Heantos-4 is a refined combination of plant extracts currently approved to treat opiate addiction in Vietnam. In addition to its beneficial effects on withdrawal and prevention of relapse, reports of sedation during clinical treatment suggest that arousal networks in the brain may be recruited during Heantos administration. T-type calcium channels are implicated in the generation of sleep rhythms and in this study we examined whether a Heantos-4 extraction modulates T-type calcium channel currents generated by the Cav3.1, Cav3.2 and Ca3.3 subtypes. Utilizing whole-cell voltage clamp on exogenously expressed T-type calcium channels we find that Heantos inhibits Cav3.1 and Cav3.3 currents, while selectively potentiating Cav3.2 currents. We further examined the effects of Heantos-4 extract on low-threshold burst-firing in thalamic neurons which contribute to sleep oscillations. Using whole-cell current clamp in acute thalamic brain slices Heantos-4 suppressed rebound burst-firing in ventrobasal thalamocortical neurons, which express primarily Cav3.1 channels. Conversely, Heantos-4 had no significant effect on the burst-firing properties of thalamic reticular neurons, which express a mixed population of Cav3.2 and Cav3.3 channels. Examining Heantos-4 effects following oral administration in a model of absence epilepsy revealed the potential to exacerbate seizure activity. Together, the findings indicate that Heantos-4 has selective effects both on specific T-type calcium channel isoforms and distinct populations of thalamic neurons providing a putative mechanism underlying its effects on sedation and on the thalamocortical network. Opiate dependence is estimated to affect 15 million people worldwide and opiate overdose is believed to result in approximately 69,000 mortalities per year , 3. As sHeantos-4, which is the Greek term for “Plants” is a mixture of organic herbs developed in Vietnam and recently approved for the clinical alleviation of withdrawal symptoms in individuals dependent upon opiates . FurtherV3.1- CaV3.3) are a sub-class of calcium channels that activate at more hyperpolarized membrane potentials than their High Voltage-Activated (HVA) counterparts (CaV1.1- CaV1.4 and CaV2.1- CaV2.3). As a result, T-type calcium channels open in response to smaller depolarizations than HVA calcium and sodium channels, endowing them with a unique the ability to modulate cellular excitability at near-resting membrane potentials [Voltage-gated calcium channels are a class of membrane bound proteins that regulate the cellular entry of calcium ions upon depolarization . T-type tentials . Low-thrtentials , 9. Thaltentials . While btentials , 11 it itentials –14.V3.1- CaV3.3). Further, we correlated the effects observed for cloned channels with Heantos-mediated modulation of ventrobasal (VB) and reticular thalamic nucleus (TRN) neurons in acute rat brain slices. Finally, we examined the effects of Heantos-4 oral administration on seizure activity in the absence epilepsy model, Genetic Absence Epilepsy Rats from Strasbourg (GAERS).In this study we examined whether Heantos-4 can directly modulate exogenously expressed T-type calcium channels , whereas, CaV3.2 currents were significantly potentiated , and a higher concentration (1 mg/ml) in the CaV3.1 and CaV3.3 channels  and CaV3.2 , but not for CaV3.3.   between −35 mV and 10 mV following Heantos-4 application  Fig. . In addi mV Fig. . The tauion Fig. .Fig. 2VoV3.1 and CaV3.3 currents but potentiated CaV3.2 currents, we sought to evaluate its effects in a native system wherein these T-type isoforms are differentially expressed. VB neurons are glutamatergic, sensory thalamocortical neurons that primarily express the CaV3.1 channel [Given that Heantos-4 inhibited Ca channel –17 and w channel , 18. If P < 0.05 paired T-test; Fig. P > 0.05, paired T-test). This equated to a significant % increase in burst threshold in Heantos-4 treated neurons compared to control . Heantos-4 also induced a modest but significant depolarization of the resting membrane potential . Heantos-4 had no effect on the number of action potentials per burst , 250 mg/kg = 12.0 ± 1.4 s (n = 3), 500 mg/kg = 18.6 ± 2.3 s (n = 3); control vs 250 mg/kg P = 0.986, control vs 500 mg/kg P = 0.004, 250 mg/kg vs 500 mg/kg P = 0.011 ANOVA); Fig. V3.2 channels within the cortical seizure initiation focus.Heantos-4 in its dry powdered form suspended in CMC/saline see  was oralV3.1 and CaV3.3 but potentiates CaV3.2 channels, and subsequently found that burst-firing was differentially modulated in two distinct classes of thalamic neurons. In VB neurons that primarily express CaV3.1 burst firing was inhibited by Heantos, but in TRN neurons that express a combination of CaV3.2 and CaV3.3 burst-firing was not affected.Heantos-4 is approved by the Vietnamese Food and Drug Administration and is used clinically to treat opiate addicts in Vietnam , we sougV3.2 channels display a degree of redox sensitivity due to the presence of extracellular cysteine residues and/or a histidine residue found only in CaV3.2, but not CaV3.1 or CaV3.3 channels [V3.2 channels are inhibited by oxidation and potentiated by reduction of at least one of these residues. In addition, CaV3.2 displays a higher efficacy to inhibition by nickel (<100 μM) and other trace metals due to the presence of a high affinity binding and metal-catalyzed oxidation site provided by the same extracellular histidine residue [V3.2 could occur as a result of one or more active components. Of note, a shift in the activation curve to hyperpolarized potentials is observed in CaV3.1 and CaV3.2 channels, but not in CaV3.3 channels. Redox modulation of CaV3.2 channels induces only nominal effects on the voltage-dependence of activation [V3.2 channels with L-cysteine has been reported to cause an approximate 5 mV shift towards hyperpolarized potentials [V3.2 currents via reduction of redox-sensitive channel residues. However, the mechanism underlying the Heantos-mediated differential modulation of voltage-dependence of activation in CaV3.1 and CaV3.3 channels is unknown. It is possible that Heantos contains certain components that inhibit both CaV3.1 and CaV3.3, and other components that modulate the gating dynamics of CaV3.1 only. Alternately, a single component of Heantos may have a differential effect on the gating of CaV3.1 and CaV3.3, while simultaneously inhibiting both channels. Once analysis of the active components in Heantos has been performed it will be possible to dissect out how this differential modulation occurs. Further research is required to determine the exact compound(s) that induce the potentiation of CaV3.2 and also the inhibition of CaV3.1 and CaV3.3. To this end, we are currently conducting mass spectrographic analyses of Heantos-4 compounds that pass into the cerebrospinal fluid compartment in vivo. Preliminary analyses confirm the presence in brain CSF of > 98% of phytochemical classes identified with the components of Heantos-4 (data not shown). In the meanwhile, given that existing T-type channel pharmacological tools are limited to the redox-mediated inhibition or potentiation of CaV3.2 without affecting CaV3.1 or CaV3.3, Heantos-4 provides a novel pharmacological tool to inhibit CaV3.1 or CaV3.3 and potentiate CaV3.2.To date only a few pharmacological agents have been discovered that can selectively distinguish between the three distinct T-type calcium channel isoforms. Cachannels , 28, 29. residue . Heantos residue , 5 and ttentials . This isAgents that block all three T-type channel isoforms abolish both depolarizing burst-firing in TRN neurons and rebound burst-firing in thalamocortical neurons , 31–34. V3.1 due to its fast activation/inactivation kinetics and hyperpolarized voltage-dependence of activation [in situ hybridization [Cacna1g gene that encodes CaV3.1 in mice abolishes burst-firing in thalamocortical neurons [The T-type calcium currents that underlie low-threshold burst-firing in thalamic neurons have been studied extensively . Intivation . This isdization  and quandization , 36. In  neurons .V3.2/ CaV3.3 currents underlie the LTS since CaV3.2 channels activate quickly and CaV3.3 channels inactivate slowly [V3.2 is redox sensitive and TRN neurons display enhanced or suppressed burst-firing upon application of reducing and oxidizing agents, respectively [Cacna1i gene, encoding CaV3.3 the T-type current is approximately 40% of the total T-type current in TRN neurons of wild-type mice [V3.3 channel, T-type calcium currents were attenuated by approximately 80% in comparison to wild-type mice, although low threshold bursts could still be elicited in 75% of animals [V3.2/CaV3.3 knockout mice [V3.2 currents are predicted to contribute 20-40% of the total T-type calcium channel current in TRN neurons, with the remaining current contributed by the CaV3.3 isoform. This assumption is supported by our finding that Heantos-4 has no effect on burst-firing in TRN neurons and agrees with our in vitro data showing potentiation of CaV3.2 and inhibition of CaV3.3 currents and predicted to result in opposing modulation that cancels out any global effect on the total T-type current.In TRN neurons the burst duration activates quickly but is of longer duration than in VB neurons indicating that the combined Cae slowly , 37. Thie slowly  and by rectively . In suppype mice . In a se animals . Of noteout mice . Taken tV3.1 mRNA is expressed at higher levels in burst-firing PVN neurons than the other T-type channel isoforms, the PVN LTS is abolished by 50 μM nickel implicating CaV3.2 since this concentration would not block CaV3.1 currents [V3.2, not CaV3.1 appears to be involved in pain transduction in the PVN [To date, Heantos-4 has been used clinically solely for the purpose of treating aspects of drug addiction. The paraventricular thalamic nucleus (PVN) has been implicated in drug-seeking behaviour  and T-tycurrents . Further the PVN . With fu the PVN . This suIn addition to addiction, T-type calcium channels are implicated in a number of other neurological disorders including in epileptic seizures , 46–48, V3.1 and CaV3.3 but potentiates CaV3.2 T-type calcium currents. In addition, Heantos-4 selectively inhibits burst-firing in VB thalamic neurons while having no significant effect on TRN neurons. This supports the data from exogenously expressed T-type calcium channel isoforms and their corresponding differential expression in distinct VB and TRN thalamic nuclei. That Heantos-4 appears to exacerbate absence seizure activity in GAERS suggest that caution may be warranted in the clinical usage of Heantos-4 in seizure-prone individuals. Determining whether the effect of Heantos-4 on T-type calcium channels is related to its role in the treatment of drug-craving and withdrawal will require additional studies. Regardless, our findings that Heantos-4 differentially modulates distinct neuronal populations expressing T-type calcium channel isoforms may be relevant with respect to a number of neurological disorders in which T-type calcium channels are implicated [In summary, Heantos-4 is a clinically utilized treatment for drug addiction and is currently the subject of studies aimed at determining its mechanism(s) of action . Here weplicated , 68.V3.1, hCaV3.2 or hCaV3.3 were grown at 37 °C in DMEM supplemented with 10% heat inactivated fetal bovine serum. Cells expressing T-type calcium channel isoforms were selected by incubation with hygromycin. Cells were seeded on poly-D-Lysine (0.1 mg/ml) coated glass coverslips and hygromycin removed from media 48 h before voltage clamp recordings.Flp-In 293 cells (Invitrogen), stably expressing pcDNA5/FRT plasmids containing hCa2, 5 MgATP and 0.3 NaGTP (pH 7.2) External: 2 CaCl2, 1 MgCl2, 10 HEPES, 40 TEACl, 92 CsCl and 10 Glucose (pH 7.4). Fire polished patch pipettes (borosilicate glass) had typical resistances of 3 to 5 MΩ when containing internal solution. The recording chamber was grounded with a Ag/AgCl pellet. Whole-cell currents were recorded at room temperature using an Axopatch 200B amplifier . Data was acquired with pClamp software package version 9 (Axon Instruments Inc.). Series resistance (Rs) was compensated by 65–75% and seals with Rs values higher than 20MΩ or cells with peak current lower than 100pA were discarded. Currents sampled at 10 kHz and filtered at 2 kHz. Data analysis was carried out using Clampfit 9 (Axon Instruments Inc.) and software Origin version 7.5 .Calcium currents were recorded at 22–24 °C using whole-cell voltage clamp with the following solutions containing in mM: Internal: 120 Cs-Methanesulphonate, 11 EGTA, 10 HEPES, 2 MgClTimecourse of drug action was obtained by depolarizing the membrane with a 200 msec pulse to −30 mV from a holding potential of −100 mV every 5 s. The peak calcium current was taken from each trace for analysis. A two minute stable baseline was acquired before initiating a five minute Heantos application via the perfusate.V3.1 and CaV3.2 = 150 msec, CaV3.3 = 450 msec) from a holding potential of −110 mV. Test pulses from −90 to +10 mV were applied at 5 mV steps. Peak amplitude of calcium currents was plotted against test pulse potential and I-V curves were fitted using a modified Boltzmann equation: I = (Gmax*(Vm-Er))/(1 + exp((Vm-V50)/k)), where Gmax is the maximum value of membrane conductance, Vm is the test potential, Er is the extrapolated reversal potential, V50 is the half-activation potential, and k  reflects the voltage sensitivity. Activation curves were obtained by calculating conductance from the I-V curves and plotting the normalized conductance as a function of the membrane potential. The data was fitted with the Boltzmann equation: G/Gmax = A2 + (A1-A2)/(1 + exp((Vm-V50)/k)), where A1 is minimum normalized conductance, A2 is maximum normalized conductance, Vm is the test potential, V50 is the half-activation potential, and k value the slope of the activation curve (Slope constant).The current-voltage (I-V) relationship was obtained before and after the timecourse protocol by depolarizing the membrane with pulses  were used in acute brain slice experiments in accordance with Canadian Council for Animal Care guidelines.2), sacrificed by decapitation, the brains rapidly removed and transferred to ice cold sucrose-aCSF containing in mM: 214 sucrose, 26 NaHCO3, 1.25 NaH2PO4, 11 glucose, 2.5 KCl , 0.5 CaCl2, 6 MgCl2, bubbled with 95% O2:5% CO2. Brain tissue was glued to a cutting chamber in a vibrating microtome , which was then filled with ice cold sucrose-aCSF. Horizontal brain slices containing the whole thalamus (350 μm thick) were cut from the level of the ventral TRN/VB and incubated for a minimum of 30 min at 34 °C in a current clamp recording solution containing in mM: 126 NaCl, 2.5 KCl, 26 NaHCO3, 1.5 NaH2PO4, 2 CaCl2, 2 MgCl2, 10 glucose; bubbled with 95% O2:5% CO2. Slices were transferred to a recording chamber superfused with current clamp recording solution and maintained at 33–35 °C. VB and TRN neurons were visualized using a DIC microscope  and infrared camera  and visually identified by their location, morphology and orientation. All recordings were undertaken using a Multiclamp 700B amplifier and pClamp software version 9 (Molecular devices). The recording chamber was grounded with a Ag/AgCl pellet.Animals were anesthetized using isoflurane  filled with the following solution containing in mM: 120 K-gluconate, 10 HEPES, 1 MgClAdult GAERS (4–6 month old) were anesthetized with isoflurane and implanted with skull screw electrodes on the somatosensory cortex surface  and a reference electrode in the cerebellum . Electrodes were connected to a custom EEG interface implanted on the skull. Following a 2 week recovery period the interface was connected to a wireless headstage  and EEG signals acquired in the animals’ home cage for 1 h.Heantos-4, a brown, tea-like powder was provided by Dr. Sung at the Institute of Chemistry, Vietnam Academy of Science and Technology . For in vitro electrophysiology experiments Heantos-4 was prepared at 1 mg/ml by immersing in aCSF, vortexing for several seconds and incubating at 50 °C for 10 min followed by sonication at 22–24 °C for 5 min. Insoluble components of Heantos were then filtered and the flow-through used in all experiments at this concentration (1 mg/ml) or as a serial dilution.For in vivo experiments Heantos-4 was mixed into 0.5% carboxymethylcellulose (CMC) in 0.9% saline and administered via oral gavage 30 min prior to EEG recording.P value < 0.05 as significant. Data was plotted as mean values ± standard error.Data followed a normal distribution and statistical significance was calculated using Student’s T-test (paired where appropriate) or ANOVA with Tukey's Post-hoc test taking


Plasmodium falciparum M1 family aminopeptidase is currently considered as a promising target for anti-malarial chemotherapy. Several series of inhibitors developed by various research groups display IC50/Ki values down to nM range on native PfA-M1 or recombinant forms and block the parasite development in culture at µM to sub-µM concentrations. A handful of these inhibitors has been tested on murine models of malaria and has shown anti plasmodial in vivo activity. However, most of these inhibitors do also target the other neutral malarial aminopeptidase, PfA-M17, often with lower Ki values, which questions the relative involvement and importance of each enzyme in the parasite biology.P. falciparum strains, 3D7 and FcB1, respectively chloroquino-sensitive and resistant. Evaluated in vivo, on the murine non-lethal model of malaria Plasmodium chabaudi chabaudi, this amino-benzosuberone derivative was able to reduce the parasite burden by 44 and 40% in a typical 4-day Peters assay at a daily dose of 12 and 24 mg/kg by intraperitoneal route of administration.An amino-benzosuberone derivative from a previously published collection of chemicals targeting specifically the M1-aminopeptidases has been identified; it is highly potent on PfA-M1 (Ki = 50 nM) and devoid of inhibitory activity on PfA-M17 (no inhibition up to 100 µM). This amino-benzosuberone derivative (T5) inhibits, in the µM range, the in vitro growth of two Plasmodium parasites in vitro and in vivo, highlights the relevance of PfA-M1 in the biological development of the parasite as well as in the list of promising anti-malarial targets to be considered in combination with current or future anti-malarial drugs.The evaluation of a highly selective inhibitor of PfA-M1, over PfA-M17, active on The online version of this article (doi:10.1186/s12936-017-2032-4) contains supplementary material, which is available to authorized users. Plasmodium genus, Plasmodium falciparum being responsible for the most severe lethal forms , is presented on Fig. Murine models of malaria are currently used to test anti-malarial drug candidate in vivo. In this study, a non-lethal sistance –62. Intesistance , 47. Pridatabase ). The enP. c. chabaudi, therefore, appeared suitable to evaluate the in vivo effect of T5.A 3D- model for PcA-M1 was generated based on the 3D-structure of PfA-M1 (PDB: 4J3B) to assess the structural identity between the two enzymes . A superP. falciparum growth was examined in parasite cultures. The 50% inhibitory concentrations (IC50) of T5 against asynchronous 3D7 and FcB1 strains were 11.2 ± 3.4 and 6.5 ± 2.4 µM, respectively  allowed to calculate the selectivity index (SI) corresponding to the ratio of cytotoxicity and anti-malarial activity for both strains. T5 revealed a SI of 12.5 and 21.7 for 3D7 and FcB1, respectively.The ability of the benzosuberone derivative T5 to inhibit P. falciparum was confirmed in vitro, T5 was further evaluated in a detailed in vitro ADME screen  then exposed to three T5 doses corresponding to 2, 10 and 20 times the T5 IC50  is the dual inhibitor phosphinic compound Co4 (compound 4) that targets both PfA-M1 (Ki 79 nM) and PfA-M17 (Ki 13.2 nM). Nevertheless, it should be noted that this compound has been tested in vivo following a different protocol, namely using 100 mg/kg doses, twice a day for 7 days, which corresponds to daily doses sixteen times higher than those used here with T5, and to a much longer treatment timing, close to doubled (7 days versus 4 days in the current study) [According to current literature, malarial M1/M17 inhibitors present interesting in vitro potency against t study) . Anothert study) .P. falciparum strains grown in vitro, displaying IC50 of 6.5 ± 2.4 µM (FcB1) and 11.2 ± 3.4 µM (3D7) and showing a significant antiparasitic effect in vivo. Note that there are several orders of magnitude discrepancies between the Ki value of T5 on PfA-M1 and the IC50 value on parasite growth in vitro. Such a situation is currently encountered in the malaria field  versus PfA-M17 (Ki ≫ 100 µM). This compound was active against two The morphological alterations induced by T5-treatments, notably in both trophozoite and schizont stage parasites confirm a key role of PfA-M1 during the breakdown of haemoglobin but indicate a possible additional role in late stages. Importantly, T5 had no effect on the morphology of ring stage parasites. The digestive vacuole swelling observed at the three tested T5 concentrations, at 36 h post-invasion, was however lower than what was observed by Harbut and collaborators using the PfA-M1-inhibitor BTA on the 3D7 strain , which iIn conclusion, this is the first report denoting in vitro and in vivo inhibitory activity for a new compound targeting PfA-M1 but not PfA-M17, further strengthening the potential of this target for anti-malarial research.


Their optical properties were analysed, and they was mixed with TiO2, sintered, and used as a photoelectrode (PE) in dye-sensitized solar cells (DSSCs). The as-prepared photoelectrode was characterized by photoluminescence spectroscopy, diffuse reflectance, electrochemical impedance spectroscopy (EIS) and X-ray diffraction. The photoelectric conversion efficiency of the DSSC with TiO2:Y1.86Eu0.14WO6 (100:2.5) was 25.8% higher than that of a DSCC using pure TiO2 as PE. This high efficiency is due to the ability of the luminescent material to convert ultraviolet radiation from the sun to visible radiation, thus improving the solar light harvesting of the DSSC.Y DSSCs4f transitions in RE-doped luminescent materials can be efficiently used as either NIR-to-visible up-convertors or ultraviolet (UV)-to-visible down-convertors for applications in DSSCs.The spectral response of conventional DSSCs is very narrow compared to the whole wavelength range of sunlight. The band between 400 and 700 nm only accounts for approximately 43% of the sun's total radiant energy –8. All oar cells ,10. A noet al. described how the use of the inorganic phosphors LAVO4:Dy3+ introduced into a DSSC, not only improves the electrical conversion efficiency (ca 23%), but also expands considerably the lifetime of the solar cell [On the other hand, the literature reports that exposure to UV light produces serious damage to the DSSC since the iodine present in the electrolyte is irreversibility consume under UV light . Down-colar cell .3+-doped Y2W06 and its application in photoelectrodes (PE) in DSSCs in order to investigate the possibility of increasing the spectral response of DSSCS using down-shifting luminescent materials.This paper is part of our continuing research on the synthesis, applications, characterization and luminescent properties of inorganic phosphors containing rare-earth cations –16. In t2.2.1.2O3, Y2O3, WO3, TiO2 nanopowder (21 nm), I2, LiI, tetrabutylammonium iodide, and t-octylphenoxypolyethoxyethanol as emulsification agent (Triton X-100) were analytical grade and supplied by Sigma-Aldrich. Conducting glass plates of indium tin oxide (ITO) with a surface resistivity of 8–12 Ω sq−1 and the sensitizing dye N-719 (RuL2(NCS)2:2TBA ) were also purchased from Sigma-Aldrich and used without further treatment.All chemical reagents including Eu2.2.2WO6:Eu3+ phase was prepared via a solid-state reaction at high temperature [2O3, Eu2O3 and WO3, in stoichiometric proportions. The mixtures were placed in an alumina boat and heated to 973 K for 10 h before cooling below 400 K. The sample was removed from the crucible, ground into a powder, and reheated for 10 h at 1273 K. This procedure was repeated, and the sample was finally heated to 1373 K for another 10 h. All of these synthetic processes were performed under an air atmosphere. Optical inspection of the products showed homogeneous white powders.The Yperature ,18. All α radiation (λ = 1.54057 Å) across the range of 10° ≤ 2θ ≤ 60° to determine the phase purity. The experimental PXRD patterns for the doped and un-doped samples were almost in perfect agreement with those reported in the inorganic crystal structure database (ICSD) database [Powder X-ray diffraction (PXRD) data were collected using a Bruker D8 Advance diffractometer fitted with a graphite monochromator using Cu Kdatabase .2.3.1.86Eu0.14WO6  were dispersed in a TiO2 sol, ultrasonicated, stirred for about 30 min and finally used as slurry. The PE was prepared by the doctor-blade technique [−4 M in ethanol) for 24 h. For comparison, a PE without Y1.86Eu0.14WO6 (i.e. pure TiO2) was also prepared. Finally, the DSSC was assembled by filling an electrolyte solution  between the dye-sensitized Y1.86Eu0.14WO6/TiO2 electrode and a platinized conducting glass electrode. Both electrodes were clipped together, and a cyanoacrylate adhesive was used as a sealant to prevent the electrolyte solution from leaking. For comparison, a DSSC was prepared with the pure TiO2 PE using the same method.Different amounts of Yechnique ,21. Afte2.4.1.86Eu0.14WO6 phosphor surface morphologies were determined via scanning electron microscopy . The photoluminescence (PL) spectra were measured using a JASCO FP-6500 spectrofluorometer. All of the spectra were collected at room temperature. The sample quantities were identical across all experiments so that the photoluminescence intensities could be compared. A Perkin-Elmer Lambda 20 UV-Vis spectrophotometer equipped with a Labsphere RSA-PE-20 diffuse reflectance accessory was used to measure the diffuse reflectance of the samples over the range of 200--600 nm (6.2--2.1 eV). The photovoltaic test of the DSSCs was performed using a PET cell tester , under simulated AM 1.5 solar illumination of one sun (100 mW cm–2) from a 300 W Xe lamp. The intensity was adjusted using a calibrated c-Si solar cell. The I–V measurements were attained using a Keithley Sourcemeter model 2400. The electrochemical impedance spectroscopy (EIS) analysis were carried out with Solartron 1280 impedance analyser. Nyquist plots were measured at open circuit voltage (Voc) over a frequency range of 1–104 under one-sun illumination. To determine the amount of dye desorbed from the photoelectrode a 1 M solution of NaOH was used and the UV-Vis spectra were recorded in a Perkin-Elmer Lambda 25 spectrophotometer.The phase purities were determined via PXRD analysis (vide supra). The nanocrystalline Y3.1.86Eu0.14WO6 was inspected via SEM. The results revealed that the samples are spherical and exhibit some tendency to aggregate, as shown in 2 and the doped samples. No remarkable differences were observed. The phosphor was embedded within the structure of TiO2, which is an important characteristic to obtain a good dye distribution [The crystallite morphology of Yribution .Figure D = 0.90λ/βcosθ) can estimate the crystallite size of the luminescent material from the PXRD results if the crystals are smaller than 1000 Å. In this equation, D is the average grain size, λ is the wavelength of the radiation used in the diffraction experiments (vide supra), θ is the diffraction angle and β is the full width at half maximum (FWHM) of the observed peak [θ = 29.5° was used to calculate the grain size of Y1.86Eu0.14WO6. Our results indicate that the crystallite size for the sample is approximately 62 nm. The PXRD patterns of pure TiO2 and Y1.86Eu0.14WO6  dispersed in TiO2 and pure Y1.86Eu0.14WO6 are displayed in Additionally, the Scherrer equation  dispersed in TiO2 are superimposed in the 2 is 3.2 eV [2 were not obstructed by the inorganic phosphor.The corresponding UV-Vis diffuse reflectance spectra of the pure TiOs 3.2 eV . Ground 7F0 → 5L6), 465 nm (7F0 → 5D2) and 540 nm (7F0 → 5D1). The broad, intense band consists of overlapping O2−-Eu3+ and O2−-W6+ charge transfer bands. Upon excitation at 300 nm, the resulting emission spectrum originates from the transition between the 5D0 excited state to the 7Fj ground states  for the 4f6 configuration of Eu3+, with the most prominent electric dipole transition, 7D0 → 5F2, centred at about 610 nm. 1.86Eu0.14WO6 compared with the absorbance of N-719 dye.The synthesized phosphor shows the typical excitation and emission bands. The photoluminescence excitation spectrum (PLE) exhibits a broad charge transfer band centred at about 300 nm along with very weak f-f transitions at about 395 nm (2:Y1.86Eu0.14WO6 photoelectrodes were prepared using the doctor-blade technique [2:Y1.86Eu0.14WO6 (100:2.5) electrode  and pure TiO2 PEs. To obtain significant data, five DSSCs were prepared with and without 2.5 wt% of the luminescent material. When the PE consists of pure TiO2, the DSSC has a short-circuit current density (Jsc) of 8.6 mA cm–2, an open circuit voltage (Voc) of 0.76 V, a field-factor (FF) of 0.48 and a photoelectric conversion efficiency (PCE) of 3.1%. The DSSCs with TiO2:Y1.86Eu0.14WO6 (100:2.5) photoelectrode show a Jsc of 12.3 mA cm–2, a Voc of 0.76 V, a FF of 0.43 and a PCE of 3.9%. The DSSC with the down-shifting material exhibits a higher Jsc due to the increase in photo-generated photons in the visible region converted from the UV region of the solar spectrum. By the other hand, the Rs and Rsh values for the DSSC using the mixed TiO2:Y1.86Eu0.14WO6 (100:2.5) photoelectrode were 9.8 Ω and 1657 Ω, respectively. Whereas for the DSSC using bare TiO2 the values were and 10.8 Ω cm2 and 2036 Ω cm2, respectively.2 and TiO2:Y1.86Eu0.14WO6), the photoelectrodes were soaked in a 1 M NAOH solution for 12 h. The absorption spectra of the desorbed dye represented the amount of dye absorbed for the photoanodes. −9 and 2.94 × 10−9 mol cm−2 for TiO2 and TiO2:Y1.86Eu0.14WO6, respectively. Remarkable is the fact that the TiO2:Y1.86Eu0.14WO6 (100:2.5) show less absorption than the pristine TiO2, their photovoltaic properties are better. The DSSC parameters obtained in our study are shown in To determine the amount of dye loading in both photoanodes . The higher IPCE is attributed to the down-shifting from the ultraviolet light to visible light due to the presence of Y1.86Eu0.14WO6 in the photoelectrode [The incident photon-to-current efficiency (IPCE) for the dye-sensitized solar cell of DSSC with down-shifting material and with pure TiOlectrode . The ultlectrode . Our resVoc. The recorded spectra show a circle at low frequency corresponds to the impedance of TiO2/electrolyte interface. 2:Y1.86Eu0.14WO6 (100:2.5) (3.6 Ω) than that using the bare TiO2 (4.8 Ω). This also indicates less impedance suggesting a faster electron transport, and consequently an improvement in the conversion efficiency. If the concentration of DS phosphor exceeds 2.5%, the charge carrier transportation is hindered. According to Yin et al., the TiO2:Y1.86Eu0.14WO6 (100:2.5) must have the smaller Rct value. This implies that the electron transfer from the TiO2:Y1.86Eu0.14WO6 to the electrolyte is more efficient than that of the pristine TiO2 electrode [On the other hand, the electrochemical impedance spectroscopy (EIS) is an important tool to understand the transport properties within an electrochemical system –34. The lectrode .Figure 2−xEuxWO6/TiO2 core-shell nanoparticles as photoelectrode (PE) for use in dye sensitized solar cells (DSSCs) is in progress.More work in order to prepare Y4.2:Y1.86Eu0.14WO6 (100:2.5) mixture was used as a photoelectrode to fabricate DSSCs that were more efficient than DSSCs based on pure TiO2 photoactive electrodes. The solar conversion efficiency for a DSSC with the mixed PE reached 3.9%, which is 25.8% higher than that of a DSCC using pure TiO2 as the PE.The optimized TiO2. Finally, in this work we demonstrated that the down-shifted luminescence could be an effective way to improve the sunlight harvesting of solar cells. The last result is supported by electrochemical impedance spectroscopy and optical measurements.On the other hand, ground state diffuse reflectance absorption measurements indicated that the addition of the down-shifting phosphor does not affect the semiconductor character of TiO


Understanding the molecular basis underlying the formation of bone-forming osteocytes and lipid-storing adipocytes will help provide insights into the cause of disorders originating in stem/progenitor cells and develop therapeutic treatments for bone- or adipose-related diseases. In this study, the role of RGS2 and RGS4, two members of the regulators of G protein signaling (RGS) family, was investigated during adipogenenic and osteogenenic differentiation of human mesenchymal stem cells (hMSCs).RNA against RGS2 resulted in decreased differentiation efficiency during both adipogenesis and osteogenesis. On the other hand, expression knock-down of RGS4 also resulted in decreased adipogenic differentiation but increased osteogenic differentiation.Expression of RGS2 and RGS4 were found to be inversely regulated during adipogenesis induced by dexamethasone (DEX) and 3-isobutyl-methylxanthine, regardless if insulin was present, with RGS2 up-regulated and RGS4 down-regulated in response to adipogenic induction. RGS2 expression was also up-regulated during osteogenesis at a level similar to that induced by treatment of DEX alone, a shared component of adipogenic and osteogenic differentiation inducing media, but significantly lower than the level induced by adipogenic inducing media. RGS4 expression was down-regulated during the first 48 h of osteogenesis but up-regulated afterwards, in both cases at levels similar to that induced by DEX alone. Expression knock-down using small interfering RGS2 and RGS4 are differentially regulated during adipogenic and osteogenic differentiation of hMSCs. In addition, both RGS2 and RGS4 play positive roles during adipogenesis but opposing roles during osteogenesis, with RGS2 as a positive regulator and RGS4 as a negative regulator. These results imply that members of RGS proteins may play multifaceted roles during human adipogenesis and osteogenesis to balance or counterbalance each other’s function during those processes.The online version of this article (10.1186/s40659-017-0148-1) contains supplementary material, which is available to authorized users. Advancement in understanding adipose and bone tissue biology will help develop new strategies for the prevention and intervention of adipose- and bone-related diseases, including obesity and osteoporosis. Adipogenesis and osteogenesis are processes in which uncommitted stem cells differentiate into mature adipocytes or osteocytes, respectively. Over the past two decades or so, many individual adipogenic regulators have been independently uncovered, which include specific signaling pathways , growth factors or cytokines , transcription factors , GTPase proteins and its regulators (RHO and ROCK etc.), epigenetic regulators and microRNAs etc. . SimilarsiRNA hits identified through a high throughput screen were found to promote osteogenic differentiation but inhibited adipogenesis, and cAMP was identified to play opposing roles in osteogeneis vs. adipogenesis [While the adipogenic and osteogenic lineage commitment clearly involve distinct master transcriptional regulators and downstream genetic cascades, in many cases, they are also regulated by the same signaling pathways  and genes , 18. Forogenesis . In addiogenesis . Some meogenesis –23. Aginogenesis –28. ThesHuman mesenchymal stem cells (hMSCs) are a type of adult stem cell that exist in multiple tissues in the body, including adipose tissue, bone marrow and peripheral blood, and play important roles in maintaining normal tissue homeostasis. They can be isolated, expanded and differentiated in vitro into a number of specialized cell types including adipocytes and osteocytes, which makes them an excellent in vitro cell model for studying human adipogenesis and osteogenesis . Using aq-class Gα subunits, whereas RGS4 has intrinsic GAP activity for both Gq and Gi/o-class Gα subunits [Using adipose tissue derived hMSCs as an in vitro model for adipogenic differentiation, we identified through microarray analysis two members of the regulator of G protein signaling (RGS) family, RGS2 and RGS4, which were differentially regulated upon adipogenic induction (unpublished data). Both RGS2 and RGS4 belong to the B/R4 subfamily of RGS proteins family characterized by a conserved 120 aas RGS domain flanked by short amino and carboxyl termini . They arsubunits , 39. As subunits –45. Asidsubunits .RGS2−/− mice showed lower expression levels of adipogenic markers including PPARYγ, CEBPα, and leptin. In another study using NIH-3T3 mouse preadipocyte cells, RGS2 overexpression promoted adipogensis in the presence of a ligand for PPAR γ [RGS4 knockout mice also showed a significantly lower body weight compared to wild type mice [Despite knowledge in a wide array of biological events involving RGS proteins, our current understanding of the role of RGS proteins during human adipogenesis and osteogenesis is very limited. No study thus far has directly examined the role of RGS in osteogenesis, though past studies have revealed an important role of RGS proteins during bone remodeling by modulating osteoclastogenesis , 48. RGSr PPAR γ . RGS4 knype mice , though ype mice . The rolIn this study, we characterized the temporal expression patterns of both RGS2 and RGS4 genes during adipogenic and osteogenic differentiation of hMSCs, as well as their function during both processes. Our results demonstrated that RGS2 and RGS4 are differentially regulated during adipogenic and osteogenic differentiation of hMSCs, with both playing positive roles during adipogenesis but opposite roles during osteogenesis.The adipose-derived hMSCs used in this study were obtained from a commercial source see “”. PrevioOf 3 independent repeats, the average clonogenicity (number of cells per clone is > 50) was determined to be 8% (± 0.67%)  family began with a microarray analysis aimed at identifying novel regulators of human adipogenesis (unpublished data). Briefly, the expression profiles of hMSCs exposed to adipogenic differentiation condition (IBMX + DEX + insulin in growth media) was compared to undifferentiated hMSCs at 36 and 72 h post induction. Through this analysis, RGS2 and RGS4 were found to be significantly up- and down-regulated respectively during early adipogenesis, with RGS2 up regulated by 15-folds, and RGS4 down regulated by 100-folds at 72 h post induction. Because adipose and bone cells share common progenitor cells and there are shared regulators between adipogenesis and osteogenesis , 18, we RGS2 and RGS4 during both osteogenic and adipogenic differentiation of hMSCs. For adipogenesis, hMSCs were cultured in hyclone growth media (CM) supplemented with IBMX (0.45 µM), DEX (1 µM), and insulin (10 µg/ml), which is abbreviated as adipogenic inducing media (AIM). For osteogenesis, cells were cultured in CM supplemented with DEX (0.2 µM), β-glycerophosphate (10 mM), and ascorbic acid-2-phosphate (0.05 mM), which is abbreviated as osteogenic inducing media (OIM). DEX is a common component used in hMSC differentiation into adipocyte, osteocyte, and chondrocytes, and is thought to be necessary to potentiate differentiation and prevent apoptosis [RGS2 and RGS4, RT-PCR was performed on both genes in hMSCs cultured in 8 different media treatments that include CM (control group), DEX (0.2 µM), DEX (1 µM), DEX (0.2 µM) + IBMX, AIM with 0.2 µM DEX, AIM with 1 µM DEX, OIM with 0.2 µM DEX, and OIM with 1 µM DEX, at eight different time points including D0.5, D1, D1.5, D2, D4, D5, D6 and D7 post initial treatment, with media change at 48-h intervals. All treatment group media was made in CM. Transcript level of RGS2 and RGS4 in each treatment group were normalized to that of a housekeeping gene HSP90  and compared to its normalized level in CM control at corresponding time point.We first sought to determine the temporal expression pattern of poptosis , 57. TheC/EBPα and PPARγ, two well-known master regulators for adipogenic lineage commitment, were both highly up regulated in AIM treated cells  vs. osteogenic induction (OIM)  Fig. a. StartiRGS2 is upregulated by all treatment types relative to its expression in control CM, though the degree of changes differs between adipogenic conditions and the other treatment conditions at certain time points  compared to the other treatment conditions between D1 and D4.Expression of RGS2 and RGS4 in OIM treatment was regulated in parallel to that by dexamethasone treatment alone, regardless of DEX concentrations, indicating that the other two components in OIM media, AA-2-P and β-glycerophosphate, had no significant effect on RGS2 and RGS4 expression. RGS2 was upregulated by both DEX and OIM starting as early as D0.5 and subsiding by D6, whereas RGS4 was slightly downregulated by DEX and OIM during D0.5 to D2 but up-regulated afterwards. On the contrary, expression of both RGS2 and RGS4 differs significantly in adipogenic conditions as compared to in DEX alone, regardless of DEX concentration, indicating that IBMX and/or insulin exerted additional effect on their expression. Since their expression in IBMX + DEX inducing media is highly similar as in AIM, it suggests that IBMX, and not insulin, exerts such effect. Similar to DEX treatment, adipogenic treatment enhanced RGS2 expression until D6, but at significantly higher level (around twofolds) than DEX alone during D1 to D4. For RGS4 expression however, adipogenic treatment not only significantly inhibited the expression of RGS4 at a much greater level than DEX alone during D0.5 to D2 (near undetectable level), but also continued to downregulate its expression throughout the remaining course when it was being upregulated by DEX alone. Hence regulation of RGS2 and RGS4 expression were completely opposite to each other in adipogenic conditions.In conclusion, expression of both RGS2 and RGS4 would remain similar in AIM based on a different growth media. Temporal expression pattern of RGS2 and RGS4 was then re-examined in parallel in culture conditions based on Hyclone CM, heat-inactivated fetal bovine serum (FBS) in DMEM complete media (HI-FBS CM), or FBS in DMEM complete media (FBS CM). The conditions included Hyclone CM or HI-FBS CM, Hyclone CM DEX (1 µM) or HI-FBS CM DEX (1 µM), Hyclone CM based AIM or HI-FBS CM based AIM or FBS CM based AIM , and Hyclone CM based OIM (with 1 µM DEX) at D0.5, D1, D1.5, D2, D3 and D4 post adipogenic initiation  used in composing the AIM media for adipogenic induction is a proprietary product that might contain unknown growth factor supplement, we wondered whether expression of RGS4 was downregulated by both DEX alone and AIM from D0.5 to D3, regardless whether they were Hyclone CM or HI-FBS CM based, however, its level was significantly higher in Hyclone CM based AIM than in HI-FBS CM based AIM at day 1 and day 2  than that in HI-FBS CM based AIM , 84% (day 2), 83% (day 6), and 72% (day 12) compared to siCON, without any noticeable cytotoxic effect in siCON treated cells  and RGS2 mRNA (siRGS2-2 and siRGS2-3) were tested. SiRNA was transfected into cells at 2 days (D-2) prior to adipogenic differentiation initiation (D0 AIM). Expression of RGS2 and RGS4 in transfected ad-hMSCs were examined at day 1, 3, 5, 7, and 12 post adipogenic initiation with AIM containing 1.0 µM DEX or osteogenic initiation with OIM containing 0.2 µM DEX  lower at day 3 (39%), 5 (32%), and 7 (48%) in siRGS4-8 treatment groups compared to siCON controls (100%)  lower at day 3 (56%), 5 (36%), 7 (31%) and 12 (65%) compared to controls (100%)  lower in siRGS4-8 samples at day 1 (51%), 3 (39%), 7 (41%) and 12 (78%) compared to siCON (100%)  lower at day 1 (65%), and 3 (23%), 7 (57%) and 14 (76%) , except for day 7, when expression of RGS4 isoform 3 was consistently, thought slightly, down regulated in siRGS4-8 and siRGS4-10 transfected cells as compared to in siCON transfected cells by about 30 and 20% in AIM and OIM treatment condition, respectively  lower in siRGS2-2 samples at day 1 (84%), 3 (73%), 5 (83%), and 7 (80%) compared to siCON (100%)  lower at day 1 (71%), and 3 (56%), 5 (63%), 7 (68%) and 12 (79%)  lower in siRGS2-2 samples at day 1 (73%), and non-significantly lower by day 3 (86%), 7 (80%), and 12 (83%)  down regulated at day 1 (51%), day 3 (63%), day 7 (75%), but resumed to control level by day 12 (103%) , we wondered whether siRGS2 would have greater knockdown in HI-FBS CM based AIM due to lower basal level of RGS2 expression, and hence greater phenotypic effect. Similar to previous studies, expression knockdown of RGS2 by siRGS2-2 and siRGS2-3 was evaluated at days 1, 3, 5, 7, and 14 post treatment initiation with HI-FBS CM based AIM at 48 h after siRNA transfection. In siRGS2-2 samples, expression of RGS2 was significantly lower (p < 0.05) at day 3 (69%), 5 (60%) and 7 (51%), but resumed to control levels (97%) at day 12 as compared to siCON samples (100%)  at day 1 (43%), 3 (34%), 5 (32%), and 7 (49%) and resumed to control level by day 12 (94%) compared to siCON (100%)  compared to siRGS2-2 (by 30–50%) at the RNA level, both of which are greater than their respective silencing effect in Hyclone CM based AIM. Next, we examined the effect of expression knockdown induced by siRGS2 and siRGS4 on adipogenic and osteogenic differentiation of hMSCs.In conclusion, during early adipogenic and osteogenic treatments in Hyclone CM based media, both siRNA on several metrics of adipogenesis. As described previously, ad-hMSCs were reverse transfected with 16.5 nM of control (siCON) or targeted siRNA in Hyclone growth media (CM). After 48 h, adipogenesis was initiated by AIM with 1.0 μM DEX. After 12 days of AIM treatment, with media change at 48-h intervals, cells were fixed and stained with DAPI (nuclear stain) and OilRedO (oil droplet staining). Overlapping images of DAPI and OilRedO stained cells were taken from multiple wells of each treatment group for total cell counting, adipocytes counting and area measurements of stained lipid droplets in OilRedO images, and OilRedO dye was subsequently extracted with isopropanol and quantified by absorbance reading at 515 nm  compared to siCON controls (100%), and the difference between siRGS4-8 treatment group (98%) and siCON control (100%) was insignificant  compared to siCON (100%), but insignificantly lower in RGS4-8 (85%) compared to siCON (100%)  and siRGS4-10  treatment groups compared to siCON controls (100%)  and RGS4-10  treatment groups compared to siCON controls (100%)  and siRGS4-10  treatment groups compared to siCON controls (100%)  or siRGS2-3 (91%) treatment groups and siCON controls (100%) neither  measurements of OilRedO stained oil droplets trended lower in both siRG2-2 (81%) and siRGS2-3 (80%) treatment groups compared to siCON controls (100%), but the difference was not statistically significant  and siRGS2-3 (97%) slightly but consistently trended lower than siCON controls (100%), though statistically insignificant neither (data not shown). Overall, siRGS2 did not significantly affect total fat areas or total cell numbers as compared to siCON treatment in Hyclone CM based AIM condition.For ity Fig. a. OilRedher Fig. b. Consisant Fig. c, d. NucsiRGS2 on adipogenesis induced by HI FBS CM based AIM was also analyzed. In contrary to Hyclone CM based AIM condition, OilRedO stain intensity in both siRGS2-2 and siRGS2-3 treatments appeared visually reduced compared to siCON  but not in siRGS2-3 (96%) treatment groups as compared to siCON controls (100%)  of stained oil droplets was significantly lower in siRGS2-2  and only trended lower in siRGS2-3  treatments as compared to in siCON controls (100%)  but not in siRGS2-3 (92%) treatment groups compared to siCON controls (100%)  and siRGS2-3 (86%) treatments compared to siCON controls (100%)  and siRGS2-3 (90%) groups were not significantly different from siCON controls (100%)  in hMSCs that had been subjected to siRGS2/siRGS4 or siCON transfection. Similar to previous expression analyses, expression of those genes in siRGS2/siRGS4 treated cells was measured by RT-PCR and compared to its value in siCON treated samples at the same time point, after normalization to the expression level of internal control HSP90.Expression knockdown of siRGS4 samples, expression levels of PPARγ and C/EBPα in siRGS4-8 were not significantly different from siCON controls at all time points examined, except for PPARγ upregulation at day 1  and C/EBPα downregulation at day 12  compared to siCON (100%)  compared to siCON controls (100%)   Fig. a, b. How0%) Fig. a, b. ExpsiRGS2 samples, expression level of PPARγ was slightly but significantly lower in siRGS2-2 treatments (84–90%) as compared to siCON (100%) on day 5, 7 and 12, and similar difference between siRGS2-3 and siCON was observed on day 1 and day 12  and siRGS2-3 (77%) on day 3 compared to siCON (100%), but remained unchanged at the other time points  compared to siCON controls (100%), but was not significantly changed at the other time points. In siRGS2-3 samples, levels of LPL were slightly but significantly lower at day 3 (83%) and 5 (72%) compared to siCON samples (100%), but remained insignificantly different at the other time points  and day 5  but not by siRGS2-3 as compared to siCON (100%)  and 12 , but slightly downregulated in siRGS2-3 at day 3  compared to siCON (100%)  at day 5 but not by siRGS2-3 compared to siCON (100%)  than siRGS2. In addition, siRGS4-10 downregulated all three genes whereas siRGS4-8 only inhibited LPL, which is also consistent with the more disruptive effect of siRGS4-10 on adipogenic differentiation of hMSCs as compared to siRGS4-8.In conclusion, consistent with their different levels of inhibitory effect on the adipogenic outcome of hMSCs, siRNA transfection approach. Briefly, even number of ad-hMSCs were reverse transfected with xtremeGENE/siRNA complex at 16.5 nM in Hyclone control (CM) media. After 48 h, osteogenesis was induced by 0.2 μM DEX OIM media, which was subsequently changed every 48 h. After 18–26 days of OIM media treatment, cells were fixed and stained with alizarin red S, which specifically stains for calcific deposit (hydroxylapatite) by osteocytes. Alizarin Red S dye was subsequently extracted with acetic acid and quantified calorimetrically at 405 nm as a measurement of osteogenic differentiation efficiency  present in siRGS4-10 treatment groups but not so much in siRGS4-8 groups    and siRGS2-3  treated samples compared to siCON controls (100%) .In comparison to siRGS4 and siRGS2 had opposing effect on osteogenic differentiation of hMSCs, with the former promoting while the latter inhibiting the process, without affecting total cell numbers. In addition, similar to a more disruptive effect of siRGS4-10 on adipogenic differentiation of hMSCs as compared to siRGS4-8, the former also demonstrated a greater enhancing effect on osteogenic differentiation of hMSCs compared to the latter.In conclusion, RGS4 promoted osteogenic differentiation of hMSCs while siRGS2 inhibited it without affecting total cell numbers, it implied a direct effect on osteogenic differentiation. Effect of siRGS4 and siRGS2 on the temporal expression of known osteogenic markers, Runx2, Osteocalcin (OC) and alkaline phosphatase (ALP) was further evaluated. Runx2 is an osteogenic master regulator [OC encodes a bone specific protein synthesized by osteoblast and serves as a marker of osteogenic maturation [ALP encodes an enzyme that function to promote mineralization by increasing phosphate concentrations [Since siegulator . OC encoturation , while Atrations , 61. Sintrations , primerssiRGS4 OIM treatments, Runx2 expression was upregulated in both siRGS4-8 and siRGS4-10 treated cells as compared to siCON controls starting on D3 post OIM treatment initiation, but the enhancement is clearly much stronger in siRGS4-10 samples, at about 2- to 4-fold higher level than in siRGS4-8   in siCON (100%)  and day 5  in siRGS2-2 and at day 14  in siRGS2-3 compared to siCON (100%)  and siRGS2-3 (87%) but then was transiently upregulated at day 3  and day 5  in siRGS2-2 and at day 14  in siRGS2-3 compared to siCON (100%) . Both RGS2 and RGS4 belong to the R4/B subfamily, along with RGS1, 3, 5, 8, 13, 16, 18 and 21 . RGS proαq, Gα12 . These Gαq, Gα12 , 69. RGSsubunits , 39. HowRGS4 was expressed in high level in hMSCs but was quickly down regulated to near undetectable level within 24 h post adipogenic initiation. Expression knockdown of RGS4 by siRGS4 resulted in significantly reduced total cell numbers, indicating that it normally plays a role in regulating cell proliferation, and possibly a role in hMSCs self-renewal. Interestingly, down regulation of RGS4 also inhibited adipogenic differentiation of hMSCs, indicating that it plays a positive role during adipogenesis, which seems to contradict with its down regulation in response to adipogenic induction. One could speculate that its down regulation during the first 3 days of adipogenic induction might be necessary for hMSCs to exit its ‘stem cell’ mode and prepare for differentiation, however, subsequent adipogenic commitment (day 3 and day 6 post adipogenic initiation) and maturation (after day 6) would benefit from up-regulation of RGS4 expression. Molecular study indeed demonstrated that siRGS4 inhibited the expression of PPARγ and C/EBPα, whose normal upregulation in response to adipogenic induction is concomitant with the onset of adipogenic commitment, suggesting that RGS4 expression is beneficial to the upregulation of those two master control genes. It is also possible that the down regulation of RGS4 during normal adipogenic differentiation of hMSCs might have exerted a negative effect throughout and maintaining its high level of expression as in undifferentiated hMSCs could have significantly facilitated the differentiation process. To distinguish the two different scenarios would require additional future study to effectively overexpress RGS4 during adipogenic differentiation. Nevertheless, our observation of the positive role that RGS4 plays during adipogenesis is consistent with past study of RGS4 knockout mice, which showed a significantly lower body weight compared to wild type mice [ype mice , though ype mice .PPARγ, C/EBPα and LPL, suggesting that RGS2 might normally regulate adipogenesis through a different route. Nevertheless, the overall effect of siRGS2 on adipogenesis is consistent with past findings from RGS2 knockout mice, which have lower weights, reduced fat deposits, decreased serum lipids, and lower leptin levels [siRGS2 in HI FBS CM based AIM compared to Hyclone CM based AIM, which corresponded to a greater level of RGS2 expression knockdown by siRGS2 in the former vs. the latter condition. It is possible that expression knockdown is context dependent and may be more effective when the target gene’s overall expression level is lower.On the other hand, expression of RGS2 was very low in hMSCs but escalated to high level within 24 h of adipogenic initiation, which lasted until day 6 post adipogenic initiation before subsiding to the same expression level as in hMSCs. Our functional study of RGS2 also demonstrated that it normally played a positive role during adipogenic differentiation of hMSCs, as its expression knockdown led to decreased differentiation. However, unlike RGS4, knockdown of RGS2 did not appear to exert significant effect on the expression of known adipogenic marker genes including n levels , 51. It q- and Gi-class Gα proteins can activate Rho  regulated signaling pathways involved in cytoskeletal remodeling, cell movement and organelle development [αi and/or Gαq activity by RGS4 or RGS2 is important for downregulating RhoA-ROCK signaling and allowing subsequent activation of PPARγ. Additionally, Gi-class Gα proteins can inhibit adenylyl cyclase (AC) activity required for cAMP production. Increased cAMP level as the result of IBMX induction plays a key role in adipogenic commitment [αi, it is conceivable that RGS4 may allow the activation of AC and subsequent intracellular increase of cAMP to promote adipogenesis.While future studies are needed to understand the molecular mechanisms by which RGS2 and RGS4 might regulate adipogenesis, one could hypothesize a couple of potential mechanisms. Both Gelopment –72. RhoAelopment . Inductielopment . It is pmmitment , 74. By αq-PLC and Gαs-AC signaling [q-class Gα proteins can activate the phospholipase C-β (PLC-β) pathway that leads to the cleavage of phosphatidylinosiatol 4,5-biphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG), and regulate intracellular Ca2+ release as well as protein kinase C (PKC) activity [2+ and functions to maintain intracellular Ca2+ homeostasis through the Gαq/11 pathway [2+ pathway  and planar polarity pathway. All three pathways are implicated in bone formation [2+ pathway leading to intracellular Ca2+ increase [In addition, few past studies investigated the role of RGS proteins in osteogenesis. Bone remodeling involves bone reabsorption mediated by osteoclasts and bone production mediated by osteocytes. Several past studies have suggested a role of RGS proteins in bone remodeling. For example, RGS18 promotes osteoclastogenesis whereas RGS12 and RGS10 impairs it , 75, 76.ignaling , 78. Gq-activity , 79. CaS pathway . Frizzleormation , 82, witormation , Ca2+ paincrease , and theincrease . We demomRNA expression level and protein expression level in response to siRGS2 transfection, down regulation of RGS4 appeared much delayed and weaker at the protein level as compared to the mRNA level in response to siRGS4 transfection. This implies different expression regulation kinetics between RGS2 and RGS4, with the latter likely having long half-life that could mask the effect of total reduced RGS4 transcripts. It is also possible that there might be unknown post-transcriptional regulation that hinders the progress of translation or post-translational protein modifications that might render the RGS4 antibodies used incapable of recognizing modified forms of RGS4, obscuring the actual total protein level. Poor correlation between expression levels of mRNA and protein level has been well documented [Lastly, it is interesting to point out that unlike RGS2, which demonstrated a clear correlation between its cumented –88, althGPCRs compose the largest family of membrane receptors and as a result, they are also the most widely targeted membrane proteins, with estimated 40% of clinical drugs targeting this system. As downstream regulators of these proteins, RGS proteins are likely to play essential roles during a wide range of developmental processes as well. During normal development, hMSCs residing in the adipose tissue as well as in the bone marrow would respond to different external stimuli by self-renewing or undergoing adipogenic or osteogenic differentiation. Some of these signals are mediated through the GPCR proteins. It is conceivable that RGS proteins might serve as factors of a feedback regulatory loop, in which that active differentiation would lead to expression change in these proteins such as RGS2 and RGS4, which in return would modulate the activity of their respective GPCR proteins in order to help attenuate/augment the cells’ further response to the external stimuli. How these proteins achieve regulatory specificity with different GPCRs and how they may interact with each other to fine tune specific biological event would be of great interest for future studies, as they are potentially druggable molecular targets for treating various physiological diseases.RNA against RGS2 or RGS4 both resulted in decreased adipogenic differentiation, though only knock-down of RGS4 appeared to have significant effect on the expression of examined adipogenic markers including C/EBPα and LPL; and lastly, (V) expression knock-down of RGS2 and RGS4 resulted in decreased and increased osteogenic differentiation respectively, indicating that RGS2 is normally a positive regulator while RGS4 is a negative regulator during osteogenesis. Our study demonstrates for the first time that RGS2 and RGS4 are inversely regulated during human adipogenesis even though they both play positive roles, and on the other hand, both genes were also inversely regulated during early human osteogenesis (first 48 h) but play opposing roles. This implies that members of RGS proteins may play multifaceted roles during human adipogenesis and osteogenesis to balance or counterbalance each other’s function during those processes.In summary, our results demonstrate that RGS2 and RGS4 are differentially regulated during adipogenic and osteogenic differentiation of hMSCs, with both playing positive roles during adipogenesis but opposing roles during osteogenesis. We demonstrated: (I) expression of RGS2 and RGS4 were found to be inversely regulated during adipogenesis, with RGS2 up-regulated and RGS4 down-regulated in response to adipogenic induction; (II) RGS2 expression was also up-regulated during osteogenesis, whereas RGS4 expression was down-regulated during the first 48 h of osteogenesis followed by up-regulation afterwards; (III) expression of RGS2 and RGS4 was regulated by DEX and IBMX independent of Insulin during adipogenesis, but only by DEX during osteogenesis; (IV) expression knock-down using si2 incubator at 37 °C. Cells were expanded at 1:5 splitting ratio using 0.05% trypsin–EDTA  and used at passage 4 for all assays.Human adipose-derived mesenchymal stem cells  were cultured using Hyclone Advance STEM Mesenchymal Expansion Kit  and grown and cultured in a 5% COhMSCs at P4 passage were plated at 100 cells per 10-cm plate or 96 cells per 96-well plate (1 cell/well) and cultured continuously for 21 days, with media change every 3 days. Cells were then rinsed with PBS after medium removal and stained with 0.5% crystal violet  dissolved in 20% methanol for 30 min at room Temperature (RT). Colonies containing in excess of 50 cells were counted using a Leica dissecting microscope.hMSCs at P4 passage were fixed with 4% paraformaldehyde for 10 min, rinsed three times with PBS, permeated with 0.25% Triton X-100 in PBS for 10 min at RT, washed four times with PBS, 5 min each with gentle shaking, blocked with 1% BSA in PBS for 1 h at RT, incubated with primary antibodies diluted in blocking solution overnight at 4 °C, followed by incubation with secondary antibodies also diluted in blocking solution for 1 h at RT. Images were obtained using Olympus IX50 fluorescence microscope. Primary antibody against CD73 (dilution 1:5) and CD105 (dilution 1:12.5) were from Thermofisher (cat# 41-0200 and PA5-16895 respectively). Goat anti-mouse Alexa Fluor Plus 488 secondary antibody was from Thermofisher (cat# A32723) (dilution 1:500) and Donkey anti-Rabbit FITC secondary antibody (dilution 1:500) was from R&D (cat# 711-095-152).5 cells in 100 μl were aliquoted into each FACS tube (coated with 1% BSA overnight at 4 °C prior), and 5 μl of each labeled primary antibody was added in each tube for staining for 30 min at 4 °C. Cells were stained with FITC anti-human CD90  alone, Pacific Blue anti-human CD73  alone, or both together for 30 min at 4 °C. FITC Mouse IgG1  was used as isotype controls. Unstained hMSCs were also used as negative controls. Cells were then fixed with 2% paraformaldehyde for 30 min at RT, washed with PBS once before flow cytometry analysis. Flow cytometry data was acquired through a Gallios flow cytometer (Beckman Coulter) at the City of Hope Analytical cytometry core and analyzed using the FlowJo software by Tree Star Inc.Detailed procedure can be found in our previously published study . Brieflyl-ascorbic acid  for 18–26 days.For all experimental assays, 64,000 cells were evenly plated in 24 well plates and media was changed every 48 h during differentiation induction unless specified otherwise. For adipogenic induction, ad-hMSCs were cultured in growth media supplemented with 0.2 or 1.0 μM dexamethasone , 0.45 μM 3-isobutyl-1-methylxanthine , and 10 μg/ml insulin  for 12–16 days. Depending on experimental needs, three different types of growth media were used: Hyclone CM, Heat-Inactivated Fetal Bovine Serum (FBS)-based complete media (HI-FBS CM), or FBS-based complete media (FBS CM). Hyclone CM refers to Hyclone Advance STEM Mesenchymal Expansion Kit used to grow and expand ad-hMSCs , whose composition is undisclosed. HI-FBS CM and FBS CM were composed of High Glucose Dulbecco’s Modified Eagle’s Medium (DMEM), GlutaMAX , 1% non-essential amino acids , with 10% fetal bovine serum  that was heat-inactivated or untreated, respectively. Heat-activation was performed in 57 °C water bath for 30 min. For osteogenic differentiation, ad-hMSCs were cultured in Hyclone CM supplemented with 0.2 or 1.0 μM dexamethasone, 10 mM β-glycerolphosphate , and 0.05 mM 2-phospho-siRNA-transfection agent complex at designed time point. Reverse transfection was achieved by introduction of siRNA-transfection reagent complex to culture vessel followed by equal plating of cells. Lyophilized siRNA at 1 nmol quantity was suspended in 100 µl of RNAase-free water obtaining a stock concentration of 10 µM, which was further diluted to 2 µM working stock . Transfection was done in 24-well plates. In reverse transfection, for each individual well, siRNA-transfection reagent complexes were prepared by adding 2.24 μl xtremeGENE siRNA transfection reagent  into a tube containing 224 μl MEM basal media , followed by the addition of 1.6 μl of 2 µM siRNA within 5 min. The complex was incubated for 25–30 min inside a cell culture hood at room temperature before transferring to a designated well. A total of 64,000 cells in 640 μl of growth media would be added to the mixture, followed by 24-h incubation in CO2 incubator before the media was exchanged for Hyclone CM growth media. The cells were further incubated for 24 h before subjecting to adipogenic or osteogenic differentiation induction. The following siRNA were used in this study: siCON: AllStars Neg. siRNA ; siRGS2-2: Hs_RGS2_2 ; siRGS2-3: Hs_RGS2_3 ; siRGS4-8: Hs_RG4_8 ; and siRGS4-10: Hs_RG4_10 .Two transfection methodologies, referred to as reverse and forward transfection, were examined to optimize transfection efficiency. Forward transfection was achieved by equal plating of cells into culture vessel followed by introduction of Nuclear staining was achieved using DAPI  nucleic acid stain . A working solution was prepared by diluting 1:3000 of a 14.3 mM DAPI stock in 1× PBS. Briefly, cells were fixed with 10% buffered formalin phosphate, washed three times with distilled water, and stained with the diluted DAPI solution for 30 min at room temperature. Cultures were then washed three times with distilled water and images were acquired using Olympus IX50 fluorescence microscope (7 images per well at 100× magnification were taken). Nuclear counts of each image were done using CellProfiler Image Analysis Software .g for 15 min. Supernatant was then transferred to a new microcentrifuge tube. Optical densities of solutions were measured at 405 and 690 nm in an ELx800 96 well plate reader (BioTek).Mature osteocytes secrete calcium phosphate were detected 18–24 days after osteogenic induction using a 2% Alizarin Red S Staining solution  (pH 4.1–4.3 adjusted with 0.5% Ammonium hydroxide). Cells were first fixed by 10% formalin-PBS, rinsed twice with water, incubated with Alizarin Red S solution for 10 min, washed four times with distilled water with 5 min intervals between wash, and air dried for later imaging and quantification. For quantification, a modified manufacturer’s protocol of an osteogenesis quantitation kit  was used. Briefly, dried stained cultures were incubated with 10% acetic acid for 20 min at room temperate. The loosely attached monolayer was then scrapped and total well content was transferred to microcentrifuge tubes. The mixtures were vortexed vigorously, parafilmed, and incubated at 85 °C for 10 min, followed by incubation on ice for 5 min and subsequent centrifugation at 20,000×Lipid droplets in mature adipocytes can be identified using Oil-Red-O staining solution. After fixing with 10% buffered formalin phosphate , cells were incubated with 100% propylene glycol  for 5 min at room temperature, followed by its removal and staining with Whatman filter paper-filtered Oil Red O solution  for 30 min to 2 h with gentle rocking. Next, staining solution was removed and cells were incubated with 85% propylene glycol  for 5 min, followed by rinsing in distilled water three times. Stained cells would remain covered in distilled water, parafilmed and stored at 4 °C refrigerator. Whole-well images were taken using a Leica dissection microscope. For Oil-Red-O staining quantification, cells samples were air dried overnight after removing water and Oil-Red-O stain was extracted using 150 μl/well (24-well plate) 100% isopropyl alcohol for 1 min. Optical density readings for extracted stain solution were measured at 510 and 690 nm in an ELx800 96 well plate reader (BioTek).To determine total adipocyte and total cell counts, wells were imaged using an Olympus IX50 microscope at 100× magnification after double staining cultures with both DAPI and OilRedO stain. Images were taken starting from the bottom to the top of each individual well, resulting in seven separate fields of view spanning the entire well. DAPI stain showing nuclear stain were imaged using UV-light as excitation light (emission light: blue fluorescence) and OilRedO stain was imaged using green light as excitation light (emission light: red fluorescence). For adipocyte count, DAPI and OilRedO images taken in the same field of view were merged using Adobe Photoshop and mature adipocytes (identified by large concentration of oil droplets) were manually counted in Photoshop CS6 (Adobe).For total area measurements of stained oil droplets, phase contrast images of OilRedO stained cells were taken and processed through an imaging analysis software (ImagePro Plus 7.2), which outlined the stained oil droplets of mature and immature adipocytes resulting in quantitative area measurements. Total area measurements of stained oil droplets were summed for each well and the average area measurement for each treatment group (12 wells/group) was then calculated and graphed relative to control group.For all cell pelleting, cells were detached with 0.05% Trypsin–EDTA, washed twice with 1× PBS and stored at – 80 °C until RNA extraction. Total RNA was isolated from all treatment groups using RNeasy Kit . Equal concentrations of RNA (50 or 100 ng) was then reverse transcribed into cDNA using Superscript III Reverse Transcriptase Kit . PCR amplification was conducted using HotStarTaq Polymerase . Primers for HPS90-beta (control), RGS2, RGS4, adipogenic markers , osteogenic markers  and cell cycle markers  are listed in Table 5 cells per treatment group on day 1, 2, 3, 4, 5 or 7 (for RGS4 detection), or day 1, 2 or 3 (for RGS2 detection) post AIM or OIM treatment initiation. Briefly, Protein was extracted using M-PER reagent (Thermofisher cat# 78501) containing 1× Halt protease inhibitor (Thermofisher cat# 87785). About 20 μg of protein per sample was loaded and separated in NuPAGE 10% bis–tris gel (Thermofisher cat# NP0301BOX). After transferring to nitrocellulose membrane, the blot was incubated in antigen pretreatment solution  for 10 min before incubating in StartingBlock blocking buffer (Thermofisher cat# 37543) for 30 min at RT, followed by incubation with primary antibody raised in mouse  and HRP-conjugated anti-mouse secondary antibody  for 1 h each at RT with gentle shaking. RGS4 H-12 detects isoform 3 (34 kDa) whereas RGS D-8 detects isoforms 1 and 2 (23 kDa). To detect protein bands, blot was incubated in chemiluminescent substrate working solution  overnight at 4 °C, and images were acquired and bands were quantified using Gel Doc XR+ system (BioRad).Protein was extracted from whole cell lysis from 5.6 × 10siCON as shown in graphs.All graph data is presented as the average ± standard deviations (SD). Differences in data was considered significant if p < 0.05 as determined by student’s unpaired t test. p value was determined by comparing original raw data sets between treatment and control groups, prior to percentage calculation set relative to Additional file 1: Figure S1. Clonogenic Assay of Adipose-Derived hMSCs. The number of clones with more than 50 cells after 21 days of culture was counted and representative images of clones and non-clones are included as well. Error bars represent variation among triplicates in each independent repeat (n = 3).Additional file 2: Figure S2. Expression Profile of hMSC markers CD73, CD90 and CD105 by Immunostaining and Flow Cytometry. Expression of both CD73 and CD105 were detected in ≥ 95% of the cells. Expression of CD73 was further confirmed by flow cytometry and co-staining with CD90, which also showed expression in ≥ 95% of the cells.Additional file 3: Figure S3. Temporal Expression of RGS4 and RGS2 in HI-FBS CM based AIM and OIM Treatments. Expression of RGS2 and RGS4 was examined by RT-PCR in hMSCs cultured in 8 different media treatments, including Hyclone CM, HI-FBS CM, Hyclone CM based DEX media (Hyclone 1.0 µM DEX), HI-FBS CM based DEX media (HI-FBS 1.0 µM DEX), Hyclone CM based AIM media with 1.0 µM DEX (Hyclone 1.0 µM DEX AIM), FBS CM based AIM media with 1 µM DEX (FBS 1.0 µM DEX AIM), HI-FBS CM based AIM media with 1.0 µM DEX (HI-FBS 1.0 µM DEX AIM), and Hyclone CM based OIM media with 1.0 µM DEX (Hyclone 1.0 µM DEX OIM). Expression in each treatment condition was examined at six different time points, including D0.5, D1, D1.5, D2, D3 and D4 post initial treatment. A Graph of RGS4 expression. B Graph of RGS2 expression. Graphs represent average gene expression level normalized to that of HSP90 and set relative to CM control at each given time point (n = 2).Additional file 4: Figure S4. Efficiency of siRNA transfection in adipose-derived hMSCs. Ad-hMSCs were reverse transfected with either a scrambled siCON or siTOX at 16.5 nM. A Bright field images of transfected cells at day 1, 2, 6 and 12 post siRNA transfection. B Total live cells were determined using an automated cell counter at day 1, 2, 6 or 12 days post siRNA transfection. Graphed data is shown as mean ± SD (n = 3). Asterisks represent significant differences between siTOX and siCON treated cells (**p < 0.01).Additional file 5: Figure S5. Expression knockdown of RGS4 and RGS2 at the protein level by siRGS4 and siRGS2, respectively. A. Western blot demonstrating expression of RGS4 detected by two different antibodies that recognized isoform 3 (34 kDa) and isoforms 1 & 2 (23 kDa) respectively in both siRGS4 and siCON treatment groups on day 7 post OIM initiation. (n = 2). B Western blot demonstrating expression of RGS2 detected by its antibody that recognized all isoforms at around 26 kDa in both siRGS2 and siCON treatment groups on day 2 post OIM initiation (n = 2).Additional file 6: Figure S6. Expression knockdown of RGS2 mRNA by siRGS2 during adipogenic differentiation of hMSCs induced by HI-FBS CM based adipogenic media. Expression of RGS2 was examined at day 1, 3, 5, 7, and 14 after differentiation initiation at 48 h post siRGS2 transfection. A Expression level of RGS2 in each treatment group was determined relative to their expression in siCON control group, after normalization against internal control HSP90 at each given time point. B Agarose gel images of RGS2 and HSP90 RT-PCR products were shown. Error bars represent variation between independent repeats (n = 2). *p < 0.05, **p < 0.01. Expression comparison was made between siCON and siRGS2 treatment groups at each time point.


The reactions of enantiomeric glycerol-based 3-armed lactide oligomers (H3DLAO and H3LLAO) and a diethylene glycol-based 2-armed ɛ-caprolactone oligomer (H2CLO) with hexamethylene diisocyanate (HDI) produced polyesterurethane copolymer networks  with different feed ratios of stereocomplex (sc) lactide oligomer  and H2CLO. Thermal and mechanical properties of the copolymer networks were compared with those of a simple homochiral (hc) network (PEU-3DLAO) produced by the reaction of H3DLAO and HDI. X-ray diffraction and differential scanning calorimetric analyses revealed that sc crystallites are formed without any hc crystallization for PEU-3scLAO/2CLOs, and that PEU-3DLAO is amorphous. The melting temperatures of sc crystallites for PEU-3scLAO/2CLOs were much higher than that of hc crystallites of H3DLAO. The polarized optical microscopic analysis revealed that the nucleation efficiency is enhanced with increasing feed of H3scLAO fraction, whereas the spherulite growth rate is accelerated with increasing feed H2CLO fraction over 100/0-50/50 networks. PEU-3scLAO/2CLO 100/0  exhibited a higher tensile strength and modulus than PEU-3DLAO. The elongation at break and tensile toughness for PEU-3scLAO/2CLOs increased with an increasing feed amount of H2CLO. It is an interesting subject whether the high melt stability is reduced or not by changing the 4-armed enantiomeric lactide oligomers to 3-armed oligomers.Stereocomplex (sc) formation between enantiomeric poly were prepared by the ring-opening polymerizations of LLA and DLA, initiated at the three hydroxy groups of glycerol, and crosslinked with H2CLO using hexamethylene diisocyanate (HDI) to produce polyesterurethane copolymer networks (PEU-3scLAO/2CLOs) with various feed ratios of stereocomplex lactide oligomer  and H2CLO . The infMn) ~2000 was purchased from Sigma-Aldrich Corporation . The H2CLO is a diethylene glycol-initiated 2-armed ɛ-caprolactone oligomer with a degree of polymerization per arm of m = ca. 8.4 (2) was purchased from KISHIDA CHEMICAL Co. Ltd. . l-lactide  and d-lactide  were purchased from Musashino Chemical Laboratory, Ltd. . All the reagents were used without further purification.Glycerol (GC) and 1,1,2-trichloroethane (TCE) were purchased from Kanto Chemical Co. Inc. . Hydroxy-terminated 2-armed ɛ-caprolactone oligomer (H2CLO) of which the catalogue name is polycaprolactone diol with a number-averaged molecular weight , LLA  and chlorobenzene (60 mL) were put into a nitrogen purged three-necked flask. The mixture was heated to 150 °C, and then Sn(Oct)2  was added into the flask. The resulting mixture was stirred at 150 °C under a nitrogen atmosphere for 24 h. Hexane (500 mL) was added to the cooled reaction mixture with stirring, and then the supernatant was decanted off. This process was three times repeated and the separated precipitate was filtered, dried at 80 °C in a vacuum oven to give H3LLAO as a white powder . The degree of polymerization (n) per arm measured by 1H-NMR method was 17.8. H3DLAO was also synthesized as a white powder in 95% yield by the same procedure as that of H3LLAO except to use DLA instead of LLA. The value of n for H3DLAO was 15.2.A typical synthetic procedure of H3LLAO with a theoretical degree of polymerization of lactate unit  per arm, A typical synthetic procedure of PEU-3scLAO/2CLO 75/25 is as follows: A solution of H3LLAO , H3DLAO , H2CLO  and HDI  in TCE (60 mL) was poured into a petri dish (diameter: 97 mm) made of poly(tetrafluoroethylene). The molar ratio of OH/NCO in the mixture was fixed to 1/1.2. The mixture was dried at 60 °C for 24 h, and then 130 °C for 4 h in an electric oven. The obtained PEU-3scLAO/2CLO with the feed weight ratio of (H3LLAO + H3DLAO)/H2CLO 75/25 (thickness: ca. 0.3 mm) was peeled off from the petri dish. PEU-3scLAO/2CLO  films were also prepared by a similar method. PEU-3scLAO/2CLO 100/0 and PEU-3scLAO/2CLO 0/100 were also abbreviated as PEU-3scLAO and PEU-2CLO, respectively. The reaction products of H3DLAO with HDI (PEU-3LLAO) and a stereocomplex of H3LLAO and H3DLAO (H3scLAO) were also prepared by a similar method for comparison.1H-NMR) spectra were recorded on a Bruker AV-400 (400 MHz)  using CDCl3 and tetramethylsilane as a solvent and an internal standard, respectively.Proton nuclear magnetic resonance  FT-IR 8400s by the attenuated total reflectance (ATR) method. The IR spectra were acquired using 50 scans at a resolution of 4 cm−1. Degree of swelling (Ds) was measured by dipping a film (10 × 5 × 0.3 mm3) in chloroform, N,N-dimethylformamide (DMF) or ethanol at room temperature for 2 days according to the following equation: w0 is the initial weight of the film and w1 is the weight of the swollen film after dipping. In addition, the film dipped in chloroform for 3 days was dried at 40 °C in a vacuum oven for 24 h, and the weight reduction percentage by the chloroform extraction was calculated by the equation: Weight reduction percentage (%) = w2 is the weight of the dried film.Fourier transform infrared (FT-IR) spectra were recorded at room temperature in the range from 4000 to 700 cm−1, using Cu Kα radiation  at 40 kV and 14 mA. All scans were in the range 5° ≤ 2θ ≤ 30° at a scanning rate of 1.0° min−1 and a step size of 0.01°.X-ray diffraction (XRD) analysis was performed at ambient temperature on a Rigaku  RINT-2100 X-ray diffractometer at a scanning rate of 2.0° min−1, held at the temperature for 3 min to eliminate a thermal history of the sample, and then cooled to −100 °C at a cooling rate of 100 °C·min−1. After being held at −100 °C for 3 min, the second heating scan was monitored at a heating rate of 20 °C·min−1. Glass transition temperature , cold crystallization temperature , enthalpy of cold crystallization , melting temperature  and enthalpy of melting  for each component (x = LAO or CLO) were determined from the first and second heating curves. The crystallinities  of hc or sc oligolactide crystallites and oligocaprolactone crystallites were calculated using the following equation:w is the weight fraction of (H3LLAO + H3DLAO) and −1) or sc-PLA (142 J·g−1) in case of x = LAO [w is the weight fraction of H2CLO and −1) in case of x = CLO [Differential scanning calorimetry (DSC) measurements were performed on a Perkin-Elmer  Diamond DSC in a nitrogen atmosphere. The as-prepared samples (8–12 mg) were heated from −100 °C to 200 °C at a heating rate of 20 °C·min x = LAO ,23, and  x = CLO .Morphology of fractured surfaces of the copolymer networks was observed by field emission-scanning electron microscopy (FE-SEM), using a Hitachi S-4700 machine . All samples were fractured after immersion in liquid nitrogen for about 5 min. The fracture surfaces were sputter-coated with gold to provide enhanced conductivity.−1 and the growing of spherulites was monitored over time at that temperature.Polarized optical microscopy was performed on an Olympus BXP polarizing microscope equipped with a Japan High-tech hot-stage RH-350 and a Sony CCD-IRIS color video camera. After a sample was heated to 220 °C on the hot-stage and held at 220 °C for 10 min, it was cooled to a specified temperature (120–150 °C) at a cooling rate of 50 °C·min3) was performed on a Rheolograph Solid instrument  under an atmosphere of air with a chuck distance of 20 mm, a frequency of 1 Hz and a heating rate of 2 °C·min−1, based on ISO 6721-4:1994 .Dynamic mechanical analysis (DMA) of the rectangular plates (40 × 8 × 0.3 mmT5) was measured on a Shimadzu TGA-50 thermogravimetric analyzer. A sample of about 5 mg was heated from room temperature to 500 °C at a heating rate of 20 °C·min−1 in a nitrogen purge stream at a flow rate of 50 mL·min−1.The 5% weight loss temperature  was performed at 25 °C using a Shimadzu Autograph AG-1, based on the standard method for testing the tensile properties of plastics (JIS K7161:1994 (ISO527-1:1993)). Span length and testing speed were 25 mm and 3 mm·min1H-NMR spectrum of H3LLAO in CDCl3. Methine proton signals (Hc and Hc′) of repeating and terminal lactate units of H3LLAO were separately observed at δ 5.18 and 4.36 ppm, respectively. The two signals overlapped with the methine (Ha) and methylene (Hb) proton signals of the GC moiety, respectively. Additionally, methyl proton signals (Hd and Hd′) of repeating and terminal lactate units of H3LLAO were closely observed at δ 1.59 and 1.49 ppm, respectively. The degree of polymerization (n) of lactate unit  per arm for H3LLAO was calculated to be 17.8, based on the integral ratio of Ha,c/Hb,c′. The n value measured by the same 1H-NMR method for H3DLAO was 15.2. Although the n value of H3LLAO was a slightly higher than that of H3DLAO, the difference should not be attributed to a difference of LLA and DLA but to experimental error, because the n value evaluated by 1H-NMR method deviates by ca. 15% in several experiments. If the n values of H3LLAO and H3DLAO were lower than 15, the stereocomplexation ability becomes worse [n values were much higher than 15, the subsequent crosslinking reaction becomes difficult because of a much lower terminal hydroxy content. The molecular weights calculated from the n values (17.8 and 15.2) for H3LLAO and H3DLAO were 3940 and 3378, respectively. These values were used for calculation of their feed amounts for the reaction with HDI. PEU-3scLAO/2CLO films were prepared by the crosslinking reaction of H3LLAO, H3DLAO, H2CLO and HDI  observed for H3LLAO and the strong band due to NCO stretching vibration (νNCO) at 2250 cm−1 observed for HDI were almost nonexistent for PEU-3scLAO/2CLOs. Although absorption bands characteristic of urethane bond for PEU-3scLAO/2CLOs are not so strong because the fraction of urethane bonds formed in the polymer network is slight, new absorption bands of N−H stretching vibration (νN–H) and N−H bending vibration (δN−H) certainly appeared at 3350 and 1535 cm−1, respectively. The band of urethane C=O stretching vibration (νC=O) overlapped with the ester νC=O band of the lactide oligomer (LAO) and caprolactone oligomer (CLO) segments of PEU-3scLAO/2CLOs at ca. 1745 cm−1. These results suggest that the reaction of hydroxy group of H3LLAO/H3DLAO and isocyanate group of HDI proceeded smoothly to generate a polymer network having bridged urethane linkages.Ds values of PEU-3scLAO/2CLOs dipped in chloroform, DMF or ethanol for 2 days. The Ds increased with increasing CLO fraction, indicating that crosslinking density decreases with increasing feed of 2-armed H2CLO. In the case of using ethanol, the Ds values (13%–62%) in ethanol were much lower than those in chloroform and DMF, suggesting that the network has a low affinity toward ethanol. Furthermore, the weight reduction percentages of PEU-3scLAO/2CLOs  after extracted with chloroform for 3 days were 1.64%, 2.51%, 2.39% and 16.7%, respectively. The fact that 25/75 network had a slightly higher weight reduction percentage than the other networks should be caused by the extraction of branched polymers, with a higher fraction of H2CLO-based moiety. PEU-3scLAO/2CLO 0/100  completely dissolved in chloroform because the sample is a linear polymer.The formation of polymer networks for PEU-3scLAO/2CLOs  were also confirmed by swelling and extraction experiments. Tg,LAO and a Tm,LAO of hc crystals at 59.9 °C and 118.0 °C, respectively. PEU-3DLAO showed only a Tg,LAO at 64.7 °C, whose value was higher than that of H3DLAO. This result suggests that the formation of a polymer network disturbed the chain mobility. The fact that no Tm,LAO was observed for PEU-3DLAO indicates that the hc polymer networks are amorphous, which in agreement with the results of XRD. Among the as-prepared samples, only H3scLAO displayed a Tc,LAO at 88.9 °C , implying that the sc-crystallization was not completed during the sample preparation. Considering that the as-prepared H3scLAO is a powder sample, it is supposed that imperfect sc-crystallites are precipitated after mixing of H3LLAO and H3DLAO in TCE. PEU-3scLAO/2CLOs  displayed the Tm,LAO at 173.1–178.5 °C, which were much higher than the Tm,LAO (118.0 °C) of H3DLAO, and were a little lower than the Tm,LAO (186.5 °C) based on sc crystals of H3scLAO. The χc,LAOs (24%–30%) of sc-crystallites for PEU-3scLAO/2CLOs  were comparable to those (28.2%) for H3scLAO. The χc,LAO for PEU-3scLAO/2CLOs did not largely reduce with an increasing CLO fraction, indicating that sc crystallization is not totally disturbed by the presence of CLO moiety. Additionally, the Tm,LAO (173.1–178.5 °C) for PEU-3scLAO/2CLOs was a little lower than the already-reported Tm,LAO (179.5–180.3 °C) for PEU-4scLAO/2CLOs [c,LAO (23.9%–29.6%) for PEU-3scLAO/2CLOs was a little higher than the already-reported χc,LAO (12.1%–22.5%) for PEU-4scLAO/2CLOs, suggesting that a lower crosslinking density for PEU-3scLAO/2CLOs leads to a higher chain mobility, facilitating the crystallization. We could not observe clear Tg,LAOs in the first heating DSC thermograms of PEU-3scLAO/2CLOs .AO/2CLOs . This re−1 from 200 °C to −100 °C. The second heating DSC curves are also shown in Hc,LAO and ∆Hm,LAO were close to each other indicates that the sample before the second heating is almost amorphous. The Tc,LAOs and Tm,LAOs of PEU-3scLAO/2CLOs were observed at 69–92 °C and 175–183 °C, respectively. The Tm,LAOs estimated from the second heating scans were comparable to those (173–179 °C) from the first heating scans, indicating that almost the same sc-crystallites are regenerated after melting at 200 °C. It is noteworthy that PEU-3scLAO/2CLOs  showed no Tc,LAOs and Tm,LAOs based on hc crystallites. This is probably caused by the possibility that a melted LLA oligomer segment is relatively close to the position of a melted DLA oligomer segment, because both the segments are incorporated in the polymer network. The Tm,LAO (176.6 °C) and χc,LAO (16.4%) for PEU-3scLAO/2CLO 100/0 were lower than those (186.5 °C and 26.6%) of H3scLAO, respectively, implying that the urethane crosslinking disturbed the sc crystallization to some extent. The fact that the χc,LAO of PEU-3scLAO/2CLO 75/25 was higher than that of the 100/0 sample indicates that the incorporation of CLO segments enhanced cold crystallization of LAO segments.E″s) corresponding to Tgs for PEU-3scLAO and PEU-2CLO were 51.7 and −51.4 °C. The E″ peak temperature (−26.8 °C) for PEU-3scLAO/2CLO 75/25 was much higher than that for PEU-2CLO, suggesting that the LAO and CLO segments are compatibilized in agreement with the results of FE-SEM. The E″ peak temperatures (−49.0 and −51.4 °C) for CLO-rich phase of PEU-3scLAO/2CLOs 50/50 and 25/75 were close to that (−51.4 °C) of PEU-2CLO, whereas we could not observe the E″ peak temperatures related to the LAO-rich phase because the DMA measurements stopped at around 40 °C due to melting of the CLO segment, suggesting that the CLO segments are phase-separated, in agreement with the FE-SEM. The storage modulus (E′) of PEU-3scLAO dropped at around 45 °C due to a glass transition of the LAO-segment. The E′ of PEU-2CLO was reduced at around −50 °C due to a glass transition of the CLO segment, and dropped at around 30 °C due to melting of the CLO segment. The fact that PEU-3scLAO/2CLOs 50/50 and 25/75 exhibited similar E′ curves to PEU-2CLO reflects that the CLO segments are phase-separated. On the other hand, when the E′ values at the same temperature are compared, the E′ of PEU-3scLAO/2CLO 75/25 was lower than that of PEU-3scLAO, and higher than that of PEU-2CLO, in agreement with the fact that the LAO and CLO segments in PEU-3scLAO/2CLO 75/25 are compatibilized.T5 for PEU-3scLAO/2CLOs increased with increasing CLO fraction in agreement with the fact that the T5 (297.7 °C) of H2CLO was much higher than those (173.0 and 188.0 °C) of H3LLAO and H3DLAO. There is little difference in T5 between PEU-3DLAO and PEU-3scLAO in agreement with the fact that there is little difference in thermal stability between sc-PLA and PLLA or PDLA [−3), it is given by the area under the stress-strain curve [c,CLO of the 50/50 sample  modulus and elongation at break [Mw 1.3 × 106 [Mw not higher than 5 × 104 has a high stereocomplexation ability, which is brittle, and that a linear sc-PLA with a Mw not lower than 1 × 105 displays a higher tensile strength and modulus than the corresponding hc-PLA does, while it has a relatively lower stereocomplexation ability [at break . It is n.3 × 106 . Althoug ability . In a si−1 from 200 °C. No melting temperatures of hc crystallites were observed for PEU-3scLAO/2CLOs in the first and second heating DSC curves. The polarized optical microscopic analysis revealed that the nucleation efficiency was enhanced with increasing LAO fraction, whereas the spherulite growth rate was accelerated with increasing CLO fraction over 100/0–50/50 networks. The change of E″ peak temperatures measured by DMA was in agreement with the compatibilizaion for PEU-3scLAO/2CLO 75/25 and phase-separated morphology of PEU-3scLAO/2CLOs 50/50 and 25/75. Tensile strength, modulus and elongation at break of PEU-3scLAO were higher than those of PEU-3DLAO, sc-PLA and PEU-4scLAO. Elongation at break and tensile toughness for PEU-3scLAO/2CLOs increased with increasing CLO fraction. It was noteworthy that the PEU-3scLAO based on 3-armed lactide oligomers exhibited much better tensile properties than the PEU-4scLAO based on 4-armed lactide oligomers without any reduction of the high stereocomplexation ability and melt stability. The sc copolymer networks in this study are expected to be used as biodegradable and biocompatible materials requiring better tensile properties than those of PLA or sc-PLA.The stereocomplex formation, thermal and mechanical properties of PEU-3scLAO/2CLOs  prepared by reactions of H3LLAO, H3DLAO and H2CLO with HDI were compared with those of H3scLAO, PEU-3DLAO. Although H3scLAO was obtained as a powder sample, PEU-3scLAO/2CLOs and PEU-3DLAO became relatively tough films. The formation of network structure by reactions of the hydroxy and isocyanate groups for PEU-3scLAO/2CLOs was confirmed by the FT-IR analysis, extraction and swelling experiments using chloroform. XRD analysis revealed that sc crystallites are preferentially formed for PEU-3scLAO/2CLOs and H3scLAO, while hc crystallites are formed for H3LLAO and H3DLAO, and that PEU-3DLAO is amorphous. The melting temperatures of sc crystallites for PEU-3scLAO/2CLOs were much higher than those of hc crystallites of H3LLAO and H3DLAO, while the values were slightly lower than that of H3scLAO. Although cold crystallization temperatures for PEU-3scLAO/2CLOs were not observed during the first heating scan, but the temperatures were observed during the second heating scans after cooling at a rate of 100 °C·min