Inhibition of MMP8 effectively alleviates manic-like behavior and reduces neuroinflammation by modulating astrocytic CEBPD.

There is an intrinsic relationship between psychiatric disorders and neuroinflammation, including bipolar disorder. Ouabain, an inhibitor of Na+/K+-ATPase, has been implicated in the mouse model with manic-like behavior. However, the molecular mechanisms linking neuroinflammation and manic-like behavior require further investigation. CCAAT/Enhancer-Binding Protein Delta (CEBPD) is an inflammatory transcription factor that contributes to neurological disease progression. In this study, we demonstrated that the expression of CEBPD in astrocytes was increased in ouabain-treated mice. Furthermore, we observed an increase in the expression and transcript levels of CEBPD in human primary astrocytes following ouabain treatment. Transcriptome analysis revealed high MMP8 expression in human primary astrocytes following CEBPD overexpression and ouabain treatment. We confirmed that MMP8 is a CEBPD-regulated gene that mediates ouabain-induced neuroinflammation. In our animal model, treatment of ouabain-injected mice with M8I (an inhibitor of MMP8) resulted in the inhibition of manic-like behavior compared to ouabain-injected mice that were not treated with M8I. Additionally, the reduction in the activation of astrocytes and microglia was observed, particularly in the hippocampal CA1 region. Excessive reactive oxygen species formation was observed in ouabain-injected mice, and treating these mice with M8I resulted in the reduction of oxidative stress, as indicated by nitrotyrosine staining. These findings suggest that MMP8 inhibitors may serve as therapeutic agents in mitigating manic symptoms in bipolar disorder.

Volatility Measurements of Individual Components in Organic Aerosol Mixtures Using Temperature-Programmed Desorption-Direct Analysis in Real Time-High Resolution Mass Spectrometry.

Atmospheric organic aerosols (OA) have profound effects on air quality, visibility, and radiative forcing of climate. Quantitative assessment of gas-particle equilibrium of OA components is critical to understand formation, growth, distribution, and evolution of OA in the atmosphere. This study presents a novel ambient pressure measurement approach developed and tested for untargeted screening of individual components in complex OA mixtures, followed by targeted chemical speciation of identified species and assessment of their physicochemical properties such as saturation vapor pressure and enthalpies of sublimation/evaporation. The method employs temperature-programmed desorption (TPD) experiments coupled to "direct analysis in real time" (DART) ionization source and high resolution mass spectrometry (HRMS) detection. Progression of the mass spectra is acquired in the TPD experiments over a T = 25-350 °C temperature range, and extracted ion chromatograms (EIC) of individual species are used to infer their apparent enthalpies of sublimation/evaporation (ΔHsub*) and saturation vapor pressure (pT*, Pa, or CT*, µg m-3) as a function of T. We validate application of this method for analysis of selected organic compounds with known ΔHsub and CT values, which showed excellent agreement between our results and the existing data. We then extend these experiments to interrogate individual components in complex OA samples generated in the laboratory-controlled ozonolysis of α-pinene, limonene, and ß-ocimene monoterpenes. The abundant OA species of interest are distinguished based on their accurate mass measurements, followed by quantitation of their apparent ΔHsub* and CT* values from the corresponding EIC records. Comparison of C298K* values derived from our experiments for the individual OA components with the corresponding estimates based on their elemental composition using a "molecular corridors" (MC) parametrization suggests that the MC calculations tend to overestimate the saturation vapor pressures of OA components. Presented results indicate very promising applicability of the TPD-DART-HRMS method for the untargeted analysis of organic molecules in OA and other environmental mixtures, enabling rapid detection and quantification of organic pollutants in the real-world condensed-phase samples at atmospheric pressure and without sample preparation.

Molecular and Structural Characterization of Isomeric Compounds in Atmospheric Organic Aerosol Using Ion Mobility-Mass Spectrometry.

Secondary organic aerosol (SOA) formed through multiphase atmospheric chemistry makes up a large fraction of airborne particles. The chemical composition and molecular structures of SOA constituents vary between different emission sources and aging processes in the atmosphere, which complicates their identification. In this work, we employ drift tube ion mobility spectrometry with quadrupole time-of-flight mass spectrometry (IM-MS) detection for rapid gas-phase separation and multidimensional characterization of isomers in two biogenic SOAs produced from ozonolysis of isomeric monoterpenes, d-limonene (LSOA) and α-pinene (PSOA). SOA samples were ionized using electrospray ionization (ESI) and characterized using IM-MS in both positive and negative ionization modes. The IM-derived collision cross sections in nitrogen gas (DTCCSN2 ) for individual SOA components were obtained using multifield and single-field measurements. A novel application of IM multiplexing/high-resolution demultiplexing methodology was employed to increase sensitivity, improve peak shapes, and augment mobility baseline resolution, which revealed several isomeric structures for the measured ions. For LSOA and PSOA samples, we report significant structural differences of the isomer structures. Molecular structural calculations using density functional theory combined with the theoretical modeling of CCS values provide insights into the structural differences between LSOA and PSOA constituents. The average DTCCSN2 values for monomeric SOA components observed as [M + Na]+ ions are 3-6% higher than those of their [M - H]- counterparts. Meanwhile, dimeric and trimeric isomer components in both samples showed an inverse trend with the relevant values of [M - H]- ions being 3-7% higher than their [M + Na]+ counterparts, respectively. The results indicate that the structures of Na+-coordinated oligomeric ions are more compact than those of the corresponding deprotonated species. The coordination with Na+ occurs on the oxygen atoms of the carbonyl groups leading to a compact configuration. Meanwhile, deprotonated molecules have higher DTCCSN2 values due to their elongated structures in the gas phase. Therefore, DTCCSN2 values of isomers in SOA mixtures depend strongly on the mode of ionization in ESI. Additionally, PSOA monomers and dimers exhibit larger DTCCSN2 values (1-4%) than their LSOA counterparts owing to more rigid structures. A cyclobutane ring is present with functional groups pointing in opposite directions in PSOA compounds, as compared to noncyclic flexible LSOA structures, forming more compact ions in the gas phase. Lastly, we investigated the effects of direct photolysis on the chemical transformations of selected individual PSOA components. We use IM-MS to reveal structural changes associated with aerosol aging by photolysis. This study illustrates the detailed molecular and structural descriptors for the detection and annotation of structural isomers in complex SOA mixtures.

Metabolic Remodeling Promotes Cardiac Hypertrophy by Directing Glucose to Aspartate Biosynthesis.

RATIONALE: Hypertrophied hearts switch from mainly using fatty acids (FAs) to an increased reliance on glucose for energy production. It has been shown that preserving FA oxidation (FAO) prevents the pathological shift of substrate preference, preserves cardiac function and energetics, and reduces cardiomyocyte hypertrophy during cardiac stresses. However, it remains elusive whether substrate metabolism regulates cardiomyocyte hypertrophy directly or via a secondary effect of improving cardiac energetics. OBJECTIVE: The goal of this study was to determine the mechanisms of how preservation of FAO prevents the hypertrophic growth of cardiomyocytes. METHODS AND RESULTS: We cultured adult rat cardiomyocytes in a medium containing glucose and mixed-chain FAs and induced pathological hypertrophy by phenylephrine. Phenylephrine-induced hypertrophy was associated with increased glucose consumption and higher intracellular aspartate levels, resulting in increased synthesis of nucleotides, RNA, and proteins. These changes could be prevented by increasing FAO via deletion of ACC2 (acetyl-CoA-carboxylase 2) in phenylephrine-stimulated cardiomyocytes and in pressure overload-induced cardiac hypertrophy in vivo. Furthermore, aspartate supplementation was sufficient to reverse the antihypertrophic effect of ACC2 deletion demonstrating a causal role of elevated aspartate level in cardiomyocyte hypertrophy. 15N and 13C stable isotope tracing revealed that glucose but not glutamine contributed to increased biosynthesis of aspartate, which supplied nitrogen for nucleotide synthesis during cardiomyocyte hypertrophy. CONCLUSIONS: Our data show that increased glucose consumption is required to support aspartate synthesis that drives the increase of biomass during cardiac hypertrophy. Preservation of FAO prevents the shift of metabolic flux into the anabolic pathway and maintains catabolic metabolism for energy production, thus preventing cardiac hypertrophy and improving myocardial energetics.

Oxidative stress diverts tRNA synthetase to nucleus for protection against DNA damage.

Tyrosyl-tRNA synthetase (TyrRS) is known for its essential aminoacylation function in protein synthesis. Here we report a function for TyrRS in DNA damage protection. We found that oxidative stress, which often downregulates protein synthesis, induces TyrRS to rapidly translocate from the cytosol to the nucleus. We also found that angiogenin mediates or potentiates this stress-induced translocalization. The nuclear-localized TyrRS activates transcription factor E2F1 to upregulate the expression of DNA damage repair genes such as BRCA1 and RAD51. The activation is achieved through direct interaction of TyrRS with TRIM28 to sequester this vertebrate-specific epigenetic repressor and its associated HDAC1 from deacetylating and suppressing E2F1. Remarkably, overexpression of TyrRS strongly protects against UV-induced DNA double-strand breaks in zebrafish, whereas restricting TyrRS nuclear entry completely abolishes the protection. Therefore, oxidative stress triggers an essential cytoplasmic enzyme used for protein synthesis to translocate to the nucleus to protect against DNA damage.

Increasing Fatty Acid Oxidation Prevents High-Fat Diet-Induced Cardiomyopathy Through Regulating Parkin-Mediated Mitophagy.

BACKGROUND: Increased fatty acid oxidation (FAO) has long been considered a culprit in the development of obesity/diabetes mellitus-induced cardiomyopathy. However, enhancing cardiac FAO by removing the inhibitory mechanism of long-chain fatty acid transport into mitochondria via deletion of acetyl coenzyme A carboxylase 2 (ACC2) does not cause cardiomyopathy in nonobese mice, suggesting that high FAO is distinct from cardiac lipotoxicity. We hypothesize that cardiac pathology-associated obesity is attributable to the imbalance of fatty acid supply and oxidation. Thus, we here seek to determine whether further increasing FAO by inducing ACC2 deletion prevents obesity-induced cardiomyopathy, and if so, to elucidate the underlying mechanisms. METHODS: We induced high FAO in adult mouse hearts by cardiac-specific deletion of ACC2 using a tamoxifen-inducible model (ACC2 iKO). Control and ACC2 iKO mice were subjected to high-fat diet (HFD) feeding for 24 weeks to induce obesity. Cardiac function, mitochondria function, and mitophagy activity were examined. RESULTS: Despite both control and ACC2 iKO mice exhibiting a similar obese phenotype, increasing FAO oxidation by deletion of ACC2 prevented HFD-induced cardiac dysfunction, pathological remodeling, and mitochondria dysfunction, as well. Similarly, increasing FAO by knockdown of ACC2 prevented palmitate-induced mitochondria dysfunction and cardiomyocyte death in vitro. Furthermore, HFD suppressed mitophagy activity and caused damaged mitochondria to accumulate in the heart, which was attenuated, in part, in the ACC2 iKO heart. Mechanistically, ACC2 iKO prevented HFD-induced downregulation of parkin. During stimulation for mitophagy, mitochondria-localized parkin was severely reduced in control HFD-fed mouse heart, which was restored, in part, in ACC2 iKO HFD-fed mice. CONCLUSIONS: These data show that increasing cardiac FAO alone does not cause cardiac dysfunction, but protects against cardiomyopathy in chronically obese mice. The beneficial effect of enhancing cardiac FAO in HFD-induced obesity is mediated, in part, by the maintenance of mitochondria function through regulating parkin-mediated mitophagy. Our findings also suggest that targeting the parkin-dependent mitophagy pathway could be an effective strategy against the development of obesity-induced cardiomyopathy.

Hyperthermic intraperitoneal chemotherapy enhances antitumor effects on ovarian cancer through immune-mediated cancer stem cell targeting.

PURPOSE: We aimed to determine the effects and possible mechanisms of hyperthermic intraperitoneal chemotherapy (HIPEC) in targeting ovarian cancer stem-like cells (CSCs). METHODS: Murine ovarian cancer cell lines presenting CSC surface markers were grown intraperitoneally in both immunocompetent and immunodeficient mice, which were then treated by intraperitoneal hyperthermia with the chemotherapeutic agents: paclitaxel and cisplatin. Tumor growth was measured by non-invasive luminescent imaging. Intraperitoneal immune cells, such as CD4+, CD8+ T cells, macrophages, and dendritic cells, were evaluated through flow cytometry analysis. RESULTS: Combined hyperthermia and chemotherapy exhibited an efficient therapeutic effect in the immunocompetent mice. However, a similar effect was not observed in the immunodeficient mice. Intraperitoneal hyperthermia increased the number of Intraperitoneal macrophages and dendritic cells that were lost due to chemotherapy. Compared with ovarian cancer bulk cells, CSCs were more susceptible to phagocytosis by macrophages. CONCLUSION: We demonstrated that the superior therapeutic efficacy and reduced proportion of CSCs associated with intraperitoneal hyperthermic chemotherapy were immune-related. Hyperthermia recruits the phagocytes that target surviving CSCs after chemotherapy. These results provide a novel mechanism for the efficacy of HIPEC in treating ovarian cancer.

The Role of Oxidative Stress and Autophagy in Blue-Light-Induced Damage to the Retinal Pigment Epithelium in Zebrafish In Vitro and In Vivo.

Age-related macular degeneration (AMD) is the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris among elderly individuals and is the leading cause of blindness worldwide. Thus, a better understanding of the underlying mechanisms in retinal tissue activated by blue light exposure is important for developing novel treatment and intervention strategies. In this study, blue-light-emitting diodes with a wavelength of 440 nm were applied to RPE cells at a dose of 3.7 ± 0.75 mW/cm2 for 24 h. ARPE-19 cells were used to investigate the underlying mechanism induced by blue light exposure. A trypan blue exclusion assay was used for the cell viability determination. Flow cytometry was used for apoptosis rate detection and autophagy analysis. An immunofluorescence microscopy analysis was used to investigate cellular oxidative stress and DNA damage using DCFDA fluorescence staining and an anti-γH2AX antibody. Blue light exposure of zebrafish larvae was established to investigate the effect on retinal tissue development in vivo. To further demonstrate the comprehensive effect of blue light on ARPE-19 cells, next-generation sequencing (NGS) was performed for an ingenuity pathway analysis (IPA) to reveal additional related mechanisms. The results showed that blue light exposure caused a decrease in cell proliferation and an increase in apoptosis in ARPE-19 cells in a time-dependent manner. Oxidative stress increased during the early stage of 2 h of exposure and activated DNA damage in ARPE-19 cells after 8 h. Furthermore, autophagy was activated in response to blue light exposure at 24-48 h. The zebrafish larvae model showed the unfavorable effect of blue light in prohibiting retinal tissue development. The RNA-Seq results confirmed that blue light induced cell death and participated in tissue growth inhibition and maturation. The current study reveals the mechanisms by which blue light induces cell death in a time-dependent manner. Moreover, both the in vivo and NGS data uncovered blue light's effect on retinal tissue development, suggesting that exposing children to blue light could be relatively dangerous. These results could benefit the development of preventive strategies utilizing herbal medicine-based treatments for eye diseases or degeneration in the future.

Cyclodextrin-micellar electrokinetic chromatography of apolipoproteins on human very low-density lipoprotein.

The apolipoproteins (APOs) of human very low-density lipoprotein (VLDL) were investigated by an optimized cyclodextrin-micellar electrokinetic chromatography (CD-MEKC) method. The separation buffer consisted of 20 mM sodium phosphate, 40 mM bile salts (50% sodium cholate and 50% sodium deoxycholate), 25 mM carboxymethyl-ß-cyclodextrin (CM-ß-CD) (pH 7.0). For CD-MEKC separation, a sample injection time of 12 s, a separation voltage of 15 KV, and a capillary temperature of 15°C were chosen. The optimal CD-MEKC method showed good resolution and repeatability for VLDL APOs. Identification and quantitation of VLDL APOs CI, CIII, and E were based on comparison with human APO standards. Good linear relationships with correlation coefficient (R2 ) 0.99 were obtained for APOs CI, CIII, and E standards. For these three APOs, the linear ranges were within 0.01-0.54 mg/mL, and the concentration limits of detection (LODs) were lower than 0.02 mg/mL. Moreover, VLDL APOs from four uremic patients and four healthy subjects were compared. The uremic and healthy CD-MEKC profiles showed dramatic difference. The levels of APO CIII were significantly higher for two patients, and the level of APO E was significantly higher for one patient. This study might be helpful for following the disease development of uremia and cardiovascular disease (CVD) in the future.

Classification of the Multiple Stages of Parkinson's Disease by a Deep Convolution Neural Network Based on 99mTc-TRODAT-1 SPECT Images.

Single photon emission computed tomography (SPECT) has been employed to detect Parkinson's disease (PD). However, analysis of the SPECT PD images was mostly based on the region of interest (ROI) approach. Due to limited size of the ROI, especially in the multi-stage classification of PD, this study utilizes deep learning methods to establish a multiple stages classification model of PD. In the retrospective study, the 99mTc-TRODAT-1 was used for brain SPECT imaging. A total of 202 cases were collected, and five slices were selected for analysis from each subject. The total number of images was thus 1010. According to the Hoehn and Yahr Scale standards, all the cases were divided into healthy, early, middle, late four stages, and HYS I~V six stages. Deep learning is compared with five convolutional neural networks (CNNs). The input images included grayscale and pseudo color of two types. The training and validation sets were 70% and 30%. The accuracy, recall, precision, F-score, and Kappa values were used to evaluate the models' performance. The best accuracy of the models based on grayscale and color images in four and six stages were 0.83 (AlexNet), 0.85 (VGG), 0.78 (DenseNet) and 0.78 (DenseNet).

Effect of hyperthermia on improving neutrophil restoration after intraperitoneal chemotherapy.

Purpose: Intraperitoneal (IP) chemotherapy has several benefits but also can have severe hematologic side effects. We compared the effects of hyperthermic intraperitoneal chemotherapy (HIPEC) and conventional IP chemotherapy on bone marrow suppression and evaluated whether HIPEC increased neutrophil recovery.Methods: HIPEC or IP chemotherapy was administered to ovarian cancer-bearing mice. Bone marrow progenitor cell colony-forming unit (CFU) count, serum cytokine levels, and peripheral leukocyte count after HIPEC and IP chemotherapy were compared.Results: Peripheral neutrophil count, cytokine (G-CSF and CXCL1/KC) levels, and bone marrow progenitor cell CFU count were significantly higher after HIPEC than after IP chemotherapy.Conclusions: Hyperthermia increased the serum neutrophil-recruiting cytokine levels and reduced the magnitude of chemotherapy-induced neutropenia. Thus, HIPEC improved neutrophil and bone marrow recovery compared with conventional IP chemotherapy.

Feasible Classified Models for Parkinson Disease from 99mTc-TRODAT-1 SPECT Imaging.

The neuroimaging techniques such as dopaminergic imaging using Single Photon Emission Computed Tomography (SPECT) with 99mTc-TRODAT-1 have been employed to detect the stages of Parkinson's disease (PD). In this retrospective study, a total of 202 99mTc-TRODAT-1 SPECT imaging were collected. All of the PD patient cases were separated into mild (HYS Stage 1 to Stage 3) and severe (HYS Stage 4 and Stage 5) PD, according to the Hoehn and Yahr Scale (HYS) standard. A three-dimensional method was used to estimate six features of activity distribution and striatal activity volume in the images. These features were skewness, kurtosis, Cyhelsky's skewness coefficient, Pearson's median skewness, dopamine transporter activity volume, and dopamine transporter activity maximum. Finally, the data were modeled using logistic regression (LR) and support vector machine (SVM) for PD classification. The results showed that SVM classifier method produced a higher accuracy than LR. The sensitivity, specificity, PPV, NPV, accuracy, and AUC with SVM method were 0.82, 1.00, 0.84, 0.67, 0.83, and 0.85, respectively. Additionally, the Kappa value was shown to reach 0.68. This claimed that the SVM-based model could provide further reference for PD stage classification in medical diagnosis. In the future, more healthy cases will be expected to clarify the false positive rate in this classification model.

[Project to Decrease the Incidence of Neonatal Hypothermia in the Newborn Center].

BACKGROUND & PROBLEMS: An investigation found that 66.7% of the neonatal hypothermia (body temperature < 36.5°C) cases diagnosed within one hour of transfer from the delivery room in our hospital were affected by a significantly increased risk of physiological abnormalities, which subsequently increased their risk for mortality. Therefore, monitoring and maintaining the normal body temperature of newborn infants are vital in infant care. PURPOSE: This project aimed to improve the current situation of neonatal hypothermia. RESOLUTION: This project was implemented from Oct. 1, 2016 to Oct. 31, 2017 and used several approaches to improve neonatal hypothermia. A neonatal hypothermia caring protocol was developed and the infant admission materials were standardized; the infant hypothermia alert card and posters were displayed in easy-to-notice locations; an in-service training course on neonatal hypothermia was provided; and an infant hypothermia care checklist was tabulated for examination and recognition. RESULTS: After the implementation of this project, the average time required to raise the body temperature of infants to normal (36.5°C) was 1.5 hours, which was 2 hours faster than the pre-project time of 3.5 hours. Moreover, the time needed to raise the body temperature to normal was one hour for newborn infants with birthweights ≥ 2,500 grams, which was one hour faster than the pre-project time of two hours, and 1.5 hours for newborn infants with birthweights < 2,500 grams, which was three hours faster than the pre-project time of 4.5 hours. The goals of this project were effectively achieved in both groups. CONCLUSIONS: Neonatal hypothermia is an important issue affecting the health status of newborn infants. This project strengthened the awareness of nurses regarding neonatal hypothermia and is worthwhile to be implemented in clinical neonatal care.

A compartmentalized culture device for studying the axons of CNS neurons.

We report here the development of a compartmentalized culture device that allows the spatial separation of the somatodendrites and axons of central nervous system (CNS) neurons. The device consists of two compartments separated by a septum constructed by attaching a porous polycarbonate track etch (PCTE) filter on top of a microchannel-filled polydimethylsiloxane (PDMS) membrane. The surface and microchannels of the septum are coated and filled, respectively, with materials that support neuron growth and neurite migration. When rat hippocampal neurons are cultured in the top compartment, axons are the only processes that can migrate through the septum to the bottom compartment. The axons in the bottom compartment can be studied directly in real-time or through immunofluorescence staining after fixation. Axons containing ∼3 µg protein can be isolated from each device for biochemical analyses. In addition, the septum also impedes the movement of small molecules between the top and bottom compartments. This feature allows the somatodendrites and axons of neurons, which occupy the top and bottom compartments of the device, respectively, to be manipulated independently. The potential applications of the device as a tool in diverse studies concerning neuronal axons and in screening reagents that regulate axonal functions have also been discussed.

A 4-Phenoxyphenol Derivative Exerts Inhibitory Effects on Human Hepatocellular Carcinoma Cells through Regulating Autophagy and Apoptosis Accompanied by Downregulating α-Tubulin Expression.

Hepatocellular carcinoma (HCC) is a leading cancer worldwide. Advanced HCCs are usually resistant to anticancer drugs, causing unsatisfactory chemotherapy outcomes. In this study, we showed that a 4-phenoxyphenol derivative, 4-[4-(4-hydroxyphenoxy)phenoxy]phenol (4-HPPP), exerts an inhibitory activity against two HCC cell lines, Huh7 and Ha22T. We further investigated the anti-HCC activities of 4-HPPP, including anti-proliferation and induction of apoptosis. Our results showed that higher dosage of 4-HPPP downregulates the expression of α-tubulin and causes nuclear enlargement in both the Huh-7 and Ha22T cell lines. Interestingly, the colony formation results showed a discrepancy in the inhibitory effect of 4-HPPP on HCC and rat liver epithelial Clone 9 cells, suggesting the selective cytotoxicity of 4-HPPP toward HCC cells. Furthermore, the cell proliferation and apoptosis assay results illustrated the differences between the two HCC cell lines. The results of cellular proliferation assays, including trypan blue exclusion and colony formation, revealed that 4-HPPP inhibits the growth of Huh7 cells, but exerts less cytotoxicity in Ha22T cells. Furthermore, the annexin V assay performed for detecting the apoptosis showed similar results. Western blotting results showed 4-HPPP caused the increase of pro-apoptotic factors including cleaved caspase-3, Bid and Bax in HCC cells, especially in Huh-7. Furthermore, an increase of autophagy-associated protein microtubule-associated protein-1 light chain-3B (LC3B)-II and the decrease of Beclin-1 and p62/SQSTM1 were observed following 4-HPPP treatment. Additionally, the level of γH2A histone family, member X (γH2AX), an endogenous DNA damage biomarker, was dramatically increased in Huh7 cells after 4-HPPP treatment, suggesting the involvement of DNA damage pathway in 4-HPPP-induced apoptosis. On the contrary, the western blotting results showed that treatment up-regulates pro-survival proteins, including the phosphorylation of protein kinase B (Akt) and the level of survivin on Ha22T cells, which may confer a resistance toward 4-HPPP. Notably, the blockade of extracellular signal-regulated kinases (ERK), but not Akt, enhanced the cytotoxicity of 4-HPPP against Ha22T cells, indicating the pro-survival role of ERK in 4-HPPP-induced anti-HCC effect. Our present work suggests that selective anti-HCC activity of 4-HPPP acts through induction of DNA damage. Accordingly, the combination of ERK inhibitor may significantly enhance the anti-cancer effect of 4-HPPP for those HCC cells which overexpress ERK in the future.

Membrane-Bound Thrombomodulin Regulates Macrophage Inflammation in Abdominal Aortic Aneurysm.

OBJECTIVE: Thrombomodulin (TM), a glycoprotein constitutively expressed in the endothelium, is well known for its anticoagulant and anti-inflammatory properties. Paradoxically, we recently found that monocytic membrane-bound TM (ie, endogenous TM expression in monocytes) triggers lipopolysaccharide- and gram-negative bacteria-induced inflammatory responses. However, the significance of membrane-bound TM in chronic sterile vascular inflammation and the development of abdominal aortic aneurysm (AAA) remains undetermined. APPROACH AND RESULTS: Implicating a potential role for membrane-bound TM in AAA, we found that TM signals were predominantly localized to macrophages and vascular smooth muscle cells in human aneurysm specimens. Characterization of the CaCl2-induced AAA in mice revealed that during aneurysm development, TM expression was mainly localized in infiltrating macrophages and vascular smooth muscle cells. To investigate the function of membrane-bound TM in vivo, transgenic mice with myeloid- (LysMcre/TM(flox/flox)) and vascular smooth muscle cell-specific (SM22-cre(tg)/TM(flox/flox)) TM ablation and their respective wild-type controls (TM(flox/flox) and SM22-cre(tg)/TM(+/+)) were generated. In the mouse CaCl2-induced AAA model, deficiency of myeloid TM, but not vascular smooth muscle cell TM, inhibited macrophage accumulation, attenuated proinflammatory cytokine and matrix metalloproteinase-9 production, and finally mitigated elastin destruction and aortic dilatation. In vitro TM-deficient monocytes/macrophages, versus TM wild-type counterparts, exhibited attenuation of proinflammatory mediator expression, adhesion to endothelial cells, and generation of reactive oxygen species. Consistently, myeloid TM-deficient hyperlipidemic mice (ApoE(-/-)/LysMcre/TM(flox/flox)) were resistant to AAA formation induced by angiotensin II infusion, along with reduced macrophage infiltration, suppressed matrix metalloproteinase activities, and diminished oxidative stress. CONCLUSIONS: Membrane-bound TM in macrophages plays an essential role in the development of AAA by enhancing proinflammatory mediator elaboration, macrophage recruitment, and oxidative stress.

The Aggregation Pheromone of Phyllotreta striolata (Coleoptera: Chrysomelidae) Revisited.

Aggregations of the striped flea beetle Phyllotreta striolata on their crucifer host plants are mediated by volatiles emitted from feeding males. The male-specific sesquiterpene, (6R,7S)-himachala-9,11-diene (compound A), was shown previously to be physiologically and behaviorally active, but compound A was attractive only when combined with unnaturally high doses of the host plant volatile allyl isothiocyanate (AITC) in field trapping experiments. This indicated that our understanding of the chemical communication in this species is incomplete. Another male-specific sesquiterpenoid, (3S,9R,9aS)-3-hydroxy-3,5,5,9-tetramethyl-5,6,7,8,9,9a-hexahydro-1H-benzo[7]annulen-2(3H)-one (compound G), has been reported from an American P. striolata population. We confirmed the presence of compound G, and investigated its interaction with compound A and AITC in a P. striolata population in Taiwan. Compound G was attractive to Taiwanese P. striolata in laboratory bioassays, but significantly more beetles were attracted to a blend of compounds A and G. Under the same conditions, P. striolata showed no preference for the blend of A and G combined with a range of doses of AITC over the sesquiterpenoid blend alone. The sesquiterpenoid blend was tested further in field trapping experiments and attracted significantly more beetles than traps baited with compound A and ecologically relevant amounts of AITC. We conclude that A and G are components of the male-specific aggregation pheromone of P. striolata in Taiwan, and that the attractiveness of the pheromone is not reliant on the presence of AITC. Our results further indicate that the male-specific sesquiterpenoid blends differ qualitatively between the Taiwanese and American populations of P. striolata.

Role of the dorsal medial habenula in the regulation of voluntary activity, motor function, hedonic state, and primary reinforcement.

The habenular complex in the epithalamus consists of distinct regions with diverse neuronal populations. Past studies have suggested a role for the habenula in voluntary exercise motivation and reinforcement of intracranial self-stimulation but have not assigned these effects to specific habenula subnuclei. Here, we have developed a genetic model in which neurons of the dorsal medial habenula (dMHb) are developmentally eliminated, via tissue-specific deletion of the transcription factor Pou4f1 (Brn3a). Mice with dMHb lesions perform poorly in motivation-based locomotor behaviors, such as voluntary wheel running and the accelerating rotarod, but show only minor abnormalities in gait and balance and exhibit normal levels of basal locomotion. These mice also show deficits in sucrose preference, but not in the forced swim test, two measures of depression-related phenotypes in rodents. We have also used Cre recombinase-mediated expression of channelrhodopsin-2 and halorhodopsin to activate dMHb neurons or silence their output in freely moving mice, respectively. Optical activation of the dMHb in vivo supports intracranial self-stimulation, showing that dMHb activity is intrinsically reinforcing, whereas optical silencing of dMHb outputs is aversive. Together, our findings demonstrate that the dMHb is involved in exercise motivation and the regulation of hedonic state, and is part of an intrinsic reinforcement circuit.