Numerical Simulation of Gas-Liquid Gravity Displacement in Vertical Fractures during Drilling of Carbonate Formations.

In petroleum drilling, carbonate formations characterized by natural fractures can result in troublesome gas-liquid gravity displacement, which refers to the phenomenon that the drilling mud leakage and gas kick are simultaneously triggered. This work focuses on clarifying the mechanism of gas-liquid displacement in vertical fractures during the drilling of carbonate formations and investigating the characteristics of gas-liquid displacement under various conditions. First, the bottom hole pressure allowing for gas-liquid gravity displacement is analyzed, which determines the coexistence condition of leakage and kick in vertical fractures. Then, a theoretical model of gas-liquid displacement flow in a vertical fracture is established. To verify the reliability and accuracy of the model, the results of numerical simulation are compared with those of a visualization experiment. The development process and flow characteristics of gas-liquid displacement in the fracture under different conditions are numerically simulated. The effects of pressure difference, drilling mud property, and fracture geometry on the gas-liquid displacement rate are analyzed. It is found that the drilling mud leakage rate increases with the increase of fracture width, fracture height, and drilling mud density, while it decreases with the increase of pressure difference and fracture length. The gas invasion rate increases with the increase of fracture width, fracture height, and pressure difference, while it decreases with the increase of drilling mud density and fracture length. The equations for leakage rate and gas invasion rate are derived by the response surface method, and the methods for mitigating gas-liquid gravity displacement are discussed. It is expected that the present work provides a better understanding of the gas-liquid gravity displacement in carbonate formations.

Low-intensity pulsed ultrasound promotes cell viability and inhibits apoptosis of H9C2 cardiomyocytes in 3D bioprinting scaffolds via PI3K-Akt and ERK1/2 pathways.

The aim of this study was to investigate whether low-intensity pulsed ultrasound (LIPUS) promotes myocardial cell viability in three-dimensional (3D) cell-laden gelatin methacryloyl (GelMA) scaffolds. Cardiomyoblasts (H9C2s) were mixed in 6% (w/v) GelMA bio-inks and printed using an extrusion-based 3D bioprinter. These scaffolds were exposed to LIPUS with different parameters or sham-irradiated to optimize the LIPUS treatment. The viability of H9C2s was measured using Cell Counting Kit-8 (CCK8), cell cycle, and live and dead cell double-staining assays. Western blot analysis was performed to determine the protein expression levels. We successfully fabricated 3D bio-printed cell-laden GelMA scaffolds. CCK8 and live and dead cell double-staining assays indicated that the optimal conditions for LIPUS were a frequency of 0.5 MHz and an exposure time of 10 min. Cell cycle analysis showed that LIPUS promoted the entry of cells into the S and G2/M phases from the G0/G1 phase. Western blot analysis revealed that LIPUS promoted the phosphorylation and activation of ERK1/2 and PI3K-Akt. The ERK1/2 inhibitor (U0126) and PI3K inhibitor (LY294002) significantly reduced LIPUS-induced phosphorylation of ERK1/2 and PI3K-Akt, respectively, which in turn reduced the LIPUS-induced viability of H9C2s in 3D bio-printed cell-laden GelMA scaffolds. A frequency of 0.5 MHz and exposure time of 10 min for LIPUS exposure can be adapted to achieve optimized culture effects on myocardial cells in 3D bio-printed cell-laden GelMA scaffolds via the ERK1/2 and PI3K-Akt signaling pathways.

Behavioral Responses to Familiar Versus Unfamiliar Older People as a Source of Disgust.

Disgust, as a part of the behavioral immune system, leads people to avoid behaviors of pathogens so as to reduce the probability of infection. Disgust also shows the source effects based on familiarity. However, these source effects have not been tested on the older population. Thus, we tested the source effects of emotional and behavioral reactions from the disgust toward older adults and the possible moderating effects of filial piety on disgust. In the first study, we employed the self-report method to test the source effects of emotional feelings of disgust amongst undergraduates. In the second study, we measured whether filial piety among community adults produced moderating effects of the disgust toward older adults. In the third study, we employed the shape discrimination task to test the source effects of behavioral avoidance to older adults among undergraduates. The first and third studies show stronger negative emotional/avoidance reactions towards unfamiliar older adults than familiar older adults, affirming the source effects of disgust towards older adults that we expected. However, we did not find moderating effects of filial piety associated with disgust. These findings can help us understand the evolutionary origin of disgust toward older adults, which is likely activated more intensely and quickly in response to unfamiliar individuals as compared with familiar individuals.

Prognostic Association Between Perioperative Red Blood Cell Transfusion and Postoperative Cardiac Surgery Outcomes.

Objective: To investigate the correlation between red blood cell transfusion and clinical outcome in patients after cardiac surgery. Methods: Demographic, clinical characteristics, treatment with/without transfusion, and outcomes of patients after cardiac surgery from the Medical Information Mart for Intensive Care-III database were collected. Patients were divided into two groups according to perioperative transfusion. A multivariable logistic regression analysis was utilized to adjust for the effect of red blood cell transfusion on outcomes for baseline and covariates and to determine its association with outcomes. Results: In total, 6,752 patients who underwent cardiac surgery were enrolled for the analysis. Among them, 2,760 (40.9%) patients received a perioperative transfusion. Compared with patients without red blood cell transfusion, transfused patients demonstrated worse outcomes in inhospital mortality, 1-year mortality, and all-cause mortality. Adjusting odds ratios (ORs) for the significant characteristic, patients with perioperative transfusion remained significantly associated with an increased risk of inhospital mortality [OR = 2.8, 95% confidence interval (CI) 1.5-5.1, P = 0.001], 1-year mortality (OR = 2.0, 95% CI 1.4-2.7, P < 0.001), and long-term mortality (OR = 2.2, 95% CI 1.8-2.8, P < 0.001). Conclusion: Perioperative red blood cell transfusion is associated with a worse prognosis of cardiac surgery patients. Optimal perioperative management and restricted transfusion strategy might be considered in selected patients.

Melatonin protects against focal cerebral ischemia-reperfusion injury in diabetic mice by ameliorating mitochondrial impairments: involvement of the Akt-SIRT3-SOD2 signaling pathway.

Diabetic patients are more vulnerable to cerebral ischemia-reperfusion (CIR) injury and have a worse prognosis and higher mortality after ischemic stroke than non-diabetic counterparts. Melatonin can exert neuroprotective effects against CIR injury in nondiabetic animal models. However, its effects on diabetic CIR injury and the underlying mechanisms remain unclarified. Herein, we found that melatonin administration improved neurological deficit, cerebral infarct volume, brain edema, and cell viability, reduced mitochondrial swelling, reactive oxygen species generation, and cytoplasmic cytochrome C release, and increased mitochondrial antioxidant enzymes activities, adenosine triphosphate production, and mitochondrial membrane potential in both streptozotocin-induced diabetic mice and high glucose-treated HT22 cells. Importantly, melatonin also activated protein kinase B (Akt) and sirtuin 3 (SIRT3)/superoxide dismutase 2 (SOD2) signaling and upregulated mitochondrial biogenesis-related transcription factors. However, these effects were largely attenuated by LY294002 (a specific Akt signaling blocker) administration. Additionally, 3-TYP (a selective SIRT3 inhibitor) and SIRT3 siRNA inhibited the above protective effects of melatonin as well as the upregulation of SIRT3 and the decrease of SOD2 acetylation but did not affect the p-Akt/Akt ratio. Overall, we demonstrate that melatonin can alleviate CIR injury in diabetic mice by activating Akt-SIRT3-SOD2 signaling and subsequently improving mitochondrial damage.

Irradiation of carotid baroreceptor with low-intensity pulsed ultrasound exerts different metabolic protection in perirenal, epididymal white adipose tissue and interscapular brown adipose tissue of obese rats.

This study was designed to clarify whether the irradiation of carotid baroreceptor (CB) with low-intensity pulsed ultrasound (LIPUS) protects against obesity by rebalancing the autonomic nervous system (ANS). Obesity was induced using a high-fat diet (HFD) for 8 weeks in Sprague-Dawley rats. Irradiation with LIPUS was daily (20 minutes a day) applied to the right CB. In our study, LIPUS significantly ameliorated metabolic disorders in obese rats. LIPUS partly restored norepinephrine (NE) and acetylcholine (ACH) levels in the perirenal white adipose tissue (PWAT), epididymal white adipose tissue (EWAT), interscapular brown adipose tissue (IBAT), and plasma of obese rats. LIPUS partially rectified the dysregulated AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor (PPAR) α/É£ pathway in the PWAT, EWAT, and IBAT of obese rats. PPARγ and PPARγ target genes respond more sensitively to HFD and LIPUS in PWAT and EWAT than in IBAT. NE, ACH, uncoupling protein-1, phosphorylated AMPK, PPARα, and PPARα target genes respond more sensitively to HFD and LIPUS in IBAT than in PWAT and EWAT. Conclusion: LIPUS irradiation of CB exerts different metabolic protection in PWAT, EWAT, and IBAT by rebalancing the ANS and rectifying the AMPK/PPARα/É£ pathway in obese rats.

Effects of Additional Intraoperative Administration of Sufentanil on Postoperative Pain, Stress and Inflammatory Responses in Patients Undergoing Laparoscopic Myomectomy: A Double-Blind, Randomized, Placebo-Controlled Trial.

PURPOSE: Although pain after laparoscopic surgery is assumed to be minor, many women still suffer from unexpected postoperative pain. Thus, we aimed to assess whether additional intraoperative administration of sufentanil could help to improve postoperative pain and related agitation, stress, and inflammation response in patients undergoing laparoscopic myomectomy. PATIENTS AND METHODS: Forty female patients with uterine myoma scheduled for laparoscopic myomectomy under general anesthesia were randomized to receive sufentanil (group T, n=20) or normal saline (group C, n=20) 1h before the end of the surgery. The postoperative pain, agitation, stress, inflammation, and adverse effects were measured. RESULTS: As the primary outcome, the visual analog scale (VAS) pain score was significantly reduced in group T as compared with group C at each measured time point in a post-anesthesia care unit (PACU), VAS 5 min (31.5 ± 2.7 vs 40.6 ± 5.6) (P<0.001), VAS 30 min (36.5 ± 4.5 vs 46.0 ± 2.9) (P<0.001), VAS 1h (37.8 ± 4.0 vs 48.6 ± 5.5) (P<0.001). The secondary outcomes, including the sedation agitation scale (SAS) scores, plasma concentrations of epinephrine and norepinephrine, and the levels of plasma interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α) in group T were remarkably lower than those in group C (P < 0.001). The cough cases in group T also showed a significant reduction in comparison with group C (P < 0.05). In addition, the anesthetic recovery time, including the spontaneous breathing recovery time and extubation time, were not significantly different between the two groups, as were the cases of respiratory depression and postoperative delirium (P > 0.05). CONCLUSION: For patients undergoing laparoscopic myomectomy, administration of sufentanil 1 h before the end of surgery shows excellent analgesic and sedative effects, alleviated postoperative stress and inflammatory responses, reduced incidence of cough, without prolonging anesthetic recovery time and increasing adverse reactions.

The Value of Plasma-Based MicroRNAs as Diagnostic Biomarkers for Ovarian Cancer.

BACKGROUND: Ovarian cancer (OC) is one of the most threatening diseases among women in the world. Plasma microRNAs (miRNAs) may serve as promising diagnostic biomarkers for patients with OC. MATERIALS AND METHODS: Using quantitative reverse transcription polymerase chain reaction (qRT-PCR) based on Exiqon panel, we identified 27 differentially expressed miRNAs from 2 OC pool samples and 1 normal control (NC) pool in the initial screening phase. Then we further validated the identified miRNAs through the training (32 OC vs. 34 NCs) and validation stages (69 OC vs. 66 NCs) using qRT-PCR. The expression levels of the miRNAs were also assessed in tissues and exosomes. RESULTS: Five plasma miRNAs (miR-205-5p, miR-145-5p, miR-10a-5p, miR-346 and miR-328-3p) were significantly overexpressed in OC in comparison with NCs. The areas under the receiver operating characteristic curve of the 5-miRNA panel were 0.788 for the training stage and 0.763 for the validation stage. The level of miR-205-5p has significantly different expression in patients with well-moderate histological grade compared with those with a poor grade (P = 0.012). The expression levels of the 5 miRNAs were also significantly upregulated in the exosomes of OC plasma samples (32 OC vs. 32 NCs). However, the expression of the 4 miRNAs (miR-145-5p, miR-10a-5p, miR-346 and miR-328-3p) was significantly lower in tumor samples than in normal tissues (22 OC vs. 22 NCs). CONCLUSIONS: The 5 plasma miRNAs may be noninvasive diagnostic biomarkers of OC. The plasma miR-205-5p level may reflect the change trend of the histological grade of OC patients.

Novel approach for quantification of endoplasmic reticulum Ca2+ transport.

The type 2a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a key role in Ca2+ regulation in the heart. However, available techniques to study SERCA function are either cell destructive or lack sensitivity. The goal of this study was to develop an approach to selectively measure SERCA2a function in the cellular environment. The genetically encoded Ca2+ sensor R-CEPIA1er was used to measure the concentration of Ca2+ in the lumen of the endoplasmic reticulum (ER) ([Ca2+]ER) in HEK293 cells expressing human SERCA2a. Coexpression of the ER Ca2+ release channel ryanodine receptor (RyR2) created a Ca2+ release/reuptake system that mimicked aspects of cardiac myocyte Ca2+ handling. SERCA2a function was quantified from the rate of [Ca2+]ER refilling after ER Ca2+ depletion; then, ER Ca2+ leak was measured after SERCA inhibition. ER Ca2+ uptake and leak were analyzed as a function of [Ca2+]ER to determine maximum ER Ca2+ uptake rate and maximum ER Ca2+ load. The sensitivity of this assay was validated by analyzing effects of SERCA inhibitors, [ATP]/[ADP], oxidative stress, phospholamban, and a loss-of-function SERCA2a mutation. In addition, the feasibility of using R-CEPIA1er to study SERCA2a in a native system was evaluated by using in vivo gene delivery to express R-CEPIA1er in mouse hearts. After ventricular myocyte isolation, the same methodology used in HEK293 cells was applied to study endogenous SERCA2a. In conclusion, this new approach can be used as a sensitive screening tool to study the effect of different drugs, posttranslational modifications, and mutations on SERCA function. NEW & NOTEWORTHY The aim of this study was to develop a sensitive approach to selectively measure sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) function in the cellular environment. The newly developed Ca2+ sensor R-CEPIA1er was used to successfully analyze Ca2+ uptake mediated by recombinant and native cardiac SERCA. These results demonstrate that this new approach can be used as a powerful tool to study new mechanisms of Ca2+ pump regulation.

3,3'-Diindolylmethane protects cardiomyocytes from LPS-induced inflammatory response and apoptosis.

BACKGROUND: 3,3'-Diindolylmethane (DIM) has been extensively studied as a potential therapeutic drug with free radical scavenging, antioxidant and anti-angiogenic effects. However, whether DIM has similar effects on cardiomyocytes remains unknown. Here we evaluated DIM's influence on inflammation and apoptosis of H9C2 cardiomyocytes induced by LPS and to explore the possible mechanism of the effects. METHODS: H9C2 cells were incubated with DIM (10, 20 and 30 µM) with or without LPS for 24 h. The cytotoxicity of DIM was detected by CCK-8. The levels of tumour necrosis factor (TNF)-α and interleukin (IL)-6 were then measured using RT-qPCR and ELISA. Cell apoptosis rate and reactive oxygen species (ROS) content after DIM treatment were measured by flow cytometry. Expressions of NFκB, P-NFκB, IκBa, P-IκBa, Bax and Bcl-2 after DIM treatment were detected by western blot. The rate of NFκB nuclear translocation after DIM treatment was determined by immunocytochemical analysis. RESULTS: LPS stimulation promoted TNF-α and IL-6 mRNA expression. After treatment with various concentrations of DIM (10, 20 and 30 µM), TNF-α and IL-6 mRNA expression was clearly impaired, especially in the LPS + DIM30(µM) group. ELISA was used to measure TNF-α and IL-6 concentrations in cellular supernatant, and the result was verified to be consistent with RT-qPCR. Additionally, DIM treatment significantly blocked LPS-induced oxidative stress and inhibited LPS-induced apoptosis in H9C2 cardiomyocytes according to the results detected by flow cytometry. Moreover, compared with LPS alone, DIM significantly inhibited the LPS-induced phosphorylation of NFκB (p-NFκB) and Bax expression and increased Bcl-2 expression. CONCLUSIONS: DIM may have a protective effect for H9C2 cardiomyocytes against LPS-induced inflammatory response and apoptosis. DIM may be a new insight into the treatment of septic cardiomyopathy.

Integrated Dissection of Cysteine Oxidative Post-translational Modification Proteome During Cardiac Hypertrophy.

Cysteine oxidative modification of cellular proteins is crucial for many aspects of cardiac hypertrophy development. However, integrated dissection of multiple types of cysteine oxidative post-translational modifications (O-PTM) of proteomes in cardiac hypertrophy is currently missing. Here we developed a novel discovery platform that encompasses a customized biotin switch-based quantitative proteomics pipeline and an advanced analytic workflow to comprehensively profile the landscape of cysteine O-PTM in an ISO-induced cardiac hypertrophy mouse model. Specifically, we identified a total of 1655 proteins containing 3324 oxidized cysteine sites by at least one of the following three modifications: reversible cysteine O-PTM, cysteine sulfinylation (CysSO2H), and cysteine sulfonylation (CysSO3H). Analyzing the hypertrophy signatures that are reproducibly discovered from this computational workflow unveiled four biological processes with increased cysteine O-PTM. Among them, protein phosphorylation, creatine metabolism, and response to elevated Ca2+ pathways exhibited an elevation of cysteine O-PTM in early stages, whereas glucose metabolism enzymes were increasingly modified in later stages, illustrating a temporal regulatory map in cardiac hypertrophy. Our cysteine O-PTM platform depicts a dynamic and integrated landscape of the cysteine oxidative proteome, through the extracted molecular signatures, and provides critical mechanistic insights in cardiac hypertrophy. Data are available via ProteomeXchange with identifier PXD010336.

Dickkopf­3 upregulation mediates the cardioprotective effects of curcumin on chronic heart failure.

Curcumin, isolated from rhizome of turmeric, has been widely studied as a potential therapeutic drug for cancer. However, protective effects of curcumin on chronic heart failure (CHF) have not been fully studied. In the present study, the effects of curcumin on CHF and the underlying mechanisms were investigated. A total of 40 rabbits were randomized into 4 groups: Control rabbits fed with placebo (Con) or curcumin (Con­cur), CHF rabbits fed with placebo (CHF) or curcumin (CHF­cur). CHF was induced by volume and pressure overload. The effects of curcumin on cardiac function and left ventricular (LV) structure were assessed by echocardiography and histology. The effects of curcumin on CHF molecular biomarkers were detected by dihydroethidium and immunohistochemical staining. The effects of curcumin on Dickkopf­related protein 3 (DKK­3), p38 mitogen­activated protein kinase (p38), c­Jun N­terminal kinase (JNK) and apoptosis signal­regulating kinase 1 (ASK1) were assessed by immunohistochemical staining and western blot analysis. Cardiac dysfunction and LV remodeling were successfully produced by ten weeks volume overload and eight weeks pressure overload in the CHF group. Compared with the Con group, the CHF group demonstrated higher levels of CHF molecular biomarkers, a lower level of DKK­3 expression and alterations of p38, JNK and ASK1 protein expression. Curcumin alleviated all those abnormalities markedly in the CHF­cur group. In summary, curcumin may exert cardioprotective effects by up­regulating DKK­3, which in turn may inhibit p38 and JNK signaling pathways in an ASK1­dependent way. The present study demonstrated that Dickkopf­3 upregulation mediates the cardioprotective effects of curcumin on chronic heart failure for the first time.

Anti-inflammatory and wound healing activities of calophyllolide isolated from Calophyllum inophyllum Linn.

Due to the high-cost and limitations of current wound healing treatments, the search for alternative approaches or drugs, particularly from medicinal plants, is of key importance. In this study, we report anti-inflammatory and wound healing activities of the major calophyllolide (CP) compound isolated from Calophyllum inophyllum Linn. The results showed that CP had no effect on HaCaT cell viability over a range of concentrations. CP reduced fibrosis formation and effectively promoted wound closure in mouse model without causing body weight loss. The underlying molecular mechanisms of wound repair by CP was investigated. CP markedly reduced MPO activity, and increased M2 macrophage skewing, as shown by up-regulation of M2-related gene expression, which is beneficial to the wound healing process. CP treatment prevented a prolonged inflammatory process by down-regulation of the pro-inflammatory cytokines-IL-1ß, IL-6, TNF-α, but up-regulation of the anti-inflammatory cytokine, IL-10. This study is the first to indicate a plausible role for CP in accelerating the process of wound healing through anti-inflammatory activity mechanisms, namely, by regulation of inflammatory cytokines, reduction in MPO, and switching of macrophages to an M2 phenotype. These findings may enable the utilization of CP as a potent therapeutic for cutaneous wound healing.

Frondoside A from sea cucumber and nymphaeols from Okinawa propolis: Natural anti-cancer agents that selectively inhibit PAK1 in vitro.

A sulfated saponin called "Frondoside A" (FRA) from sea cucumber and ingredients from Okinawa propolis (OP) have been previously shown to suppress the PAK1-dependent growth of A549 lung cancer as well as pancreatic cancer cells. However, the precise molecular mechanism underlying their anti-cancer action still remains to be clarified. In this study, for the first time, we found that both FRA and OP directly inhibit PAK1 in vitro in a selective manner (far more effectively than two other oncogenic kinases, LIMK and AKT). Furthermore, at least two major anti-cancer ingredients of OP, nymphaeols A and C, also directly inhibit PAK1 in vitro in a selective manner. To the best of our knowledge, FRA is the first marine compound that selectively inhibits PAK1. Likewise, these nymphaeols are the first propolis ingredients that selectively inhibit PAK1.

1,2,3-Triazolyl esterization of PAK1-blocking propolis ingredients, artepillin C (ARC) and caffeic acid (CA), for boosting their anti-cancer/anti-PAK1 activities along with cell-permeability.

Artepillin C (ARC) and caffeic acid (CA) are among the major anti-cancer ingredients of propolis, and block the oncogenic/melanogenic/ageing kinase PAK1. However, mainly due to their COOH moiety, cell-permeability of these herbal compounds is rather limited. Thus, in this study, in an attempt to increase their cell-permeability without any significant loss of their water-solubility, we have esterized both ARC and CA with the water-soluble 1,2,3-triazolyl alcohol through Click Chemistry. We found that this esterization boosts the anti-cancer activity of ARC and CA by 100 and over 400 folds, respectively, against the PAK-dependent growth of A549 lung cells, but show no effect on the PAK1-independent growth of B16F10 melanoma cells. Confirming this "selective" toxicity, these esters are still capable of blocking the kinase PAK1 strongly in cell culture (with IC50 around 5 µM), and the anti-PAK1 activity of 15A (ARC ester) and 15C (CA ester) appears to be 30-fold and 140-fold higher than ARC and CA, respectively. The 15A and 15C are 8-fold and 70-fold more cell-permeable (through the multi-drug resistant cell line EMT6) than ARC and CA, respectively. These data altogether suggest that both 15A and 15C would be far more useful than propolis for the treatment of a wide variety of PAK1-dependent diseases/disorders such as cancers, Alzheimer's diseases (AD), hypertension, diabetes (type 2), and hyper-pigmentation.

Hair Growth Promoting and Anticancer Effects of p21-activated kinase 1 (PAK1) Inhibitors Isolated from Different Parts of Alpinia zerumbet.

PAK1 (p21-activated kinase 1) is an emerging target for the treatment of hair loss (alopecia) and cancer; therefore, the search for PAK1 blockers to treat these PAK1-dependent disorders has received much attention. In this study, we evaluated the anti-alopecia and anticancer effects of PAK1 inhibitors isolated from Alpinia zerumbet (alpinia) in cell culture. The bioactive compounds isolated from alpinia were found to markedly promote hair cell growth. Kaempferol-3-O-ß-d-glucuronide (KOG) and labdadiene, two of the isolated compounds, increased the proliferation of human follicle dermal papilla cells by approximately 117%-180% and 132%-226%, respectively, at 10-100 µM. MTD (2,5-bis(1E,3E,5E)-6-methoxyhexa-1,3,5-trien-1-yl)-2,5-dihydrofuran) and TMOQ ((E)-2,2,3,3-tetramethyl-8-methylene-7-(oct-6-en-1-yl)octahydro-1H-quinolizine) showed growth-promoting activity around 164% and 139% at 10 µM, respectively. The hair cell proliferation induced by these compounds was significantly higher than that of minoxidil, a commercially available treatment for hair loss. Furthermore, the isolated compounds from alpinia exhibited anticancer activity against A549 lung cancer cells with IC50 in the range of 67-99 µM. Regarding the mechanism underlying their action, we hypothesized that the anti-alopecia and anticancer activities of these compounds could be attributed to the inhibition of the oncogenic/aging kinase PAK1.

The serum/PDGF-dependent "melanogenic" role of the minute level of the oncogenic kinase PAK1 in melanoma cells proven by the highly sensitive kinase assay.

We previously demonstrated that the oncogenic kinase PAK4, which both melanomas and normal melanocytes express at a very high level, is essential for their melanogenesis. In the present study, using the highly sensitive "Macaroni-Western" (IP-ATP-Glo) kinase assay, we investigated the melanogenic potential of another oncogenic kinase PAK1, which melanoma (B16F10) cells express only at a very minute level. After transfecting melanoma cells with PAK1-shRNA for silencing PAK1 gene, melanin content, tyrosinase activity, and kinase activity of PAK1 were compared between the wild-type and transfectants. We found that (i) PAK1 is significantly activated by melanogenic hormones such as IBMX (3-isobutyl-1-methyl xanthine) and α-MSH (melanocyte-stimulating hormone), (ii) silencing the endogenous PAK1 gene in melanoma cells through PAK1-specific shRNA reduces both melanin content and tyrosinase activity in the presence of both serum and melanogenic hormones to the basal level, (iii) the exogenously added wild-type PAK1 in the melanoma cells boosts the α-MSH-inducible melanin level by several folds without affecting the basal, and (iv) α-MSH/IBMX-induced melanogenesis hardly takes place in the absence of either serum or PAK1, clearly indicating that PAK1 is essential mainly for serum- and α-MSH/IBMX-dependent melanogenesis, but not the basal, in melanoma cells. The outcome of this study might provide the first scientific basis for explaining why a wide variety of herbal PAK1-blockers such as CAPE (caffeic acid phenethyl ester), curcumin and shikonin in cosmetics are useful for skin-whitening.

1,2,3-Triazolyl ester of Ketorolac: A "Click Chemistry"-based highly potent PAK1-blocking cancer-killer.

An old anti-inflammatory/analgesic drug called Toradol is a racemic form of Ketorolac (50% R-form and 50% S-form) that blocks the oncogenic RAC-PAK1-COX-2 (cyclooxygenase-2) signaling, through the direct inhibition of RAC by the R-form and of COX-2 by the S-form, eventually down-regulating the production of prostaglandins. However, due to its COOH moiety which is clearly repulsive to negatively-charged phospholipid-based plasma membrane, its cell-permeability is rather poor (the IC50 against the growth of human cancer cells such as A549 is around 13 µM). In an attempt to boost its anti-cancer activity, hopefully by increasing its cell-permeability through abolishing the negative charge, yet keeping its water-solubility, here we synthesized a 1,2,3-triazolyl ester of Toradol through "Click Chemistry". The resultant water-soluble "azo" derivative called "15K" was found to be over 500 times more potent than Toradol with the IC50 around 24 nM against the PAK1-dependent growth of A549 cancer cells, inactivating PAK1 in cell culture with the apparent IC50 around 65 nM, and inhibiting COX-2 in vitro with the IC50 around 6 nM. Furthermore, the Click Chemistry boosts the anti-cancer activity of Ketorolac by 5000 times against the PAK1-independent growth of B16F10 melanoma cells. Using a multi-drug-resistant (MDR) cancer cell line (EMT6), we found that the esterization of Ketorolac boosts its cell-permeability by at least 10 folds. Thus, the Click Chemistry dramatically boosts the anti-cancer activity of Ketorolac, at least in three ways: increasing its cell-permeability, the anti-PAK1 activity of R-form and anti-COX-2 activity of S-form. The resultant "15K" is so far among the most potent PAK1-blockers, and therefore would be potentially useful for the therapy of many different PAK1-dependent diseases/disorders such as cancers.

Evaluation and Treatment of Endoplasmic Reticulum (ER) Stress in Right Ventricular Dysfunction during Monocrotaline-Induced Rat Pulmonary Arterial Hypertension.

PURPOSE: Endoplasmic reticulum (ER) stress contributes to pulmonary artery hypertension (PAH). However, the exact roles of ER stress in right ventricular (RV) dysfunction, which is strongly associated with PAH, are largely unknown. Here, we aimed to explore how ER stress affects RV function in a rat PAH model and evaluated the effects of an ER stress inhibitor on RV dysfunction. METHODS: We examined expression changes of an ER marker: chaperone glucose-regulated protein 78 (GRP78), three ER stress sensor proteins: activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and protein kinase RNA-like endoplasmic reticulum kinase (PERK), and a key ER stress-induced apoptosis indicator: CCAAT/enhancer-binding protein homologous protein (CHOP), with inflammation indicators: interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and matrix metalloproteinases (MMPs) in RV at 3, 7, 14 and 28 days following a single dose of monocrotaline (MCT) injection, with or without a preventive treatment [4-phenylbutyric acid (PBA)]. RV function was evaluated by histological, molecular and echocardiographic analysis. RESULTS: 1) GRP78 protein expression started to increase (1.5 ± 0.06 fold change) at 3d post MCT injection, even before the formation of PAH. 2) ATF6, IRE1, and PERK showed distinctive expression patterns post MCT injection. 3) CHOP expression remained low at day 3 & 7, but significantly increased at day 14 (p < 0.05), along with the peak of RV cardiomyocytes apoptosis. 4) PBA inhibited ER stress and alleviated remodeling and dysfunction in the RV. CONCLUSIONS: The early phase of ER stress might benefit RV function, whereas the extended phase led to RV cardiomyocyte apoptosis and dysfunction. Inhibition of ER stress by PBA during PAH directly improved RV function.

Mitochondrial aldehyde dehydrogenase 2 deficiency aggravates energy metabolism disturbance and diastolic dysfunction in diabetic mice.

Diabetes causes energy metabolism disturbance and may lead to cardiac dysfunction. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) protects cardiac function from myocardial damage. Therefore, understanding of its roles in diabetic heart is critical for developing new therapeutics targeting ALDH2 and mitochondrial function for diabetic hearts. This study investigated the impact of ALDH2 deficiency on diastolic function and energy metabolism in diabetic mice. Diabetes was induced in ALDH2 knockout and wild-type mice by streptozotocin. Cardiac function was determined by echocardiography. Glucose uptake, energy status, and metabolic profiles were used to evaluate cardiac energy metabolism. The association between ALDH2 polymorphism and diabetes was also analyzed in patients. Echocardiography revealed preserved systolic function and impaired diastolic function in diabetic ALDH2-deficient mice. Energy reserves (phosphocreatine/adenosine triphosphate ratio) were reduced in the diabetic mutants and were associated with diastolic dysfunction. Western blot analysis showed that diabetes induces accumulated lipid peroxidation products and escalated AMP-activated protein kinase-LKB1 pathway. Further, ALDH2 deficiency exacerbated the diabetes-induced deficient myocardial glucose uptake and other perturbations of metabolic profiles. Finally, ALDH2 mutations were associated with worse diastolic dysfunction in diabetic patients. Together, our results demonstrate that ALDH2 deficiency and resulting energy metabolism disturbance is a part of pathology of diastolic dysfunction of diabetic hearts, and suggest that patients with ALDH2 mutations are vulnerable to diabetic damage. KEY MESSAGE: ALDH2 deficiency exacerbates diastolic dysfunction in early diabetic hearts. ALDH2 deficiency triggers decompensation of metabolic reserves and energy metabolism disturbances in early diabetic hearts. ALDH2 deficiency potentiates oxidative stress and AMPK phosphorylation induced by diabetes via post-translational regulation of LKB1. Diabetic patients with ALDH2 mutations are predisposed to worse diastolic dysfunction.