mL indicating that the micelles showed good stability. However, the zeta potential of micelles was about 0 mV. This may be due to the presence of a great quantity of PEG blocking on the surface of UA-PMs particle, which could decrease the surface tension between particles and further prevent vesicle aggregation, thereby resulting in a near-neutral zeta potential of the micelles and keeping the stability of the formulation It is known that both critical micelle concentration (CMC) value and zeta potential are regarded as the important parameters for the stability of nanoparticle drug delivery system. If the absolute values of zeta potential of nanoparticle were more than 30 mV, it would be better in terms of dynamic stability. In this study, as shown in supplementary Figure In our acute toxicity study, we found the spleen coefficients (Supplementary table It was reported that drug efficacy was seriously limited by poor solubility. The solubility problems are frequently encountered in the preparations of pharmaceutical dosage forms in vitro anticancer activity of UA-PMs was tested against HepG2 cell line. The in vitro cell growth inhibition study indicated that UA-PMs showed the obvious inhibition effect on the HepG2 cells. And the IC50 value of UA-PMs was lower than that of the native UA at 24 h or 48 h showing the higher inhibitory effect compared with free UA at the same dosage. At the same time, the scratch-healing experiment was conducted to investigate the inhibition of UA-PMs on HepG2 cells migration and the results indicated that UA-PMs showed stronger inhibitory effect on the cell migration than UA with the same concentration. These results are consistent with the previous report  in vivo was taken to evaluate the anti-hepatocarcinoma effect of UA-PMs. In this experiment, UA-PMs at dose of 25, 50, 100 mg/kg were treated by i.p. administration against H22xenograft mice every other day for 17 days. From the results, UA-PMs were more effective for inhibiting tumor growth and prolonging survival times, compared with saline and free UA groups. Although 5-FU showed a similar inhibitory effect on HCC as UA-PMs at the same concentrations, the survival time of mice was bad in the treatment of 5-FU. It might because 5-FU had a large side effect on the heart of the treated mice.To investigate whether the preformed UA-PMs could be used in anti-hepatoma therapy, the in vitro, acute toxic study and antitumor activity in vivo all proved our hypothesis that UA-PMs could overcome the disadvantages of UA and get a sustained release of UA from UA-PMs thus enhancing antitumor effect on hepatocellular carcinoma. However, for the further clinical application, the formulation should be further optimized to improve the entrapment efficiency, drug-loading rate. On the other hand, maybe there is another way to develop UA formulation to conjugate UA with the amphiphilic block copolymers to form UA-PEG-PLA and further use it to prepare micelles These results from the cytotoxicity study Supplementary figures and tables.Click here for additional data file.


Caragana intermedia. We have reported that carainterol A showed potent glucose consumption activity in C2C12 muscle cells and the db/db mouse model. However, the mechanism of the hypoglycemic effect of carainterol A remains elusive. In this article, we present a network pharmacology approach to predict the target and signaling pathway of carainterol A which was subsequently validated in HepG2 cells. It was demonstrated that carainterol A could increase the protein levels of IRS-1 and the downstream protein kinase AKT phosphorylation at a low micromolar level. These findings suggest that carainterol A can be a valuable lead compound and a promising chemical probe for the insulin signaling pathway.Carainterol A is a eudesmane sesquiterpenoid extracted from  Sesquit product , was isoetformin . Due to etformin . HoweverIdentifying functional targets and clarifying the molecular mechanism of action of bioactive natural products have been proven to be particularly challenging . NetworkIn this study, carainterol A was analyzed using the PharmMapper and ClueGO to predict the target proteins and related pathways, respectively. Furthermore, carainterol A was evaluated in the HepG2 cells in order to clarify its biochemical action. We demonstrated that carainterol A could increase insulin pathway sensitivity through increasing the protein levels of IRS-1 and the downstream protein kinase AKT phosphorylation at a low micromolar level.After the removal of duplication and standardization, 118 potential molecular targets were retrieved based on the fitting score from high to low. These potential molecular targets are involved in a variety of physiological and/or pathological processes. Among the 118 protein targets, 46 targets (39%) including IGF1R, INSR, MDM2, PPARG, RXRA, PTN1, RXRB and RARG have been reported to be related to diabetes by previous studies. Sixteen pairs of compound-target interactions were found and their fitting scores of docking were greater than 3.5 . For exaBioinformatics enrichment tools have contributed greatly in the study of gene function analysis in traditional Chinese medicine . To addrThe main molecular functions were divided into five categories: insulin receptor substrate binding, insulin receptor binding, protein tyrosine kinase activity, retinoid X receptor binding and steroid hormone receptor activity. The main pathways included in the ClueGo analysis were as follows: the PI3K-Akt signaling pathway, pathways in cancer, proteoglycans in cancer, the FOXO signaling pathway, the prolactin signaling pathway, the AMPK signaling pathway, the PPAR signaling pathway, the insulin signaling pathway and the thyroid hormone signaling pathway .2C12 muscle cells. Furthermore, it also displayed increasing glucose consumption in a db/db mouse model with a micromolar value that is equivalent to that of metformin. The present study indicated that carainterol A possesses multipharmacology attributes through different target groups such as insulin receptor substrate binding, insulin receptor binding and protein tyrosine kinase activity. Thyroid hormone receptor subtype-β agonists could improve glucose tolerance and insulin sensitivity in a dose-dependent manner [In our previous study, carainterol A showed potent glucose consumption activity in Ct manner . Therefot manner . TherefoGuided by the in silico prediction results, we explored the effects of carainterol A on the insulin signaling pathway. As shown in IRS-1 has been implicated in insulin signaling transduction from the insulin receptor to AKT phosphorylation, followed by GLUT4 translocation . To furtElucidation of the pharmacological profile of a single natural product from natural medicine has been the task for many researchers over the decades. Compared to structural optimization for inhibiting a specific target, multi-target drug approaches were employed recently to study the multi-target character of a single natural product . Among tThe dysregulation of IRS-1 has been implicated in the pathogenesis of type II diabetes and cancer . Thus, iAlthough no influence on GLUT4 translocation was observed, it was proposed that carainterol A may increase glucose consumption through the unknown downstream signaling component of IRS-1. Our results have implied that carainterol A could serve as a valuable chemical probe for the insulin signaling pathway investigation.PharmMapper, a web server for potential drug target identification, is based on the use of a pharmacophore mapping approach . The besIn this study, all targets retrieved from the above were sent to database UniProt  for targClueGO, a Cytoscape plug-in, is a professional software to enhance the biological interpretation and to analyze the functionally grouped terms in the form of networks and graphs . It was The protocol of the pathway verification experiment refers to the paper published by Shen et al. . Y018 waHepG2 cells were incubated in 12-well plates overnight and the starved cells for 4 h (maintained compound concentration). Then, the cell was stimulated for 5 min by the insulin and cells were collected for the Western blot experiments. In the experiments of the regulation of protein level of IRS-1, the compounds were tested after incubation in the serum.In the plasmid pEGFPN1-rGLUT4-myc, c-myc epitope sequence was inserted into the GLUT4 protein extracellular region of rat and the C-terminus was coupled with EGFP green fluorescent protein form a fusion protein. The plasmid was stably transfected into CHO-K1 cells. In the experiment, in the case of impermeable membrane, anti-c-myc antibody as well as the red fluorescent secondary antibodies was used for indirect immunostaining. Green fluorescence intensity indicated the total GLUT4 in the cells and red fluorescence intensity indicated the total GLUT4 on the cell membrane.We used PharmMapper and ClueGO software to predict the main disease-related targets and pathways of carainterol A. Furthermore, the effect on the insulin signaling pathway was validated as the main biological mechanism of carainterol A through increasing the protein level of IRS-1 and the downstream protein kinase AKT phosphorylation level. Further studies towards how exactly carainterol A regulates the protein level of IRS-1 are in progress and will be reported in due course.


Alzheimer's disease (AD) is the most common cause of dementia and one of the leading causes of death in aging population worldwide. It is estimated that more than 50 million people around the globe are affected with AD. The accumulation of toxic amyloid plaques, hyperphosphorylated tau tangles and extensive neuronal degeneration are hallmark features of AD . MicroRNIn AD, the principal neurotoxic factor β-amyloid is formed through the sequential cleavage by β- and γ-secretase enzymes of the amyloid precursor protein (APP) . An altein vivo to increase both SOX2 and ADAM10 levels may offer a feasible approach for AD intervention. This approach will have the advantage of both enriching the level of soluble APP-α and reducing Aβ levels. Our findings highlight the potential of combinatorial regulation of transcription factors and microRNAs in the targeted regulation of ADAM10 and provides a strong basis for future research aimed at annotating trans-acting regulatory factors as therapeutic targets in AD.Another important aspect of our study was to determine if miRNA-140-5p plays a broader role in the regulation of ADAM10, such as the regulation of ADAM10 transcription factors. Transcription factor candidates were selected based on the following criteria; whether miR-140-5p was predicted to target the transcription factor gene, there was previous evidence of transcription factor activity in neurodegenerative disorders, and whether it had been previously described as a regulator of ADAM10. SRY (sex determining region Y)-box 2 also known as SOX2 fit these criteria. Therefore, next we focused on elucidating the functional relationship between the trans-acting factors SOX2 and miRNA-140-5p in ADAM10 regulation. Overexpression of SOX2 increased ADAM10 reporter construct activity indicating that SOX2 directly regulates ADAM10 promoter activity. In addition, site directed mutagenesis creation of a mutation in the miR-140 binding site, in both the ADAM10 3’UTR and the SOX2 3’UTR, ablated miR-140-5p inhibition of SOX2 and ADAM10 reporter activity. These findings suggest that miR-140-5p inhibits the production of ADAM10 both through regulation of the SOX2 transcription factor and through direct regulation of ADAM10. This information may also have wider ramifications for other APP cleavage products since ADAM10 cleavage also produces the neuroprotective soluble APP-α. Therefore, therapeutic inhibition of miR-140-5p


Since its association with familial pulmonary arterial hypertension (PAH) in 2000, Bone Morphogenetic Protein Receptor II (BMPR2) and its related signaling pathway have become recognized as a key regulator of pulmonary vascular homeostasis. Herein, we define BMPR2 deficiency as either an inactivation of the receptor, decreased receptor expression, or an impairment of the receptor’s downstream signaling pathway. Although traditionally the phenotypic consequences of BMPR2 deficiency in PAH have been thought to be limited to the pulmonary vasculature, there is evidence that abnormalities in BMPR2 signaling may have consequences in many other organ systems and cellular compartments. Revisiting how BMPR2 functions throughout health and disease in cells and organs beyond the lung vasculature may provide insight into the contribution of these organ systems to PAH pathogenesis as well as the potential systemic manifestation of PAH. Here we review our knowledge of the consequences of BMPR2 deficiency across multiple organ systems. Nearly 20 years ago, genetic linkage studies connected Bone Morphogenetic Protein Receptor II (BMPR2) to the development of familial pulmonary arterial hypertension (PAH) ,2. In thBMPR2 is a “type 2” member of the transforming growth factor (TGF)-β superfamily of serine threonine kinase membrane-bound receptors. Unlike the TGF-β receptor, BMPR2 requires a “type 1” co-receptor to form a heterotetrameric complex and transduce signals in response to ligands. Currently, there are 20 Bone Morphogenetic Proteins (BMPs) which are secreted as homo- or heterodimers and signal via type 1 and type 2 receptor complexes. There are four type 1 receptors: Activin receptor-like kinase 1(ALK1), BMPR-1A (ALK3), BMPR-1B (ALK6), and ActR-1A (ALK2). There are two relevant type 2 receptors: BMPR2 (ActRIIA) and ActRIIB. The combinations of receptors vary across cell type, and confer specificity to given BMP ligands. Notably, ALK1 is generally expressed on endothelial cells but not on smooth muscle cells. Once a heterotetrameric complex is formed in response to a ligand, the type 2 receptor phosphorylates the type 1 receptor which in turn phosphorylates both canonical and noncanonical signal transduction targets. In the canonical pathway, a receptor “mothers against decapentaplegic” (R-SMAD) protein—typically SMAD 1/5/8, reviewed in —is phospImportantly, several of the additional mutations associated with adult-onset PAH involve key members of the canonical BMPR2 signaling pathway, including SMAD8 , CaveoliNotably, the penetrance of BMPR2 mutations is 20% , suggestIn PAH, a disease with multiple cell types involved, signaling pathways implicated, and temporal dynamics at play, the strong genetic association with BMPR2 creates a key opportunity for developing and testing therapeutic interventions. Therapies with genetically supported targets are more than twice as likely to make their way through the development pipeline to preclinical trials . It is nIn PAH, the notion of universally impaired in vivo BMPR2 signaling appears to be reliably supported ,22,31, bMuch insight into the BMPR2 pathway is derived from in vitro experiments studying cells in isolation. As multiple cell types share the same signaling pathway, this may not recapitulate the spatially, and likely temporally, heterogeneous in vivo signaling network. The spatiotemporal dynamics of BMPR2 ligands, type 1 receptors, and type 2 receptors in the lung may be extremely relevant to the evolution of cardiopulmonary disease.Finally, impaired BMPR2 signaling is best studied in endothelial and smooth muscle cells and is closely linked to vascular dysfunction, yet it is known that BMPR2 is also expressed in the adult bone marrow, heart, brain, placenta, liver, skeletal muscle, pancreas, kidney, placenta, gonads, and small intestine . Global In the sections below, we define BMPR2 deficiency as either an inactivating mutation of the receptor, or an impairment of the signaling pathway, and we will examine the consequences of BMPR2 deficiency across different cellular compartments and organ systems .There are multiple causes for downregulation of BMPR2 signaling that do not involve genetic mutations, and could contribute to reducing BMPR2 below a critical threshold needed to initiate disease . The prevalence of BMPR2 mutations in a large cohort of patients with idiopathic, heritable, and anorexigen-induced PAH was 29% . As of 2Micro-RNA (miR) regulation of BMPR2 signaling occurs in complicated networks at multiple levels of the signaling pathway, reviewed in . These mBMP access to receptors is altered by numerous antagonists and the extracellular matrix, reviewed in ,40. InteFHIT) as one such gene, which, when targeted by the drug Enzastaurin, ameliorates PAH in animal models [There is evidence for genetic regulation of BMPR2 by “BMPR2 modifier genes”. We have identified fragile histadine triad , hypercholesterolemia, and angiotensin II infusion, also decrease systemic vascular expression of BMPR2 .Study of HIV-associated PAH reveals that viral protein products Tat, Nef, and gp-120 reduce BMPR2 expression . This efInflammatory cytokines such as IL-6 and tumor necrosis factor alpha (TNF-α) can decrease BMPR2 expression via miR and by metalloprotease-mediated cleavage of the receptor ,52,53. FSince its linkage to PAH, BMPR2 deficiency has been inexorably connected to pathologic pulmonary vascular changes and worse clinical outcomes. The seminal work by Atkinson et al. in 2002 demonstrated that relative to healthy controls, BMPR2 protein was significantly decreased in the vasculature of patients with both primary (idiopathic) and familial PAH harboring a BMPR2 mutation. Importantly, those patients with secondary PAH due to either ventricular septal defect, chronic thromboembolism, or scleroderma also had a significant decrease in vascular BMPR2 . BuildinIn 1974, Meyrick et al. performed an open lung biopsy of a man with primary pulmonary hypertension and found thickening of the non-muscularized vessel endothelium by electron microscopy with luminal occlusion . PioneerBMPR2 was first implicated in endothelial cell biology in 2001, when bovine aortic endothelial cells grown in tube-forming conditions on a type 1 collagen matrix upregulated BMPR2 mRNA and protein . TeicherFurther complicating the picture, multiple pathways beyond canonical SMAD signaling are influenced by BMPR2. BMP2 ligand can increase BMPR2-mediated phosphorylation of ERK, which then inactivates Glycogen Synthase Kinase 3-β (GSK3-β) degradation of β-Catenin. This ultimately facilitates the canonical Wingless (Wnt) signaling pathway and increases PAEC proliferation. Noncanonical activation of RhoA and Rac1 is facilitated by either by a SMAD1 or SMAD3 recruitment of Disheveled (Dvl) protein, which increases cell motility . The loss of BMPR2 has direct consequences on the secretion of vasodilatory and inflammatory cytokines by endothelial cells. BMP9 signals through BMRP2 to induce the transcription of Id1, Id2, E-Selectin, IL-6, and IL-8 . In PAECEndothelial cells from PAH patients exhibit a significant decrease in mitochondrial oxidative phosphorylation with an increase in aerobic glycolysis, similar to malignancies. BMPR2 deficiency in endothelial cells can potentiate these alterations in cell metabolism. Loss of BMPR2 directly induced mitochondrial dysfunction and facilitated a transition from glucose oxidation to glycolysis . BMPR deSimilar to what is observed in the lesions of PAH patients, there is increased DNA damage and impaired DNA repair which can contribute to somatic genomic mutations and microsatellite instability, potentially leading to an apoptosis-resistant phenotype . SilenciAll effects taken together, BMPR2 deficiency in endothelial cells leads to a disrupted cytoskeletal and adhesion structure, increased apoptosis, increased migration, increased production of inflammatory cytokines and vasoconstrictors, and a loss of endothelial cell identity, all of which occur in PAH. A hallmark of pathologic vascular lesions in PAH is the proliferation of smooth muscle cells (SMCs) resulting in medial hypertrophy along with the appearance of cells expressing smooth muscle cell markers in the neo-intima. Dysregulation of BMPR2 signaling is directly tied to extracellular and intracellular signaling changes which increase pulmonary artery smooth muscle cell (PASMC) migration, proliferation, and cytokine release.Work with PASMCs has shown that active canonical BMPR2 signaling is critical for controlling the two main pathologic behaviors of PASMCs in PAH: increased proliferation and decreased apoptosis. These cells express a multitude of type 1 and type 2 BMP receptors, with BMP7 long established to decrease both PASMC proliferation and apoptosis ,74. AlthBMPR2 deficiency in muscle can potentiate proliferative paracrine signaling. Loss of BMPR2 will block the ability of BMP4 and BMP7 to repress the secretion of endothelin-1 in PASMCs. Additionally, since BMP7 antagonizes the arterial contraction caused by endothelin-1, loss of BMPR2 can generate increased endothelin-1-mediated vasoconstriction . FinallySimilar to what is seen in PAECs, BMPR2 exists at an intersection between multiple signaling pathways in PASMCs. BMPR2 can create an imbalance in these pathways, giving rise to a proliferative and migratory phenotype. The Wnt signaling pathway is, again, one example. In smooth muscle, BMP2 signals through BMPR2 to phosphorylate Akt, suppress the degradation of β-Catenin, and increase fibronectin expression. Fibronectin incorporates into the extracellular matrix, promoting motility, growth, and contraction of smooth muscle cells . Beyond BMP 2, 4, and 7 can suppress proto-oncogene Src family protein tyrosine kinase phosphorylation through interaction with the c-terminus of BMPR2 . Src kinThus, BMPR2 deficiency can achieve the phenotype of increased PASMC migration and proliferation through multiple complex signaling pathways. Yet unknown is how these downstream pathways may change temporally in early versus end-stage disease or how neighboring cell types fully influence these pathways. The adventitial layer in PAH is deranged by the infiltration of inflammatory cells, increased populations of resident and circulating fibroblasts, activated dendritic cells, overexpression of chemokines, and collagen matrix deposition . AdventiFibroblast proliferation and apoptosis are influenced by BMPR2. Human fetal lung fibroblasts are known to express BMP2, BMP4, and BMP7. Further, treatment of fetal lung fibroblasts with BMP4 activates canonical and noncanonical BMPR2 pathways, prevents serum-stimulated proliferation, and increases expression of the SMC maker alpha smooth muscle actin . In respDisruption of the elastic lamina due to expression of neutrophil elastase is a prominent feature in PAH that allows for dissociation of matrix-bound growth and inflammatory factors. Notably, silencing of BMPR2 in pulmonary adventitial fibroblasts results in decreased production of elastin and fibrillin-1 in response to TGF-β1. Additionally, fibroblasts from BMPR2 mutant PAH patients have severely impaired elastic fiber formation . Taken tFurther association is demonstrated by a mouse model for scleroderma lung and skin disease. Here, a kinase-deficient TGF-β1 receptor is overexpressed in fibroblasts using the pro-α2 (I) collagen gene as a promoter. This model counterintuitively results in an overactivation of TGF-β1 target genes within fibroblasts  and is refractory to exogenous TGF-β . These mFibroblasts exist in diverse subpopulations, orchestrate the production of the extracellular matrix of connective tissue, and play integral roles in mediating wound healing and inflammation, reviewed in . AlthougBeyond what is known with HHT, descriptions of other morphologic abnormalities of the systemic vasculature in patients harboring BMPR2 mutations are sparse. BMP signaling, however, is tied to the pathogenesis of atherosclerosis, which will be covered when discussing the immune system. One exception to this is the bronchial arteries. In idiopathic PAH, the bronchial arteries are known to form communications with both the pulmonary circulation and the vaso vasorum . AlthougRight ventricular (RV) failure is the final common pathway for end-stage PAH. Both survival and RV hemodynamics, specifically, pulmonary capacitance and right ventricular stroke work index, are worse in familial versus idiopathic PAH . It was Study of left ventricular heart failure in a transverse aortic constriction mouse model shows a strong interdependence between non-BMP TGF-β signaling, ventricular dilation, interstitial fibrosis, and a reduced capillary density. This was associated with noncanonical activation of transforming growth factor beta-activated kinase 1 (TAK1) by TGFβR2 . BMP2 waCurrently, the effect of BMPR2 deficiency on RV cardiomyocytes is primary studied with respect to metabolism. PAH can be characterized by a mitochondrial impairment that shifts metabolism away from glucose and fatty acid oxidation . It has Given the relationship between non-BMP TGF-β signaling and cardiac interstitial fibrosis in the left ventricle, it is compelling to ask if BMPR2 deficiency imparts a further maladaptive response in the hypertensive RV, which is the topic of active investigations. This raises the possibility that BMPR2 rescue therapies could benefit both the lung and the RV.Multiple lines of evidence demonstrate that inflammation is a key component in the pathogenesis of PAH, reviewed in . Two key+/− mice or in BMPR2 mutant human cell culture, BMPR2 deficiency increases IL-6 and IL-8 mRNA and protein expression in response to lipopolysaccharide administration [First, BMPR2 deficiency assists in recruitment of lymphocytes, macrophages, and neutrophils to the vessel by increasing vascular cytokine production. There is increased endothelial GM-CSF production in response to BMPR2 reduction . In the stration . Furtherstration . delx4+) in mice results in constitutive activation of bone-marrow-derived macrophages, supporting a notion that active BMPR2 signaling suppresses macrophage activation and could quell inflammation [After being recruited by cytokines, BMPR2 deficiency can aid in the diapedesis of inflammatory cells to the sub-endothelial tissue. Vascular infiltration by inflammatory cells is mediated by arterial, venous, or lymphatic transendothelial migration, a complex process orchestrated by chemokines, integrins, and adhesins, reviewed in . BMPR2 rammation . Taken tThe balance of immune cell populations is altered in PAH, a fact long known but quantified recently by Marsh and colleagues. Flow cytometry defined the distribution of all cluster of differentiation-45 (CD45) positive cells in the lungs of patients with and without idiopathic PAH. In addition to a striking expansion of T cells, neutrophils, macrophages, and dendritic cells, there was a previously unknown expansion of nonclassical plasmacytoid dendritic cells and γδT cells . Whether+/CD8+) T cells, a process which is arrested by BMP2/4 acting on the thymocyte [Foxn1 transcription factor in embryonic development, with suboptimal expression of Foxn1 resulting in thymic hypoplasia and impaired T cell development [−/CD8− double negative thymocytes [The thymus plays a central role in T cell maturation and selection. Double negative thymocytes from the bone marrow mature to double positive  and BMPR2 suppressed hepcidin expression, leading to impaired iron metabolism and iron overload . Also noBMPR2 is expressed in human oocytes, follicles, and corpus lutea , and mayIn the developing murine retina, BMPR2 is highly expressed at the vascular front and essential for retinal angiogenesis . This co+ cells [There is a high incidence of PAH in myeloproliferative disorders, generally attributed to cytotoxic chemotherapy, tumor microemboli, and bone-marrow-derived angiogenic factors . Notably+ cells . Interes+ cells . This evAfter the discovery that loss of BMPR1A leads to the development of Juvenile Polyposis , it was The expression of BMPR2 in chondrosarcoma also directly correlates to a significantly reduced relapse-free survival. BMPR2 in chondrosarcoma stabilizes XIAP, leading to apoptosis resistance. Knockout of BMPR2 in chondrosarcoma with siRNA destabilized XIAP, increased tumor apoptosis, suppressed tumor growth, and increased autophagy .On the other hand, BMPR2 is expressed in ovarian cancer, with levels correlating to improved survival . This cotrans retinoic acid [In other cases, BMP signaling may influence the severity of malignancy. In prostate cancer, BMP6 is noted to upregulate ID1 expression, increasing invasiveness . Acute poic acid . BMPR2 abnormalities are also described in GI malignancy. Somatic frameshift mutations in gastric and colorectal cancers are associated with microsatellite instability and increased epithelial growth and polyp formation ,145. Finally, BMP signaling is important in bladder cancer. The progression of urothelial cancer from carcinoma in situ to invasive carcinoma is mediated by a loss of sonic hedgehog (Shh) signaling from the bladder epithelium to the stroma. Loss of Shh from de-differentiated bladder epithelium decreases stromal BMP4/5 production. Treatment with FK506 replenished the BMP signal from stromal cells and induced differentiation of pre-malignant cells to normal urothelial cells, preventing invasive carcinoma .BMPR2 deficiency is clearly central to the pathogenesis of PAH as it both is associated with a worse clinical outcome and recapitulates many of the cellular pathologic features of PAH. Despite significant and detailed knowledge of the receptor and its pathway, there are many questions that remained unanswered.FHIT can reveal novel pharmacologic therapies [Many factors downregulate BMPR2 signaling and receptor expression outside of germ line and somatic mutations. As BMPR2 haploinsufficiency in patients alone is not sufficient to alter pulmonary vascular resistance or RV functional reserve , what geherapies . Growing evidence suggests that PAH is a disease with systemic manifestations. Metabolic derangements and insulin resistance appear to be the most clinically significant pathologic manifestation outside of the heart and lungs, with growing support for pathologic renal involvement. However, as reviewed, there is potential for BMPR2 deficiency to alter immune competency, generate RV fibrosis, and cause RV capillary rarefaction. Beyond this, understanding BMPR2 signaling in tissues outside the lung may allow us to predict whether there could be untoward effects from BMPR2 modulatory therapy or if specific levels of BMP signaling are needed to maintain homeostasis in certain tissues. As noted above, BMP activation in certain cancers such breast cancer, hepatocellular carcinoma, or chondrosarcoma may be pathologic.Still the question remains as to how BMPR2 deficiency results in dramatic plexiform lesions in a relatively specific region of a single organ. It is known that the smooth muscle proliferative response to BMP4 varies in proximal versus distal PASMCs . It may Our current, detailed knowledge of BMPR2 signaling is extrapolated from cell culture from patient cell lines, study of normal cell lines, and animal models. The BMPR2 receptor can be thought of as the tip of an iceberg over a sea of complex interwoven signaling networks. Cellular crosstalk may be extremely important, and it is not clear that cell culture experiments recapitulate the in vivo signaling dynamics between endothelium, smooth muscle cells, adventitia, and immune cells. Does the robust BMPR2 protein immunostaining in the endothelium ,149 implOur understanding of BMPR2’s role in PAH pathogenesis spans 20 years and highlights the BMPR2 pathway as a current focus of pulmonary vascular research. Understanding the many pathologic consequences of BMPR2 deficiency in other organ systems and the interaction with the pulmonary vasculature is key to fully understanding and targeting the BMPR2 pathway in PAH.


Statistical learning is the ability to learn based on transitional probability (TP) in sequential information, which has been considered to contribute to creativity in music. The interdisciplinary theory of statistical learning examines statistical learning as a mechanism of human learning. This study investigated how TP distribution and conditional entropy in TP of the melody and bass line in music interact with each other, using the highest and lowest pitches in Beethoven’s piano sonatas and Johann Sebastian Bach’s Well-Tempered Clavier. Results for the two composers were similar. First, the results detected specific statistical characteristics that are unique to each melody and bass line as well as general statistical characteristics that are shared between the melody and bass line. Additionally, a correlation of the conditional entropies sampled from the TP distribution could be detected between the melody and bass line. This suggests that the variability of entropies interacts between the melody and bass line. In summary, this study suggested that TP distributions and the entropies of the melody and bass line interact with but are partly independent of each other. Statistical learning (SL) has been considered a domain-general and implicit learning system that encodes probabilistic distribution of sequential phenomena such as music and language –3. For ePsychologists agree that computational and corpus studies on music can highlight some of the statistical properties available to musical learners by SL and implicit learning –24. Partentropy” as Shannon has done , and, andentrcertainty. In the certainty). In othRecent literature has suggested that specific developmental processes modulate SL ability in the brain. For example, both Western-classical and jazz musicians are better statistical learners in general than nonmusicians –53. FurtWestern tonal classical music has a number of specific features such as isochronic metrical grids, tonal pitch spaces, hierarchical tension, and attraction contours based on the structure of the melody and chord progression ,60. The The purpose of the present studies is to investigate how TP distributions of the melody and bass line interact with each other, and how tonal mode and keys govern the statistical distributions and the interaction between the melody and bass line. The information content of TPs in the sequences containing the highest and lowest pitches in all of the movements in Beethoven’s piano sonatas  (Study 1) and Johann Sebastian Bach’s Well-Tempered Clavier (Study 2) were calculated based on six different order Markov stochastic models . First, to investigate the statistical characteristics of the melody and bass line in each piece of music, the TP distribution was analysed using principal component analysis, based on the hypothesis that there are fundamental statistical characteristics shared between the melody and bass line, and specific statistical characteristics that are unique to each. Additionally, the detectability of these characteristics may depend on the tonal mode and the keys  and/or onth-order Markov models are based on the conditional probability of an element en+1, given the preceding n elements:I[en +1|en]) in each TP was calculated based on information theory [uncertainty” of statistical distribution. Thus, using the distributions of TPs (information content) in each melody and bass line of each piece of music, the distributional characteristics of each piece of music were analysed by principal component analysis (PCA). The present study hypothesized that a component shared within the melodies or bass lines and within major or minor keys represents a specific characteristic of TP distribution depending on voice part  and tonal mode . Based on our previous papers [conditional entropy (H(AB)) in the nth-order was calculated from the information content as follows:P(bj|ai) is a conditional probability of the sequence “ai bj”. P(ai) is the probability of event ai occurring, and P(bj|ai) is the probability of bj occurring given that ai occurs previously . The conditional entropy is the sum of the bits and is regarded as the “uncertainty” of the transitional-probability distribution. The conditional entropy of each TP distribution was compared by correlation analysis. Statistical significance levels were set at p = 0.05 for all analyses.All of the movements in Ludwig van Beethoven’s piano sonatas  and Johann Sebastian Bach’s Well-Tempered Clavier, BWV 846–893, which is a collection of two series (No.1 and No.2) of preludes and fugues in all 24 major and minor keys, were used in the present studies. Using a scorewriter software program , electronic scoring data of the sequences of highest pitch were extracted from the XML files. The highest and lowest pitches were defined as the highest and lowest pitches that can be played at a given point in time; in identifying these pitches, equivalent pitches were counted as one, and grace notes were excluded. Using all the pitch sequences in each piece of music, the TPs distributions were calculated based on zeroth- to fifth-order Markov models. In Beethoven’s piano sonatas, the weighted averages of TPs of all the movements were calculated. In Bach’s Well-Tempered Clavier, the weighted averages of TPs of the prelude and fugue in No.1 and No.2 in each key were calculated. As described in detail previously , the nthn theory  as:I.All of the results in the correlation analysis are shown in This study examined how zeroth- to fifth-order TP distributions (Markov models) and the conditional entropies in the melody and bass line correlate and interact with each other in all movements of the piano sonatas by Ludwig van Beethoven . First, we investigated how the statistical characteristics of the melody and bass line can be extracted in each order Markov model using principal component analysis. It was hypothesized that there were general statistical characteristics shared between the melody and bass line as well as specific statistical characteristics that were unique to each melody and bass line based on each order model. Thus, TP distribution in the zeroth-order Markov model detected a general component that is shared between the melody and bass line, whereas those in the first- to fifth-order Markov models detected specific components that are unique to each melody and bass line . These rSecond, we investigated the relationships of conditional entropies between the melody and bass line in each order Markov model using correlation analysis. It was hypothesized that the correlation of the variability in the entropy between the melody and bass line depends on the order of TP distribution. The results suggest that the correlation of conditional entropies between the melody and bass line could be detected in the first- to fifth- but not zeroth-order Markov models. They may suggest a correlation in the variability of entropies between the melody and bass line in higher-order TP distributions. This may suggest that the correlation between the melody and bass line depends on the length of the sequence. Compared to the zeroth-order model, the higher models could essentially construct a musical phrase. Thus it is possible that the analysis of an entire musical phrase may strengthen the perceived connection between the melody and bass line. In psychological and computational studies related to SL, predictive coding, and information theory, entropy has been interpreted as the average degree of surprise associated with an outcome . EntropyAll of the results are shown in p = 0.003; minor: r = .85, p < 0.001, second: major: r = .93, p < 0.001; minor: r = .78, p = 0.003, third: major: r = .75, p = 0.005; minor: r = .91, p < 0.001; p = 0.001; minor: r = .62, p = 0.063). In fourth-order TP distributions, the conditional entropies of the melody in major keys were moderately related while those in minor keys were strongly related to those of the bass line . In fifth-order TP distributions, the conditional entropies of the melody were strongly related to those of the bass line in minor keys , whereas no significant correlation was detected in major keys. No significant correlation was detected between major and minor keys  and the conditional entropies in the melody and bass line. First, the manner in which the statistical characteristics of the melody and bass line in each of the major and minor keys could be extracted in each order Markov model was investigated using principal component analysis. It was hypothesized that there were general statistical characteristics shared between the melody and the bass line and between the major and minor keys, as well as specific statistical characteristics that were unique to each melody and bass line and to each major or minor key. Additionally, it was hypothesized that the detectability of these characteristics depends on the tonalities of the keys and the order of TPs . Thus, TThe present studies investigated how TP distributions and the conditional entropy of the melody and bass line interact with each other, using the highest and lowest pitches in Beethoven’s piano sonatas (Study 1) and Johann Sebastian Bach’s Well-Tempered Clavier (Study 2). Our findings were similar for the two composers. First, TP distribution in each model showed a general component (component 1) that is shared between the melody and bass line. Second, TP distribution in the first- and second- but not zeroth-order models detected specific components (component 2) that were unique to each melody and bass line. These results suggest that statistical characteristics specific to each melody and bass line can be disclosed in higher-order but not in zeroth-order statistical models. From the psychological and neurophysiological viewpoints of SL in the brain, higher-order but not lower-order statistical knowledge of the melody and bass line are partially independent of each other. Additionally, Study 2 also detected specific components (component 3) that are unique to each major and minor key as well as to the melody and bass line . Thus, tIn the fields of computational and informatics studies, entropy has been used to verify the validity of computational models including SL in music . A compS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.