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This paper presents a topology optimization method to design the piezoelectric stick–slip actuator. In particular, the vertical input displacement can be converted to the oblique displacement by the flexure hinge driving mechanism, and the large-stroke motion is realized. Based on the solid isotropic material with penalization (SIMP) model and combined with the motion characteristics of the stick–slip actuator, in order to obtain a larger output displacement and limit the parasitic displacement, the ratio of output displacement to input displacement is maximized as the objective function, and the relationship between parasitic displacement and output displacement is considered as a constraint condition. The method of moving asymptotes (MMA) is used to solve the optimization problem, and the driving mechanism structure is designed by the topology optimization result. The feasibility and reliability of the driving mechanism are verified by finite element analysis (FEA), then the prototype is fabricated. Experimental test results indicate that the velocity of the actuator reaches 15.25 mm/s under the locking force of 1 N and frequency of 650 Hz, and the resolution of 96 nm is achieved. This work shows that the topology optimization method can be used to improve the performance of the actuator.

The huge global burden of atherosclerotic cardiovascular diseases (CVDs) represents an urgent unmet need for the development of novel therapeutics. Dracocephalum moldavica L. has been used as a traditional Uygur medicine to treat various CVDs for centuries. Tilianin is a major flavonoid component of D. moldavica L. and has potential for preventing atherosclerosis. However, the molecular mechanisms that tilianin attenuate atherosclerosis are far from fully understood.The purpose of this study is to investigate the efficiency and underlying mechanisms of tilianin in controlling lipid profile and preventing atherogenesis.The lipid-lowering effect of tilianin was evaluated in C57BL/6 and ApoETilianin treatment improved lipid profiles in C57BL/6 and dyslipidemic ApoEThis study demonstrated the lipid-lowering effect of tilianin through SREBP2-mediated transcriptional activation of LDLR. Our findings reveal a novel anti-atherosclerotic mechanism of tilianin and underlie its potential clinical use in modulating CVDs with good availability and affordability.

Cationic amino acid transporters (CATs) supply cells with essential and semiessential dibasic amino acids. Among them, l-arginine is the substrate for nitric oxide synthases (NOS) to produce nitric oxide (NO), a key signaling molecule and second messenger. In cardiac preparations, we showed that NO acutely and directly modulates transport activity by noncompetitively inhibiting these CATs. We hypothesize that this NO regulation occurs through modification of cysteine residues in CAT proteins. Homology modeling and a computational chemistry approach identified Cys347 as one of two putative targets for NO binding, of 15 Cys residues present in the low-affinity mouse CAT-2A (mCAT-2A). To test this prediction, mammalian cell lines overexpressing mCAT-2A were used for site-directed mutagenesis and uptake studies. When Cys347 was replaced with alanine (Cys347Ala), mCAT-2A became insensitive to inhibition by NO donors. In addition, the transport capacity of this variant decreased by >50% compared to that of the control, without affecting membrane expression levels or apparent affinities for the transported amino acids. Interestingly, replacing Cys347 with serine (Cys347Ser) restored uptake levels to those of the control while retaining NO insensitivity. Other Cys residues, when replaced with Ala, still produced a NO-sensitive CAT-2A. In cells co-expressing NOS and mCAT-2A, exposure to extracellular l-arginine inhibited the uptake activity of control mCAT-2A, via NO production, but not that of the Cys347Ser variant. Thus, the -SH moiety of Cys347 is largely responsible for mCAT-2A inhibition by NO. Because of the endogenous NO effect, this modulation is likely to be physiologically relevant and a potential intervention point for therapeutics.

Tilianin (TIL) may prevent and treat myocardial ischemia reperfusion injuries. However, its oral administration is hampered by its low bioavailability. The present study aimed to formulate lipid–polymer hybrid nanoparticles (LPHNs) as carriers for the sustained release and oral bioavailability enhancement of TIL in vitro and in vivo. A nanodrug delivery system of TIL-loaded LPHNs (TIL-LPHNs) was constructed. TIL-LPHNs were prepared via a self-assembly method, and their particle size, polymer dispersity index (PDI), zeta potential, encapsulation efficiency (EE) and morphology were investigated. In addition, pharmacokinetic studies were performed in vivo. The TIL-LPHN formulation produced a spherical, homogeneous, smooth surface and multi-lamellar structured nanoparticles. The particle size and distribution profile of TIL-LPHNs had a mean particle diameter of 54.6 ± 5.3 nm and PDI of 0.112 ± 0.017. The zeta potential was −33.4 ± 4.7 mV. The EE of TIL-LPHNs was 86.6 ± 3.6%, which was determined with the dialysis method. The TIL-LPHNs significantly enhanced the oral bioavailability of TIL with a 3.7-fold increase in the area under the concentration−time curve in comparison with the TIL solution. These findings support the potential use of LPHNs in improving the stability and bioavailability of TIL via oral administration.

Paratubal lesions comprise a large number of entities. Preoperative diagnosis is often limited to mass and location, with histology required to establish a more definitive diagnosis. The purpose of this review is to review the literature and summarize benign and malignant paratubal lesions to better understand what can arise in this area.

Targeted therapy has changed the paradigm of advanced NSCLC management by improving the survival rate of patients carrying actionable gene alterations using specific inhibitors. The epidemiologic features of these alterations vary among races. Understanding the racial differences benefits drug development, clinical trial design, and health resource allocation. Compared to Caucasian and Asian populations, current knowledge on Hispanic patients is less and no data of Hispanic patients from Puerto Rico have been reported. We retrieved and analyzed the demographic, clinical, and molecular data of Hispanic NSCLC patients from Puerto Rico with molecular tests performed in the Genoptix Medical Laboratory in Carlsbad, CA, USA between 2011 and 2018. The majority of the NSCLC patients in our study had either adenocarcinoma (75.4%) or squamous cell carcinoma (15.1%). The incidence of EGFR mutations was 24%. They were more common in female and younger patients (&lt, 60 years). The deletion of Exon 19 and Exon 21 L858R comprised 55.1% and 31.0% of all EGFR mutations, respectively. The frequency of the T790M mutation was lower compared to that of Hispanic patients reported in the literature (0.5% vs. 2.1%). In addition, 18.7% of the patients were positive for KRAS mutations, which was at the high end of that reported in Hispanic patients. Other driver gene alterations, ALK, MET, RET, ROS1, KRAS, ERBB2, etc., demonstrated similar incidences, as well as gender and age distributions to those previously reported. The KRAS/TP53 and KRAS/STK11 co-mutations were of very low frequencies (3.6%), which could potentially affect the responsiveness to PD1/PD-L1 immunotherapy. Our study demonstrated that the prevalence of NSCLC gene alterations in Hispanic patients from Puerto Rico was comparable to the reported average prevalence in Latin American countries, supporting the intermediate NSCLC gene alteration rate of Hispanic patients between Asian and Caucasian patients. Novel information of the frequencies of KRAS mutation subtypes, driver gene alterations in ROS1, BRAF, and ERBB2, and passenger gene alterations including a rare case with the FGFR2-TACC2 translocation in Hispanic NSCLC patients from Puerto Rico were also described.

Background The active components of Dracocephalum moldavica L. (TFDM) can inhibit myocardial ischemia by inhibiting oxidative stress. However, the effects of TFDM on astrocytes have not been investigated in vitro. The current study aimed to explore whether TFDM protects astrocytes against H2O2-induced apoptosis through a mitochondria-dependent pathway. Methods The human glioma cell line U87 was used to investigate the ability of TFDM to protect astrocytes against H2O2-induced apoptosis. The cell counting kit-8 assay and flow cytometry were used to detect cell viability, apoptosis, MMP, Ca2+ influx and reactive oxygen species (ROS). Lactate dehydrogenase (LDH) and malonic dialdehyde (MDA) levels were measured by ELISA. In addition, protein and mRNA expression changes were detected by Western blotting and qRT-PCR. Results TFDM (0.78~200 μg/ml) had limited cytotoxic effects on the viability of U87 cells. Compared with the model group (treated with H2O2 only), cells treated with medium- and high-dose TFDM exhibited reduced MDA concentrations (P P P P ). The percentage of annexin V-FITC-stained cells was markedly suppressed by TFDM, confirming its anti-apoptotic properties. WB results showed that protein expression of Bcl-2-associated X protein (BAX), Caspase-3, Caspase-9, Caspase-12, and B-cell leukemia/lymphoma 2 (Bcl2) was reduced in the TFDM group compared with that in the model group (P Conclusions The current study revealed the protective effects of TFDM on U87 cells under oxidative stress conditions through the inhibition of a mitochondria-dependent pathway that is associated with the CaMKII/P38MAPK/ERK1/2 and PI3K/AKT/mTOR pathways.

Objective This review aims to summarize the currently available human papillomavirus (HPV) testing methods for precancerous cervical intraepithelial lesions. Materials and methods A literature search of PubMed using key words "high-risk HPV, precancerous cervical intraepithelial lesions, FDA-approved HPV tests, p16 IHC, Ki 67 IHC, fluorescent in situ hybridization for HPV, Pap smear, HPV vaccines, HPV tests using self-collected samples, and next-generation sequencing" was performed between January 1 and June 14, 2019. The package inserts of the Food and Drug Administration-approved HPV tests were obtained from the companies' Web sites. Results Multiple morphology-based, immunohistochemical staining and nucleic acid HPV tests were reviewed, including the material required, methodologies, result interpretations, as well as their advantages, limitations, and futures. The structure of HPV and its natural history of infection and transmission were touched on as well for a better understanding of these testing methods. Conclusions Human papillomavirus tests are a critical component for cervical cancer screening, and understanding of these tests helps test results interpretation and patients' triage.

Brenner tumors arise from ovarian epithelium, accounting for approximately 5% of benign ovarian epithelial tumors. The World Health Organization classification groups them into benign, borderline, and malignant on the basis of proliferation and invasiveness, and borderline Brenner tumor is defined as “displaying epithelial proliferation beyond that seen in benign Brenner's tumor, but lacking stromal invasion.” Borderline Brenner tumors are rare. Fewer than 60 cases have been reported. The more recent articles mostly focus on pathogenesis. We reviewed the literature on borderline Brenner tumor and have summarized the clinical and pathologic findings, as well as the treatment, differential diagnoses, and recent advances in histogenesis and molecular pathogenesis.

Backward motion directly limits the output performance of piezoelectric stick-slip actuators, and it has been suppressed or eliminated by various means. To suppress the backward motion, a dual-mode excitation method (DMEM) is proposed in this paper, which utilizes a piezoelectric stack and two piezoelectric plates as the main and auxiliary excitation sources of the actuator respectively. When the piezoelectric stack contracts rapidly under the traditional sawtooth wave, two piezoelectric plates excited by high-frequency sine wave make the driving foot generate ultrasonic vibration. Accordingly, the backward motion can be suppressed based on ultrasonic friction reduction to improve the output performance of actuator. To reflect the advantage of the proposed method, a flexure hinge type piezoelectric stick-slip actuator is manufactured, and the characteristic experiments are executed. According to the experimental results, the backward rates of actuator driven only by sawtooth wave are 42 % and 28 % in the clockwise and counterclockwise directions, the corresponding velocities are 252.74 mrad/s and 316.45 mrad/s, the corresponding loads are 8 g and 9 g, respectively. By comparison, the backward rates of actuator under the DMEM decrease by 10 % and 5 % along these two directions, the velocities improve by 17 % and 9 %, and the loads increase by 13 % and 11 %, respectively.

Cytarabine (AraC) has been the primary treatment agent for acute myeloid leukemia (AML) in the past 30 years, but the precise mechanism of its action is not completely known. Here we assessed the role of ERK5 in AraC-induced cell death in AML cell lines HL60 and U937 using ERK5 inhibitors BIX02189 and XMD8-92. We report that inhibition of MEK5/ERK5 activity reduces AraC-induced cell death, DNA damage, the upregulated DNA damage biomarkers, and produced G2 phase cell cycle arrest. In addition, the pro-survival protein P-Bcl2 Ser70 was found to be associated with decreased AraC-induced cell death following XMD8-92 treatment, suggesting a regulatory role of ERK5 on Bcl2 phosphorylation. Our study shows that the full potency of AraC cytotoxicity requires optimal ERK5 activity, suggesting a novel role of ERK5 in cancer chemotherapy. J. Cell. Biochem. 118: 1583-1589, 2017. © 2016 Wiley Periodicals, Inc.

Differentiation therapy can supplement the therapy of APL, but other subtypes of AML are treated principally with cytotoxic agents, with few lasting remissions. While the induction of monocyte followed by macrophage differentiation by vitamin D derivatives (VDDs) is dramatic in cultured AML cells of all subtypes, attempts to translate this to the clinic have not been effective. Thus, better understanding of the mechanisms underlying VDD-induced differentiation may improve this approach. The key events in this form of differentiation include increased expression of CD11b, and the transcription factor PU.1 is known to be a part of this process. We show here that in the transition of monocytes to macrophages induced by a VDD, ERK5, a member of the MAPK family of signaling molecules, prevents PU.1 expression. However, upon ERK5 inhibition PU.1 protein is stabilized by HSP70. Thus, ERK5 may be a target for manipulation of the immunoregulatory actions of macrophages in cancer.

In order to research and enhance bioavailability of chlorogenic acid and rutin(CA-R) via the oral route, chitosan coated composite phospholipid liposomes (C-CPLs) were applied to study on preparation, permeability and pharmacokinetic of C-CA-R-CPLs. TheC-CA-R-CPLs were prepared by the method of ethanol injection. The entrapment efficiency (EE), average particle sizes, polymer disperse index (PDI), zeta potential, shape and in vitro drug release were investigated to characterize physicochemical parameters of C-CA-R-CPLs. The penetration properties from C-CA-R-CPLs were studied through Caco-2 cells model and the pharmacokinetics in Sprague-Dawley (SD) rats were evaluated by rat jugular vein intubation tube. The EE of C-CA-R-CPLs of CA and R was 91.3±2.13% and 92.6±2.44%, particle size of C-CA-R-CPLs was 176.7±2.3 nm, PDI was 0.207±0.014 and zeta potential of 12.61±1.33 mV. CA-R-CPLs and C-CA-R-CPLs were spherical or elliptical sphere and the bilayer of the CPL was observed obviously under transmission electron. The C

Myogenic enhancer factor2 (Mef2) consists of a family of transcription factors involved in morphogenesis of skeletal, cardiac and smooth muscle cells. Among the four isoforms (Mef2A, 2B, 2C, and 2D), Mef2C was also found to play important roles in hematopoiesis. At myeloid progenitor level, Mef2C expression favors monocytic differentiation. Previous studies from our laboratory demonstrated that ERK5 was activated in 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation in AML cells and ERK5 activation was accompanied by increased Mef2C phosphorylation. We therefore examined the role of Mef2C in 1,25D-induced monocytic differentiation in AML cell lines (HL60, U937 and THP1) and found that knockdown of Mef2C with small interfering RNA (siRNA) significantly decreases the expression of the monocytic marker, CD14, without affecting the expression of the general myeloid marker, CD11b. CCAAT/enhancer-binding protein (C/EBP) β, which can bind to CD14 promoter and increase its transcription, has been shown to be the downstream effector of 1,25D-induced monocytic differentiation in AML cells. When Mef2C was knocked down, expression of C/EBPβ was reduced at both mRNA and protein levels. The protein expression levels of cell cycle regulators, p27(Kip1) and cyclin D1, were not affected by Mef2C knockdown, nor the monopoiesis related transcription factor, ATF2 (activating transcription factor 2). Thus, we conclude that 1,25D-induced monocytic differentiation, and CD14 expression in particular, are mediated through activation of ERK5-Mef2C-C/EBPβ signaling pathway, and that Mef2C does not seem to modulate cell cycle progression.

Objectives Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy with both myelodysplastic and myeloproliferative features. The clinical and pathological features of CMML are highly heterogeneous. It was reported that Hispanic whites had an age-adjusted lower incidence rate of CMML compared to non-Hispanic whites. The aim of this study is to define the cytogenetic and genomic landscape of Hispanic CMML patients and explore their potential clinical significance. Methods Clinically relevant cytogenetic results and 40-gene molecular profiles of Hispanic CMML patients in Puerto Rico (PR) from 2009 to 2018 were obtained retrospectively. Results Total 111 Hispanic CMML patients from PR were diagnosed in our institute from 2009 to 2018. The age range was from 46 to 96 years with a median age of 74. Sixty-five were male and 46 were female. The epidemiological features are similar to that in a general CMML patient population. In total, 107 patients had karyotypes available; 17 patients had abnormal karyotype (17/107, ~16%). Compared with general CMML patients, Hispanic CMML patients had a significantly lower rate of cytogenetic abnormalities (30% vs 16%). Among total 111 Hispanic CMML patients, 40-gene myeloid molecular profiles were performed in 56 CMML patients. Fifty-five out of 56 patients had mutations identified (~98.2%). The most frequent mutated genes were TET2, SRSF2, ASXL1, NRAS, and ZRSR2. Twenty-six of 56 patients (~46.4%) had mutated TET2/wild-type ASXL1. Previous studies indicated that mutated ASXL1, NRAS, RUNX1, and SETBP1 likely associate with an unfavorable prognosis in a general CMML patient population. Mutated TET2 with wild-type ASXL1 (muTET2/wtASXL1) may associate with a favorable prognosis. Compared with general CMML patients, Hispanic CMML patients in this study had relatively lower mutational rates in ASXL1 (30.4% vs 37.0%), NRAS (10.7% vs 11.7%), RUNX1 (5.3% vs 7.9%), and SETBP1 (5.3% vs 8.9%) and a higher rate of muTET2/wtASXL1 (46.4% vs 37.8%). Conclusion Hispanic CMML patients from PR had a significantly lower rate in cytogenetic abnormalities; relatively lower mutational rates in ASXL1, NRAS, RUNX1, and SETBP1; and a higher mutational rate in muTET2/wtASXL1. The findings raise a possibility of a better prognosis in Hispanic CMML patients and could be one of the explanations of a lower incidence rate of CMML in Hispanic population.

DMD (Duchenne muscular dystrophy) is an incurable rapidly worsening neuromuscular degenerative disease caused by the absence of dystrophin. In skeletal muscle a lack of dystrophin disrupts the recruitment of neuronal NOS (nitric oxide synthase) to the sarcolemma thus affecting NO (nitric oxide) production. Utrophin is a dystrophin homologue, the expression of which is greatly up-regulated in the sarcolemma of dystrophin-negative fibres from mdx mice, a mouse model of DMD. Although cardiomyopathy is an important cause of death, little is known about the NO signalling pathway in the cardiac muscle of DMD patients. Thus we used cardiomyocytes and hearts from two month-old mdx and mdx:utrophin−/− (double knockout) mice (mdx:utr) to study key steps in NO signalling: L-arginine transporters, NOS and sGC (soluble guanylyl cyclase). nNOS did not co-localize with dystrophin or utrophin to the cardiomyocyte membrane. Despite this nNOS activity was markedly decreased in both mdx and mdx:utr mice, whereas nNOS expression was only decreased in mdx:utr mouse hearts, suggesting that utrophin up-regulation in cardiomyocytes maintains nNOS levels, but not function. sGC protein levels and activity remained at control levels. Unexpectedly, L-arginine transporter expression and function were significantly increased, suggesting a novel biochemical compensatory mechanism of the NO pathway and a potential entry site for therapeutics.

Novel NiO@ZnO heterostructured nanotubes (NTs) were fabricated by the coelectrospinning method, consisting of external hexagonal ZnO shell and internal cubic NiO NTs. They are carefully investigated by scanning electron microscopy, transmission electron microscopy, scanning transmission electron microscopy, energy-dispersive X-ray spectroscopy mapping, X-ray diffraction, and X-ray photoelectron spectroscopy techniques. A reasonable formation mechanism of the hierarchical NiO@ZnO NTs is proposed, which is discussed from the view of degradation temperature of different polymers and the amount of inorganic salts. They were then explored for fabrication of H(2)S gas sensors. The gas sensing test reveals that compared with the pure ZnO, NiO, and the ZnO-NiO mixed gas sensors, hierarchical gas sensor exhibits highly improved sensing performances to dilute hydrogen sulfide (H(2)S) gas. The response of the optimum NiO@ZnO NTs sensor to 50 ppm H(2)S increases as high as 2.7-23.7 times compared to the other sensors, whereas the response and recovery times also become shorter considerably. These enhanced gas sensing properties are closely related to the change of nanostructure and activity of ZnO and NiO nanocrystals as well as combination of homo- and heterointerfaces in the optimum gas sensor, which are confirmed by a series of well-designed experiments.

One-dimensional monoclinic SrAl2O4:Ce3+ nanowires (NWs) were prepared by the electrospinning method for the first time. By annealing PVP/SrAl2O4:Ce3+ composite nanowires (NWs) with different ratio of PVP (M w ≈ 1300000) to inorganic precursors of the electrospinning solutions, size-controllable NWs were obtained, ranging of 90–180 nm. Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), scanning electron microscopy (SEM), photoluminescence (PL) spectra, and dynamics were employed to characterize the NWs. The results demonstrate that the positions of excitation and emission bands varied uneven with the ratio of PVP to the inorganic precursors and the NWs diameter, which was attributed to the difference of the crystallinity, porosity, and local micro-structures surrounding Ce3+. Two decay time constants were observed for the PL of Ce3+, one faster (1–3 ns) and one slower (10–20 ns), which were attributed to the hole-electron capture on the luminescent ions and isolated Ce ions, respectively. It is interesting to observe that for the shorter decay process, a maximum occurred as the concentration of Ce3+ varied in the range of 1–10 mol%.

Our previous research demonstrated that tilianin protects the myocardium in a myocardial ischemia reperfusion injury (MIRI) rat model and has prominent pharmacological potential as a cardiovascular drug. Our study aimed to investigate the molecular signaling implicated in the improvement of myocardial survival induced by tilianin, a flavonoid antioxidant. Tilianin (2.5, 5, and 10 mg/kg/d) or saline was orally administered to rats for 14 days. On the 15th day, ischemia was induced by ligating the left anterior descending artery for 45 min, followed by 4 h of reperfusion. The levels of MIRI-induced serum myocardial enzymes and cardiomyocyte apoptosis as well as infarct size were examined to assess the cardioprotective effects. Cardiac tissues were collected for western blot analyses to determine the protein expression of anti-apoptotic signaling molecules. In MIRI-treated rats, our results revealed that pre-administration of high dose-tilianin the reduced release of LDH, MDA, and CK-MB and increased the plasma SOD level, and significantly attenuated the infarct size. Western blot analysis showed that a remarkable rise in expression of Bcl-2 and XIAP, and decline in expression of Bax, Smac/Diablo, HtrA2/Omi, cleaved caspase-3, caspase-7 and caspase-9 was observed in the myocardium. The apoptosis index of cardiomyocytes further supports the cardioprotective effect of tilianin. Additionally, compared with the MIRI model group, pretreatment with high dose-tilianin group upregulated phosphorylated Akt and PI3K. In contrast, using the PI3K inhibitor LY294002 to block Akt activation effectively inhibited the protective effects of tilianin against MIRI. Tilianin pretreatment was beneficial for activating the PI3K/Akt signaling pathway and inhibiting myocardial apoptosis.

Cationic L-amino acids enter cardiac-muscle cells through carrier-mediated transport. To study this process in detail, L-[(14)C]lysine uptake experiments were conducted within a 10(3)-fold range of L-lysine concentrations in giant sarcolemmal vesicles prepared from rat cardiac ventricles. Vesicles had a surface-to-volume ratio comparable with that of an epithelial cell, thus representing a suitable system for initial uptake rate studies. Two Na(+)-independent, N-ethylmaleimide-sensitive uptake components were found, one with high apparent affinity (K(m)=222+/-71 microM) and low transport capacity (V(max)=121+/-36 pmol/min per mg of vesicle protein) and the other with low apparent affinity (K(m)=16+/-4 mM) and high capacity (V(max)=4.0+/-0.4 nmol/min per mg of vesicle protein). L-Lysine uptake mediated by both components was stimulated by the presence of intravesicular L-lysine as well as by valinomycin-induced membrane hyperpolarization. Altogether, this behaviour is consistent with the functional properties of the CAT-1 and CAT-2A members of the system y(+) family of cationic amino acid transporters. Furthermore, mRNA transcripts for these two carrier proteins were identified in freshly isolated rat cardiac myocytes, the amount of CAT-1 mRNA, relative to beta-actin, being 33-fold larger than that of CAT-2A. These two transporters appear to function simultaneously as a homoeostatic device that supplies cardiac-muscle cells with cationic amino acids under a variety of metabolic conditions. Analysis of two carriers acting in parallel with such an array of kinetic parameters shows significant activity of the low-affinity component even at amino acid plasma levels far below its K(m).

ERK5, a member of MAPK family, is activated by multiple AML (Acute Myeloid Leukemia) cell differentiation inducers including 1,25D (1, 25-dihydroxyvitamin D3), TPA (12-O-tetradecanoylphorbol-13-acetate) and ATRA (All-trans retinoic acid). Inhibiting ERK5 with XMD8-92 which targets its auto phosphorylation, or BIX02188 which inhibits MEK5, a kinase that activates ERK5, leads to more advanced cell maturation compared to these inducers alone. Specifically, cells treated with 1,25D and TPA combined with MEK5/ERK5 inhibitors exhibit a macrophage-like phenotype, whereas, ATRA with MEK5/ERK5 inhibitors cause the cells to differentiate further in the granulocytic direction. An increased expression of the general myeloid marker CD11b was observed following treatments with all three inducers when MEK5/ERK5 were inhibited. In this study, we compared the underlying mechanisms of the potentiation effects of MEK5/ERK5 inhibition by these inducers. PU1 is considered to be a master transcription factor (TF) directing the cells to the myeloid lineage, and sustained PU1 expression over C/EBPα results in monocytic differentiation. The role of C/EBPα is controversial: it is generally believed to be a TF favoring granulocyte differentiation, however, in some studies, overexpression of C/EBPα, together with activated estrogen receptor, can lead to the expression of macrophage-related genes. Thus, we measured the expression of these two TFs in HL60 and U937 cells treated with differentiation inducers alone or together with MEK/ERK5 inhibitors. 1,25D alone was able to increase PU1 protein expression in both cell lines; when combined with BIX02188 or XMD8-92, PU.1 expression further increased. Interestingly, increased C/EBPα expression was also observed with XMD8-92 + 1,25D. TPA induced similar changes in PU.1 expression, as well as decreased C/EBPα protein levels in the presence of the inhibitors, which was not seen with 1,25D and the inhibitors. Although ATRA alone or with BIX02188 increased PU1 expression, ATRA with XMD8-92 completely abolished PU1 expression which resulted in dramatically increased C/EBPα over PU1 ratio. From the above data, we conclude that 1) ERK5 activation acts as a brake preventing the cells from further differentiation by all inducers studied here 2) Terminal differentiation which results from the addition of ERK5 inhibitors can be due to the combined effects of two master transcription factors. 3) The same TF can function differently in different settings. (Supported by T32 CA134268 training grant and R01 CA044722-25, both from the National Cancer Institute, NIH, and by the American Institute for Cancer Research, grant 10A049). Citation Format: Ruifang Zheng, George Studzinski. ERK5 inhibition potentiates the effects of leukemia cell differentiation inducers by augmenting the function of master transcription factors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2198. doi:10.1158/1538-7445.AM2015-2198

Diabetes mellitus is a major risk factor for the development of vascular complications. We hypothesized that hyperglycemia decreases endothelial cell (EC) proliferation and survival via phosphatidylinositol 3-kinase (PI3k) and Akt signaling pathways. We cultured human umbilical vein ECs (HUVEC) in 5, 20, or 40 mM d-glucose. Cells grown in 5, 20, and 40 mM mannitol served as a control for osmotic effects. We measured EC proliferation for up to 15 days. We assessed apoptosis by annexin V and propidium iodide staining and flow cytometry, analyzed cell lysates obtained on culture day 8 for total and phosphorylated PI3k and Akt by Western blot analysis, and measured Akt kinase activity using a GSK fusion protein. HUVEC proliferation was also tested in the presence of pharmacological inhibitors of PI3k-Akt (wortmannin and LY294002) and after transfection with a constitutively active Akt mutant. ECs in media containing 5 mM d-glucose (control) exhibited log-phase growth on days 7–10. d-Glucose at 20 and 40 mM significantly decreased proliferation versus control ( P < 0.05 for both), whereas mannitol did not impair EC proliferation. Apoptosis increased significantly in HUVEC exposed to 40 mM d-glucose. d-Glucose at 40 mM significantly decreased tyrosine-phosphorylated PI3k, threonine 308-phosphorylated-Akt, and Akt activity relative to control 5 mM d-glucose. Pharmacological inhibition of PI3k-Akt resulted in a dose-dependent decrease in EC proliferation. Transfection with a constitutively active Akt mutant protected ECs by enhancing proliferation when grown in 20 and 40 mM d-glucose. We conclude that d-glucose regulates Akt signaling through threonine phosphorylation of Akt and that hyperglycemia-impaired PI3k-Akt signaling may promote EC proliferative dysfunction in diabetes.

Myogenic enhancer factors 2 (Mef2) are members of the family of MADS (MCM1-agamous-deficiens-serum response factor)-box transcription factors known to have multiple roles in morphogenesis of skeletal, cardiac and smooth muscle cells. Recently, Mef2C was found to be also involved in hematopoiesis. Bone marrow cells isolated from Mef2C knockout mice demonstrated decreased monocytic differentiation in response to cytokine stimulation, whereas constitutive expression of Mef2C promoted monopoiesis. The above findings led us to examine the role of Mef2C in 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation. Human acute myeloid leukemia (AML) cell lines, HL60 and U937, were used in this study. We found that knockdown of Mef2C with small interfering RNA (siRNA) significantly decreased the expression of the monocytic marker CD14, as well as of nonspecific esterase (NSE), suggesting that Mef2C is required for 1,25D-induced monocytic differentiation in AML cells. Our laboratory has previously demonstrated that optimal monocytic differentiation induced by 1,25D requires ERK5, and that ERK5 expression shows positive correlation with Mef2C expression. Here we show that Mef2C knockdown demonstrates similar inhibition of CD14 expression as ERK5 knockdown. CCAAT-enhancer-binding proteins (C/EBPs) α and β are two major transcription factors that can directly regulate CD14 expression. Since 1,25D has been shown to increase both C/EBPα and β expression, and these two transcription factors form heterodimers to enhance CD14 expression, we used siRNA to knock down Mef2C and found decreased expression of 1,25D-induced C/EBPβ, but not C/EBPα. Similar results were obtained using an alternative method to titrate down Mef2C with dsDNA containing its DNA binding sequence. We conclude that Mef2C is a functional downstream target of ERK5, and that 1,25D-induced CD14 and NSE expression is mediated through ERK5/Mef2C activation. (Supported by T32 CA134268 training grant and R01 CA044722 both from the National Cancer Institute, NIH, and by the American Institute for Cancer Research grant 10A049) Citation Format: Ruifang Zheng, Xuening Wang, George P. Studzinski. Mef2C enhances 1,25-dihydroxyvitamin D3-induced monocytic differentiation of human myeloid leukemia cells by regulating C/EBPβ expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4233. doi:10.1158/1538-7445.AM2014-4233

The cationic amino acid transporter CAT-2A is a low-affinity, high-capacity carrier expressed in cardiomyocytes, liver and other tissues. CAT-2A is sensitive to N-ethylmaleimide (NEM) and nitric oxide (NO) inhibition. Both reactions involve cysteine (Cys) modification. Our previous studies showed that mutating Cys347 to alanine (C347A) in mouse CAT2A (mCAT2A) results in a ∼60% decrease in transport activity, and insensitivity to NO inhibition without affecting membrane expression levels of this transporter. The decreased activity might be due to a disruption of the Cys local environment. Alternatively, this Cys could be directly implicated in transport activity. We decided to test these possibilities by replacing Cys347 with a “more conservative” serine (C347S). Serine has similar size/polarity as Cys, but lacks the -SH group. The C347S mutant shows transport activity comparable to that of wild type; however, it retains NO insensitivity. To learn more about the involvement of Cys347 in transport, we measured the L-Lys concentration dependence of uptake in the presence of radiotracer amounts of [14C]L-Lys in both wild-type and C347A mCAT-2A variant. Hyperbolic fitting yielded not significantly different Km values (6.3 ± 2.8 vs. 8.2 ± 0.4 mM) and a substantial decrease in Vmax (2.6 ± 0.6 vs. 4.7 ± 0.1 nmol/mg prot./min) for C347A compared to wild-type control. These data suggest an impaired transporter turnover rate when Cys347 is replaced with Ala. In conclusion, Cys347 -SH side chain is required for NO modulation of mCAT-2A and the size/polarity of residue 347 is critical for the normal functioning of this transporter.