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1.  Compartmentalization of β-Adrenergic Signals in Cardiomyocytes 
Trends in cardiovascular medicine  2013;23(7):250-256.
Under β-adrenergic stimulation, the distribution of cAMP is highly restricted at distinct intracellular domains for compartmentalized activation of protein kinase A, which promotes selective phosphorylation of proteins for contractile responses in cardiomyocytes. This is primarily due to a concert effort between restrictions of cAMP distribution by a family of phosphodiesterases and locally anchored protein kinase A by a family of scaffold A kinase-anchoring proteins. Moreover, these regulatory mechanisms underlie cross talk between β̃ adrenergic signals and other receptor stimulated signaling cascades, which alters the compartmentalized β̃ adrenergic signals for proper contractility in myocardium. Maintaining integrity of compartmentalized β-adrenergic signals is critical for physiological cardiac function and for preventing development of cardiac diseases.
PMCID: PMC4264830  PMID: 23528751
2.  Variants of Uncertain Significance in BRCA Testing: Evaluation of Surgical Decisions, Risk Perception, and Cancer Distress 
Clinical genetics  2013;84(5):464-472.
Studies suggest that patients carrying a BRCA variant of uncertain significance (VUS) may have lingering confusion concerning results interpretation. Counseling for uninformative BRCA-negative (UN) results is thought to be more straightforward, despite the fact that both results lead to similar methods of empiric cancer risk counseling. This study compared surgical choices and perceptions between 71 patients with VUS results and 714 patients with UN results. All patients underwent genetic counseling because of a personal or family history of breast or ovarian cancer between 1997 and 2010, and completed a two-year follow-up survey. Risk-reducing mastectomy rates in both groups were 7% (p=1.00) and risk-reducing oophorectomy rates were 5% and 3%, respectively (p=0.42). The VUS group reported less cancer distress reduction than the UN group (23.0% versus 35.8%, respectively, p=.043). Over 90% of both groups found the counseling process helpful. Overall, the study suggests that VUS results disclosed in genetic counseling did not cause excessive surgery or exaggerated cancer distress, though patients with a VUS found counseling somewhat less informative or reassuring. Future research on communication of VUS results, including pre-and post-test counseling, is essential for full realization of the potential for genomic medicine.
PMCID: PMC3751990  PMID: 23323793
BRCA1 gene; BRCA2 gene; genetic counseling; hereditary breast and ovarian cancer syndrome; mutation missense; variant of uncertain significance
3.  Integrin-Mediated Interactions with Extracellular Matrix Proteins for Nucleus Pulposus Cells of the Human Intervertebral Disc 
The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin-mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM-111 and LM-511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM-binding integrins was quantified for human NP cells using flow cytometry. The cell-ECM attachment mechanism was determined by quantifying cell attachment to LM-111, LM-511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3 and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM-111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival and biosynthesis in cell-based therapeutics.
PMCID: PMC3826265  PMID: 23737292
intervertebral disc; nucleus pulposus; integrin; extracellular matrix; human
4.  Mechanistic Modeling Reveals the Critical Knowledge Gaps in Bile Acid–Mediated DILI 
Bile salt export pump (BSEP) inhibition has been proposed to be an important mechanism for drug-induced liver injury (DILI). Modeling can prioritize knowledge gaps concerning bile acid (BA) homeostasis and thus help guide experimentation. A submodel of BA homeostasis in rats and humans was constructed within DILIsym, a mechanistic model of DILI. In vivo experiments in rats with glibenclamide were conducted, and data from these experiments were used to validate the model. The behavior of DILIsym was analyzed in the presence of a simulated theoretical BSEP inhibitor. BSEP inhibition in humans is predicted to increase liver concentrations of conjugated chenodeoxycholic acid (CDCA) and sulfate-conjugated lithocholic acid (LCA) while the concentration of other liver BAs remains constant or decreases. On the basis of a sensitivity analysis, the most important unknowns are the level of BSEP expression, the amount of intestinal synthesis of LCA, and the magnitude of farnesoid-X nuclear receptor (FXR)-mediated regulation.
PMCID: PMC4120015  PMID: 25006780
5.  Functional RIG-I-like receptors control the survival of mesenchymal stem cells 
Cell Death & Disease  2013;4(12):e967-.
Because of their potent regenerative and immunomodulatory properties, mesenchymal stem cells (MSCs) have promising therapeutic benefits in clinical treatment of inflammatory and infectious diseases. Recent studies suggest that many biological activities of MSCs are largely determined by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs). However, the role of PRRs in regulating the survival of MSCs remains unknown. In the present study, we examined the viability of MSCs after stimulation of distinct PRRs. Activation of TLRs by direct addition with their respective ligands showed no significant effect on the survival of MSCs, whereas transfection with double-stranded RNA (dsRNA) resulted in marked cell death in MSCs. Transfection of dsRNA upregulated cytosolic retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I and melanoma differentiation-associated antigen 5 (MDA5). Moreover, transfection of dsRNA activated downstream transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB), as well as induced the expression of interferon-β (IFN-β) and pro-inflammatory cytokine interleukin 6 (IL-6) via RLR signaling. Furthermore, we found that transfection of dsRNA triggered both extrinsic and intrinsic apoptotic responses via RLRs. However, ectopic expression of RIG-I or MDA5 was not sufficient to induce apoptosis of MSCs without dsRNA transfection. Our study also revealed that IκB kinase α/β (IKKα/β) was required for RLR-mediated apoptosis in MSCs, while TANK-binding kinase 1 (TBK1)/IKKɛ served a pro-survival role. Moreover, neither overexpression of B-cell lymphoma 2 (Bcl2) nor neutralizing autocrined IFN-β reduced RLR-mediated apoptosis. In addition, autophagy was induced upon activation of RLRs, however, blocking autophagy did not rescue MSCs from the dsRNA-induced cell death. To the best of our knowledge, this is the first study to explore the role of RLRs in controlling the survival of MSCs, which may provide a clue to understand the pathogenesis of viral infection in MSCs.
PMCID: PMC3877571  PMID: 24336087
mesenchymal stem cells; RIG-I-like receptors; cell survival; apoptosis; autophagy
6.  EUS compared with endoscopy plus transabdominal US in the initial diagnostic evaluation of patients with upper abdominal pain 
Gastrointestinal endoscopy  2010;72(5):967-974.
Primary upper endoscopy (EGD) and transabdominal US (TUS) are often performed in patients with upper abdominal pain.
Primary: Determine whether the combination of EGD and EUS was equivalent to EGD plus TUS in the diagnostic evaluation of upper abdominal pain. Secondary: Compare EUS versus TUS in detecting abdominal lesions, and compare EGD by using an oblique-viewing echoendoscope versus the standard, forward-viewing endoscope in detecting mucosal lesions.
Prospective, paired design.
Six academic endoscopy centers.
This study involved patients with upper abdominal pain referred for endoscopy.
All patients had EGD, EUS, and TUS. The EGD was done using both an oblique-viewing echoendoscope and the standard, forward-viewing endoscope (randomized order) by two separate endoscopists in a blinded fashion, followed by EUS. TUS was performed within 4 weeks of EGD/EUS, also in a blinded fashion. Follow-up: telephone interviews and chart reviews.
Main Outcome Measurements
Diagnose possible etiology of upper abdominal pain and detect clinically significant lesions.
A diagnosis of the etiology of upper abdominal pain was made in 66 of 172 patients (38%). The diagnostic rate was 42 of 66 patients (64%) for EGD plus EUS versus 41 of 66 patients (62%) for EGD plus TUS, which was statistically equivalent (McNemar test; P = .27). One hundred ninety-eight lesions were diagnosed with either EUS or TUS. EUS was superior to TUS for visualizing the pancreas (P < .0001) and for diagnosing chronic pancreatitis (P = .03). Two biliary stones were detected only by EUS. Two hundred fifty-one mucosal lesions were similarly diagnosed with EGD with either the standard, forward-viewing endoscope or the oblique-viewing echoendoscope (kappa = 0.48 [95% CI, .43-.54]). EGD with the standard, forward-viewing endoscope was preferred for biopsies.
No cost analysis.
The combination of EGD with EUS is equivalent to EGD plus TUS for diagnosing a potential etiology of upper abdominal pain. EUS is superior to TUS for detecting chronic pancreatitis. EGD combined with EUS should be considered in the first-line diagnostic evaluation of patients with upper abdominal pain.
PMCID: PMC3775486  PMID: 20650452
7.  Effect of Oxygen on Phosphodiesterases (PDE) 3 and 4 Isoforms and PKA Activity in the Superior Cervical Ganglia 
The cAMP-protein kinase A (PKA) signaling pathway is involved in regulating the release of transmitters from neurons and other cells. Multiple phosphodiesterase (PDE) isoforms regulate this pathway, however, the pattern of isoform expression and stimulus response across tissues has not been fully characterized.
Using fluorescent resonance energy transfer (FRET)-based imaging in primary superior cervical ganglia (SCG) neurons and real-time qPCR, we explored the role of PDE3 and PDE4 isoforms and oxygen tension in the activation of PKA and changes in gene expression. These primary neurons were infected with an adenovirus containing A-Kinase activity reporter (AKAR3) and assayed for responses to PDE inhibitors: rolipram (ROL, 1 μM), milrinone (MIL, 10 μM) and IBMX (100 μM), and adenylyl cyclase activator forskolin (FSK, 50 m M). Different PDE activity patterns were observed in different cells: high PDE4 activity (n = 3), high PDE3 activity (n = 3) and presence of activity of other PDEs (n = 3). Addition of PKA inhibitor H89 (10 μM) completely reversed the response. We further studied the effect of oxygen in the PKA activity induced by PDE inhibition. Both normoxia (20%O2/5%CO2) and hypoxia (0%O2/5%CO2) induced a similar increase in the FRET emission ratio (14.5 ± 0.8 and 14.7 ± 0.8, respectively).
PDE3a, PDE4b and PDE4d isoforms mRNAs were highly expressed in the whole SCG with no modulation by hypoxia.
Conclusion: Using a FRET-based PKA activity sensor, we show that primary SCG neurons can be used as a model system to dissect the contribution of different PDE isoforms in regulating cAMP/PKA signaling. The differential patterns of PDE regulation potentially represent subpopulations of ganglion cells with different physiological functions.
PMCID: PMC3715042  PMID: 23080174
Superior cervical ganglia (SCG); Phosphodiesterases (PDE); Cyclic AMP (cAMP); Hypoxia; Protein kinase A (PKA)
8.  SAP97 Controls the Trafficking and Resensitization of the Beta-1-Adrenergic Receptor through Its PDZ2 and I3 Domains 
PLoS ONE  2013;8(5):e63379.
Previous studies have determined that the type-1 PDZ sequence at the extreme carboxy-terminus of the ß1-adrenergic receptor (ß1-AR) binds SAP97 and AKAP79 to organize a scaffold involved in trafficking of the ß1-AR. In this study we focused on characterizing the domains in SAP97 that were involved in recycling and resensitization of the ß1-AR in HEK-293 cells. Using a SAP97 knockdown and rescue strategy, we determined that PDZ-deletion mutants of SAP97 containing PDZ2 rescued the recycling and resensitization of the ß1-AR. Among the three PDZs of SAP97, PDZ2 displayed the highest affinity in binding to the ß1-AR. Expression of isolated PDZ2, but not the other PDZs, inhibited the recycling of the ß1-AR by destabilizing the macromolecular complex involved in trafficking and functional resensitization of the ß1-AR. In addition to its PDZs, SAP97 contains other protein interacting domains, such as the I3 sequence in the SRC homology-3 (SH3) domain, which binds to AKAP79. Deletion of I3 from SAP97 (ΔI3-SAP97) did not affect the binding of SAP97 to the ß1-AR. However, ΔI3-SAP97 could not rescue the recycling of the ß1-AR because it failed to incorporate AKAP79/PKA into the SAP97-ß1-AR complex. Therefore, bipartite binding of SAP97 to the ß1-AR and to AKAP79 is necessary for SAP97-mediated effects on recycling, externalization and functional resensitization of the ß1-AR. These data establish a prominent role for PDZ2 and I3 domains of SAP97 in organizing the ß1-adrenergic receptosome involved in connecting the ß1-AR to trafficking and signaling networks.
PMCID: PMC3656048  PMID: 23696820
9.  Single molecule pull-down for studying protein interactions 
Nature protocols  2012;7(3):445-452.
This protocol describes a single molecule pull-down (SiMPull) assay for analyzing physiological protein complexes. The assay combines the conventional pull-down assay with single molecule total internal reflection fluorescence microscopy, and allows probing single macromolecular complexes directly from cell or tissue extracts. In this method, antibodies against the protein of interest are immobilized on a passivated microscope slide. When cell extracts are applied, the surface-tethered antibody captures the protein together with its physiological interaction partners. After washing away the unbound components, single molecule fluorescence microscopy is used to probe the pulled down proteins. Captured proteins are visualized through genetically encoded fluorescent protein tags or through antibody labeling. This ultra-sensitive assay requires at least 10-fold less reagents, is significantly faster and provides quantitative data compared to western blot analysis. Furthermore, SiMPull can distinguish between multiple association states of the same protein. SiMPull is generally applicable to proteins from a variety of cellular contexts and to endogenous proteins. Starting with the cell extracts and passivated slides, the assay requires 1.5 – 2.5 hours for data acquisition and analysis.
PMCID: PMC3654178  PMID: 22322217
protein-protein interaction; single molecule; immunoprecipitation; co-immunoprecipitation pull-down
10.  Isoform-specific palmitoylation of JNK regulates axonal development 
Cell Death and Differentiation  2011;19(4):553-561.
The c-jun N-terminal kinase (JNK) proteins are encoded by three genes (Jnk1–3), giving rise to 10 isoforms in the mammalian brain. The differential roles of JNK isoforms in neuronal cell death and development have been noticed in several pathological and physiological contexts. However, the mechanisms underlying the regulation of different JNK isoforms to fulfill their specific roles are poorly understood. Here, we report an isoform-specific regulation of JNK3 by palmitoylation, a posttranslational modification, and the involvement of JNK3 palmitoylation in axonal development and morphogenesis. Two cysteine residues at the COOH-terminus of JNK3 are required for dynamic palmitoylation, which regulates JNK3's distribution on the actin cytoskeleton. Expression of palmitoylation-deficient JNK3 increases axonal branching and the motility of axonal filopodia in cultured hippocampal neurons. The Wnt family member Wnt7a, a known modulator of axonal branching and remodelling, regulates the palmitoylation and distribution of JNK3. Palmitoylation-deficient JNK3 mimics the effect of Wnt7a application on axonal branching, whereas constitutively palmitoylated JNK3 results in reduced axonal branches and blocked Wnt7a induction. Our results demonstrate that protein palmitoylation is a novel mechanism for isoform-specific regulation of JNK3 and suggests a potential role of JNK3 palmitoylation in modulating axonal branching.
PMCID: PMC3307970  PMID: 21941371
c-Jun N-terminal kinase/JNK; palmitoylation; axonal branching; isoform regulation; cytoskeleton; Wnt pathway
11.  A Multicenter, Prospective Study of a New Fully Covered Expandable Metal Biliary Stent for the Palliative Treatment of Malignant Bile Duct Obstruction 
Background and Study Aims. Endoscopic placement of self-expanding metal stents (SEMSs) is indicated for palliation of inoperable malignant biliary obstruction. A fully covered biliary SEMS (WallFlex Biliary RX Boston Scientific, Natick, USA) was assessed for palliation of extrahepatic malignant biliary obstruction. Patients and Methods. 58 patients were included in this prospective, multicenter series conducted under an FDA-approved IDE. Main outcome measurements included (1) absence of stent occlusion within six months or until death, whichever occurred first and (2) technical success, need for reintervention, bilirubin levels, stent patency, time to stent occlusion, and adverse events. Results. Technical success was achieved in 98% (57/58), with demonstrated acute removability in two patients. Adequate clinical palliation until completion of followup was achievedin 98% (54/55) of evaluable patients, with 1 reintervention due to stent obstruction after 142 days. Mean total bilirubin decreased from 8.9 mg/dL to 1.2 mg/dL at 1 month. Device-related adverse events were limited and included 2 cases of cholecystitis. One stent migrated following radiation therapy. Conclusions. The WallFlex Biliary fully covered stent yielded technically successful placement with uncomplicated acute removal where required, appropriate reduction in bilirubin levels, and low rates of stent migration and occlusion. This SEMS allows successful palliation of malignant extrahepatic biliary obstruction.
PMCID: PMC3623117  PMID: 23606835
12.  Controlled permeation of cell membrane by single bubble acoustic cavitation 
Journal of Controlled Release  2011;157(1):103-111.
Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustic, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency (7.44 MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5 MHz) ultrasound pulse (duration 13.3 or 40 µs) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d = 0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106 ± 0.032 µm (n = 70) for acoustic pressure of 1.5 MPa (duration 13.3 µs), and increased to 0.171 ± 0.030 µm (n = 125) for acoustic pressure of 1.7 MPa and to 0.182 ± 0.052 µm (n=112) for a pulse duration of 40 µs (1.5 MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level.
PMCID: PMC3258473  PMID: 21945682
Sonoporation; ultrasound; intracellular delivery; membrane permeability; video-microscopy; microbubbles; cavitation
13.  Microwave-assisted Low-temperature Growth of Thin Films in Solution 
Scientific Reports  2012;2:1003.
Thin films find a variety of technological applications. Assembling thin films from atoms in the liquid phase is intrinsically a non-equilibrium phenomenon, controlled by the competition between thermodynamics and kinetics. We demonstrate here that microwave energy can assist in assembling atoms into thin films directly on a substrate at significantly lower temperatures than conventional processes, potentially enabling plastic-based electronics. Both experimental and electromagnetic simulation results show microwave fields can selectively interact with a conducting layer on the substrate despite the discrepancy between the substrate size and the microwave wavelength. The microwave interaction leads to localized energy absorption, heating, and subsequent nucleation and growth of the desired films. Electromagnetic simulations show remarkable agreement with experiments and are employed to understand the physics of the microwave interaction and identify conditions to improve uniformity of the films. The films can be patterned and grown on various substrates, enabling their use in widespread applications.
PMCID: PMC3525940  PMID: 23256037
14.  Compartmentalization of Beta-Adrenergic Signals in Cardiomyocytes 
Circulation research  2011;109(2):231-244.
Activation of adrenergic receptors (ARs) represents the primary mechanism to increase cardiac performance under stress. Activated βARs couple to Gs proteins, leading to adenylyl cyclase (AC)-dependent increases in secondary-messenger cyclic adenosine monophosphate (cAMP) to activate activation of protein kinase A (PKA). The increased PKA activities promote phosphorylation of diversified substrates ranging from the receptor and its associated partners, to proteins involved in increases in contractility and heart rate. Recent progress with live-cell imaging has drastically advanced our understanding of the βAR-induced cAMP and PKA activities that are precisely regulated in a spatiotemporal fashion in highly differentiated myocytes. Several features stand out: membrane location of βAR and its associated complexes dictates the cellular compartmentalization of signaling; βAR agonist dose-dependent equilibrium between cAMP production and cAMP degradation shapes persistent increases in cAMP signals for sustained cardiac contraction response; and arrestin acts as an agonist dose-dependent master switch to promote cAMP diffusion and propagation into intracellular compartments by sequestrating phosphodiesterase (PDE) isoforms associated with the βAR signaling cascades. These features and the underlying molecular mechanisms of the dynamic regulation of βAR complexes with AC and PDE enzymes and the implication in heart failure will be discussed.
PMCID: PMC3493619  PMID: 21737818
adrenergic receptor; phosphodiesterase; cAMP; protein kinase A
15.  Gαs-Biased β2-Adrenergic Receptor Signaling from Restoring Synchronous Contraction in the Failing Heart 
Science Translational Medicine  2011;3(100):100ra88.
Cardiac resynchronization therapy (CRT), in which both ventricles are paced to recoordinate contraction in hearts that are dyssynchronous from conduction delay, is the only heart failure (HF) therapy to date to clinically improve acute and chronic function while also lowering mortality. CRT acutely enhances chamber mechanical efficiency but chronically alters myocyte signaling, including improving β-adrenergic receptor reserve. We speculated that the latter would identify unique CRT effects that might themselves be effective for HF more generally. HF was induced in dogs by 6 weeks of atrial rapid pacing with (HFdys, left bundle ablated) or without (HFsyn) dyssynchrony. We used dyssynchronous followed by resynchronized tachypacing (each 3 weeks) for CRT. Both HFdys and HFsyn myocytes had similarly depressed rest and β-adrenergic receptor sarcomere and calcium responses, particularly the β2-adrenergic response, whereas cells subjected to CRT behaved similarly to those from healthy controls. CRT myocytes exhibited suppressed Gαi signaling linked to increased regulator of G protein (heterotrimeric guanine nucleotide–binding protein) signaling (RGS2, RGS3), yielding Gαs-biased β2-adrenergic responses. This included increased adenosine cyclic AMP responsiveness and activation of sarcoplasmic reticulum–localized protein kinase A. Human CRT responders also showed up-regulated myo-cardial RGS2 and RGS3. Inhibition of Gαi (with pertussis toxin, RGS3, or RGS2 transfection), stimulation with a Gαs-biased β2 agonist (fenoterol), or transient (2-week) exposure to dyssynchrony restored β-adrenergic receptor responses in HFsyn to the values obtained after CRT. These results identify a key pathway that is triggered by restoring contractile synchrony and that may represent a new therapeutic approach for a broad population of HF patients.
PMCID: PMC3329869  PMID: 21918105
16.  Palmitoylation Regulates Intracellular Trafficking of β2 Adrenergic Receptor/Arrestin/Phosphodiesterase 4D Complexes in Cardiomyocytes 
PLoS ONE  2012;7(8):e42658.
β2 adrenergic receptor (β2AR) is a prototypical G-protein coupled receptor that stimulates the classic cAMP-protein kinase A (PKA) signaling pathway. Recent studies indicate that the cAMP-PKA activities are spatiotemporally regulated in part due to dynamic association of β2AR with phosphodiesterase 4D (PDE4D), a group of cAMP degradation enzymes. Here, we demonstrate that in cardiomyocytes, palmitoylation of β2AR, the covalent acylation of cysteine residue 341, plays a critical role in shaping subcellular cAMP-PKA activities in cardiomyocytes via regulating β2AR association with arrestin/PDE4D. Replacing cysteine 341 on β2AR with alanine (C341A) leads to an impaired binding to β arrestin 2. Surprisingly, the C341A mutant is able to internalize via an arrestin-independent pathway at saturated concentration of agonist stimulation; the internalization becomes caveolae-dependent and requires dynamin GTPase. However, the impaired binding to β arrestin 2 also leads to an impaired recruitment of PDE4D to the C341A mutant. Thus, the mutant C341A β2AR is transported alone from the plasma membrane to the endosome without recruiting PDE4D. This alteration leads to an enhanced cytoplasmic cAMP signal for PKA activation under β2AR stimulation. Functionally, Mutation of the C341 residue or inhibition of palmitoylation modification of β2AR enhances the receptor-induced PKA activities in the cytoplasm and increases in myocyte contraction rate. Our data reveal a novel function of palmitoylation in shaping subcellular cAMP-PKA signaling in cardiomyocytes via modulating the recruitment of β arrestin 2-PDE4D complexes to the agonist-stimulated β2AR.
PMCID: PMC3415400  PMID: 22912718
17.  Pressure-induced amorphous-to-amorphous configuration change in Ca-Al metallic glasses 
Scientific Reports  2012;2:376.
Pressure-induced amorphous-to-amorphous configuration changes in Ca-Al metallic glasses (MGs) were studied by performing in-situ room-temperature high-pressure x-ray diffraction up to about 40 GPa. Changes in compressibility at about 18 GPa, 15.5 GPa and 7.5 GPa during compression are detected in Ca80Al20, Ca72.7Al27.3, and Ca66.4Al33.6 MGs, respectively, whereas no clear change has been detected in the Ca50Al50 MG. The transfer of s electrons into d orbitals under pressure, reported for the pressure-induced phase transformations in pure polycrystalline Ca, is suggested to explain the observation of an amorphous-to-amorphous configuration change in this Ca-Al MG system. Results presented here show that the pressure induced amorphous-to-amorphous configuration is not limited to f electron-containing MGs.
PMCID: PMC3332524  PMID: 22530094
18.  Differences in biofilm formation and antimicrobial resistance of Pseudomonas aeruginosa isolated from airways of mechanically ventilated patients and cystic fibrosis patients 
Pseudomonas aeruginosa biofilms exhibit increased antimicrobial resistance compared with planktonic isolates and are implicated in the pathogenesis of both acute and chronic lung infections. Whilst antibiotic choices for both infections are based on planktonic antibiotic susceptibility results, differences in biofilm-forming ability between the two diseases have not previously been explored. The aim of this study was to compare differences in biofilm formation and antibiotic resistance of P. aeruginosa isolated from intubated patients and from patients with chronic pulmonary disease associated with cystic fibrosis (CF). The temporal evolution of antibiotic resistance in clonal P. aeruginosa strains isolated from CF patients during periods of chronic infection and acute pulmonary exacerbation was also evaluated. Biofilm formation and biofilm antibiotic susceptibilities were determined using a modified microtitre plate assay and were compared with antibiotic susceptibility results obtained using traditional planktonic culture. Clonality was confirmed using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Pseudomonas aeruginosa isolates collected from intubated patients produced substantially more biofilms compared with CF isolates. There was considerable heterogeneity in biofilm-forming ability among the CF isolates and this was unrelated to pulmonary status. Biofilm antibiotic resistance developed rapidly among clonal CF isolates over time, whilst traditional antibiotic resistance determined using planktonic cultures remained stable. There was a significant positive correlation between imipenem/cilastatin and ceftazidime resistance and biofilm-forming ability. The variability in biofilm-forming ability in P. aeruginosa and the rapid evolution of biofilm resistance may require consideration when choosing antibiotic therapy for newly intubated patients and CF patients.
PMCID: PMC3176759  PMID: 21382698
Pseudomonas aeruginosa; Bacterial biofilm; Antimicrobial resistance; Mechanical ventilation; Cystic fibrosis
19.  Bio-functionalized nanoneedles for the direct and site-selective delivery of probes into living cells 
Biochimica et biophysica acta  2010;1810(3):330-338.
Accessing the interior of live cells with minimal intrusiveness for visualizing, probing and interrogating biological processes has been the ultimate goal of much of the biological experimental development.
Scope of Review
The recent development and use of the bio-functionalized nanoneedles for local and spatially-controlled intracellular delivery brings in exciting new opportunities in accessing the interior of living cells. Here we review the technical aspect of this relatively new intracellular delivery method and the related demonstrations and studies, and provide our perspectives on the potential wide applications of this new nanotechnology-based tool in the biological field, especially on its use for high resolution studies of biological processes in living cells.
Major Conclusions
Different from the traditional micropipette-based needles for intracellular injection, a nanoneedle deploys a sub-100 nm diameter solid nanowire as a needle to penetrate a cell membrane and to transfer and deliver the biological cargo conjugated onto its surface to the target regions inside a cell. Although the traditional micropipette-based needles can be more efficient in delivery biological cargoes, a nanoneedle-based delivery system offers an efficient introduction of biomolecules into living cells with high spatiotemporal resolution but minimal intrusion and damage. It offers a potential solution to quantitatively address biological processes at the nanoscale.
General significance
The nanoneedle-based cell delivery system provides new possibilities for efficient, specific, and precise introduction of biomolecules into living cells for high resolution studies of biological processes, and it has potential application in addressing broad biological questions.
PMCID: PMC2948073  PMID: 20580773
20.  FRET-based direct detection of dynamic protein kinase A activity on the sarcoplasmic reticulum in cardiomyocytes 
The second messenger cAMP-dependent protein kinase A (PKA) plays an important role in the various cellular and physiological responses. On the sarcoplasmic reticulum (SR) in cardiomyocytes, PKA regulates the calcium cycling for exciting-contraction coupling, which is often dysfunctional in a variety of heart diseases including heart failure. Here, we have developed a novel FRET-based A-kinase activity biosensor (AKAR), termed SR-AKAR3, to visualize the PKA dynamics on the SR. Activation of adrenergic receptor induces a rapid and significant increase in SR-AKAR3 FRET ratio, which is dependent on agonist occupation of the receptor and inhibited by H-89, a PKA inhibitor. Interestingly, direct activation of adenylyl cyclases or application of a cAMP analog 8-br-cAMP induced much slower and smaller increases in SR-AKAR3 FRET ratio. These data indicate that the signaling induced by adrenergic stimulation displays a preferential access to the SR in comparison to those by direct activation of adenylyl cyclases. More, SR-AKAR3 mimics endogenous protein phospholamban on the SR for PKA-mediated phosphorylation and myocyte contraction response under adrenergic stimulation. Together, this new PKA activity biosensor provides a useful tool to directly visualize the dynamic regulation of PKA activity on the SR in cardiomyocytes under various physiological and clinical conditions.
PMCID: PMC3025313  PMID: 21130738
21.  A novel radioresistant mechanism of galectin-1 mediated by H-Ras-dependent pathways in cervical cancer cells 
Cell Death & Disease  2012;3(1):e251-.
Galectin-1 is a lectin recognized by galactoside-containing glycoproteins, and is involved in cancer progression and metastasis. The role of galectin-1 in radiosensitivity has not previously been investigated. Therefore, this study tests whether galectin-1 is involved in the radiosensitivity mediated by the H-Ras signaling pathway using cervical carcinoma cell lines. A knockdown of galectin-1 expression in HeLa cells decreased clonogenic survival following irradiation. The clonogenic survival increased in both HeLa and C33A cells with galectin-1 overexpression. The overexpression or knockdown of galectin-1 did not alter radiosensitivity, whereas H-Ras was silenced in both cell lines. Whereas K-Ras was knocked down, galectin-1 restored the radiosensitivity in HeLa cells and C33A cells. The knockdown of galectin-1 increased the high-dose radiation-induced cell death of HeLa cells transfected by constitutively active H-Ras. The knockdown of galectin-1 inhibited the radiation-induced phosphorylation of Raf-1 and ERK in HeLa cells. Overexpression of galectin-1 enhanced the phosphorylation of Raf-1 and ERK in C33A cells following irradiation. Galectin-1 decreased the DNA damage detected using comet assay and γ-H2AX in both cells following irradiation. These findings suggest that galectin-1 mediates radioresistance through the H-Ras-dependent pathway involved in DNA damage repair.
PMCID: PMC3270263  PMID: 22237208
galectin-1; cervical cancer; radiosensitivity; radioresistance; H-Ras
22.  Probing Cellular Protein Complexes via Single Molecule Pull-down 
Nature  2011;473(7348):484-488.
Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here, we describe a single molecule pull-down (SiMPull) assay that combines the principles of conventional pull-down assay with single molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signaling proteins found in cytosol, membrane, and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled down proteins are functional and are used, without further processing, for single molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analyzing protein assemblies in biological pathways.
PMCID: PMC3103084  PMID: 21614075
Neuroscience  2010;171(4):1341-1356.
Hyperalgesia in animal injury models is linked to activation of descending raphespinal modulatory circuits originating in the rostral ventromedial medulla (RVM). A neurokinin-1 (NK-1) receptor antagonist microinjected into the RVM before or after inflammation produced by complete Freund’s adjuvant (CFA) resulted in an attenuation of thermal hyperalgesia. A transient (acute) or a continuous infusion of Substance P (SP) microinjected into the RVM of non-inflamed animals led to similar pain hypersensitivity. Intrathecal pretreatment or post-treatment of a 5-HT3 receptor antagonist (Y-25130 or ondansetron) blocked the SP-induced hyperalgesia. The SP-induced hyperalgesia was both GABAA and NMDA receptor-dependent after pre- and post-treatment with selective antagonists at the spinal level. A microinjection of SP into the RVM also led to increased NMDA NR1 receptor subunit phosphorylation in spinal cord tissue. The GABAA receptor-mediated hyperalgesia involved a shift in the anionic gradient in dorsal horn nociceptive neurons and an increase in phosphorylated NKCC1 protein (isoform of the Na-K-Cl cotransporter). Following a low dose of SP infused into the RVM, intrathecal muscimol (GABAA agonist) increased SP-induced thermal hyperalgesia, phosphorylated NKCC1 protein expression, and NMDA NR1 subunit phosphorylation in the spinal cord. The thermal hyperalgesia was blocked by intrathecal gabazine, the GABAA receptor antagonist, and MK-801, the NMDA receptor channel blocker. These findings indicate that NK-1 receptors in the RVM are involved in SP-induced thermal hyperalgesia, this hyperalgesia is 5-HT3-receptor dependent at the spinal level, and involves the functional interaction of spinal GABAA and NMDA receptors.
PMCID: PMC3006078  PMID: 20888891
substance P; inflammation; brainstem; serotonin; NMDA receptor; GABAA receptor
24.  Contribution of Primary Afferent Input to Trigeminal Astroglial Hyperactivity, Cytokine Induction and NMDA Receptor Phosphorylation 
The open pain journal  2010;2010(3):144-152.
We tested the hypothesis that primary afferent inputs play a role in astroglial hyperactivity after tissue injury. We first injected complete Freund’s adjuvant (CFA, 0.05 ml, 1:1 oil/saline) into the masseter muscle, which upregulated glial fibrillary acidic protein (GFAP), a marker of astrocytes, interleukin (IL)-1β an inflammatory cytokine, and phosphorylation of serine896 of the NR1 subunit (P-NR1) of the NMDA receptor in the subnuclei interpolaris/caudalis (Vi/Vc) transition zone, an important structure for processing trigeminal nociceptive input. Local anesthetic block with lidocaine (2%) of the masseter muscle at 10 min prior to injection of CFA into the same site significantly reduced the CFA-induced increase in GFAP, IL-1β and P-NR1 (p<0.05, n=4/group). We then tested the effect of peripheral electrical stimulation (ES). The ES protocol was burst stimulation consisting of trains of 4 square pulses (10–100 Hz, 0.1–3 mA, 0.5 ms pulse width). Under pentobarbital anesthesia, an ES was delivered every 0.2 s for a total of 30 min. The Vi/Vc tissues were processed for immunohistochemistry or western blot analysis at 10–120 min after ES. Compared to naive and SHAM-treated rats, there was increased immunoreactivity against GFAP, IL-1β and P-NR1 in the Vi/Vc in rats receiving ES. Double staining showed that IL-1β was selectively localized in GFAP-positive astroglia, and P-NR1-immunoreactivity was localized to neurons. These findings indicate that primary afferent inputs are necessary and sufficient to induce astroglial hyperactivity and upregulation of IL-1β, as well as neuronal NMDA receptor phosphorylation.
PMCID: PMC3002905  PMID: 21170295
Electrical stimulation; masseter nerve; astroglia; GFAP; IL-1β; neuron-glial interaction
25.  Are they relevant? A critical evaluation of the international classification of functioning, disability, and health core sets for osteoarthritis from the perspective of patients with knee osteoarthritis in Singapore 
Annals of the Rheumatic Diseases  2006;65(8):1067-1073.
To determine the extent to which health items identified from the perspective of patients with knee osteoarthritis can be linked with the International Classification of Functioning, Disability and Health (ICF); and to evaluate critically the content validity of ICF comprehensive and brief core sets for osteoarthritis.
Items identified from a focus group study were linked independently by two researchers based on the 10 a priori linking rules. Both percentage agreement and κ statistics were calculated to measure interobserver agreement. Any disagreements were resolved by reaching a consensus among the researchers. The categories linked with all items were compared with the comprehensive core set, while the categories linked with those items reported as important by over 30% of subjects within each of three local ethnic groups (Chinese, Malay, and Indian) were compared with the brief core set. Both comparisons were made only at the second level of the ICF.
In all, 74 items were linked with 44 different ICF categories through 105 linkages with generally good interobserver agreement. The 69 items were linked with the ICF at the third or fourth levels. Both commonalities and disparities were found through comparison between the categories linked with these items and both core sets.
All items could be successfully linked with the ICF. The comprehensive core set showed good content validity, while the brief core set needs to be supported by more empirical evidence in various sociocultural contexts. This study specifically complemented the development and refinement of both core sets from the perspective of patients with knee osteoarthritis.
PMCID: PMC1798241  PMID: 16396981
osteoarthritis; knee; ICF

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