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1.  Synthesis and Anti-Renal Fibrosis Activity of Conformationally Locked Truncated 2-Hexynyl-N6-Substituted-(N)-Methanocarba-nucleosides as A3 Adenosine Receptor Antagonists and Partial Agonists 
Journal of Medicinal Chemistry  2014;57(4):1344-1354.
Truncated N6-substituted-(N)-methanocarba-adenosine derivatives with 2-hexynyl substitution were synthesized to examine parallels with corresponding 4′-thioadenosines. Hydrophobic N6 and/or C2 substituents were tolerated in A3AR binding, but only an unsubstituted 6-amino group with a C2-hexynyl group promoted high hA2AAR affinity. A small hydrophobic alkyl (4b and 4c) or N6-cycloalkyl group (4d) showed excellent binding affinity at the hA3AR and was better than an unsubstituted free amino group (4a). A3AR affinities of 3-halobenzylamine derivatives 4f–4i did not differ significantly, with Ki values of 7.8–16.0 nM. N6-Methyl derivative 4b (Ki = 4.9 nM) was a highly selective, low efficacy partial A3AR agonist. All compounds were screened for renoprotective effects in human TGF-β1-stimulated mProx tubular cells, a kidney fibrosis model. Most compounds strongly inhibited TGF-β1-induced collagen I upregulation, and their A3AR binding affinities were proportional to antifibrotic effects; 4b was most potent (IC50 = 0.83 μM), indicating its potential as a good therapeutic candidate for treating renal fibrosis.
doi:10.1021/jm4015313
PMCID: PMC3954500  PMID: 24456490
2.  Identification and Characterization of Non-Tuberculous Mycobacteria Isolated from Tuberculosis Suspects in Southern-Central China 
PLoS ONE  2014;9(12):e114353.
The incidence of non-tuberculous mycobacteria (NTM)-related death has increased globally recently. To obtain information of the species and characterization of pathogens involved in NTM pulmonary infection in Southern-central China, we identified 160 non-tuberculous infection cases from 3995 acid-fast bacilli (AFB)-positive tuberculous suspects. We then randomly selected 101 non-tuberculous patients, isolated bacteria from their sputa and genotyped the pathogens using the 16S rRNA gene and 16S-23S rRNA internal transcribed spacer sequences. M. intracellulare (32.67%, 33/101), M. abscessus (32.67%, 33/101) and M. fortuitum (7.92%, 8/101) are identified in these isolates. Surprisingly, non-mycobacteria including Gordonia (8.91%, 9/101), Nocardia (5.94%, 6/101) and Tsukamurella (0.99%, 1/101) are also discovered, and the case of Tsukamurella pulmonis infection is first discovered in Southern-central China. Moreover, species of M. mucogenicum group, M. chubuense, M. kansasii, M. gastri, M. avium, M. porcinum and M. smegmatis are identified. In addition, nine immune compromised cases (8.91%, 9/101), including type two diabetes mellitus and HIV/AIDS are found to be infected with non-tuberculous bacteria. This study revealed the distribution and characteristics of non-tuberculous AFB pathogen infection occurred in Southern-central China, and suggested that physicians should be alert of the emerging of NTM and non-mycobacteria infection in AFB positive cases and take caution when choosing chemotherapy for tuberculosis-like pulmonary infections. Generally, this study may help with the development of new strategy for the diagnosis and treatment of mycobacterial infection.
doi:10.1371/journal.pone.0114353
PMCID: PMC4252139  PMID: 25463697
3.  Agonist-bound structure of the human P2Y12 receptor 
Nature  2014;509(7498):119-122.
The P2Y12 receptor (P2Y12R), one of eight members of the P2YR family expressed in humans, has been identified as one of the most prominent clinical drug targets for inhibition of platelet aggregation. Consequently, extensive mutagenesis and modeling studies of the P2Y12R have revealed many aspects of agonist/antagonist binding1-4. However, the details of agonist and antagonist recognition and function at the P2Y12R remain poorly understood at the molecular level. Here, we report the structures of the human P2Y12R in complex with a full agonist 2-methylthio-adenosine-5′-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 Å resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5′-triphosphate (2MeSATP) at 3.1 Å resolution. Analysis of these structures, together with the structure of the P2Y12R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283)5, reveals dramatic conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions, providing the first insight into a different ligand binding landscape in the δ-group of class A G protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y12R, with only partially overlapped binding pockets. The agonist-bound P2Y12R structure answers long-standing ambiguities surrounding P2Y12R-agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example of a GPCR where agonist access to the binding pocket requires large scale rearrangements in the highly malleable extracellular region, the structural studies therefore will provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y12R and potentially for other closely related P2YRs.
doi:10.1038/nature13288
PMCID: PMC4128917  PMID: 24784220
4.  Oxidized CaMKII Triggers Atrial Fibrillation 
Circulation  2013;128(16):10.1161/CIRCULATIONAHA.113.003313.
Background
Atrial fibrillation is a growing public health problem without adequate therapies. Angiotensin II (Ang II) and reactive oxygen species (ROS) are validated risk factors for atrial fibrillation (AF) in patients, but the molecular pathway(s) connecting ROS and AF is unknown. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a ROS activated proarrhythmic signal, so we hypothesized that oxidized CaMKIIδ(ox-CaMKII) could contribute to AF.
Methods and Results
We found ox-CaMKII was increased in atria from AF patients compared to patients in sinus rhythm and from mice infused with Ang II compared with saline. Ang II treated mice had increased susceptibility to AF compared to saline treated WT mice, establishing Ang II as a risk factor for AF in mice. Knock in mice lacking critical oxidation sites in CaMKIIδ (MM-VV) and mice with myocardial-restricted transgenic over-expression of methionine sulfoxide reductase A (MsrA TG), an enzyme that reduces ox-CaMKII, were resistant to AF induction after Ang II infusion.
Conclusions
Our studies suggest that CaMKII is a molecular signal that couples increased ROS with AF and that therapeutic strategies to decrease ox-CaMKII may prevent or reduce AF.
doi:10.1161/CIRCULATIONAHA.113.003313
PMCID: PMC3876034  PMID: 24030498
atrial fibrillation; arrhythmia mechanisms; calcium/calmodulin-dependent protein kinase II; angiotensin II; reactive oxygen species
5.  The role of activated adenosine receptors in degranulation of human LAD2 mast cells 
Purinergic Signalling  2014;10(3):465-475.
Mast cell degranulation triggers hypersensitivity reactions at the body–environment interface. Adenosine modulates degranulation, but enhancement and inhibition have both been reported. Which of four adenosine receptors (ARs) mediate modulation, and how, remains uncertain. Also uncertain is whether adenosine reaches mast cell ARs by autocrine ATP release and ecto-enzymatic conversion. Uncertainties partly reflect species and cell heterogeneity, circumvented here by focusing on homogeneous human LAD2 cells. Quantitative PCR detected expression of A2A, A2B, and A3, but not A1, ARs. Nonselective activation of ARs with increasing NECA monotonically enhanced immunologically or C3a-stimulated degranulation. NECA alone stimulated degranulation slightly. Selective AR antagonists did not affect C3a-stimulated degranulation. NECA's enhancement of C3a-triggered degranulation was partially inhibited by separate application of each selective antagonist, and abolished by simultaneous addition of antagonists to the three ARs. Only the A2A antagonist separately inhibited NECA's enhancement of immunologically stimulated degranulation, which was abolished by simultaneous addition of the three selective antagonists. Immunological or C3a activation did not stimulate ATP release. NECA also enhanced immunologically triggered degranulation of mouse bone marrow derived mast cells (BMMCs), which was partially reduced only by simultaneous addition of the three antagonists or by the nonselective antagonist CGS15943. BMMCs also expressed A2A, A2B, and A3 ARs. but not A1AR detectably. We conclude that (a) A1AR is unnecessary for LAD2 degranulation or AR enhancement; (b) A2A, A2B, and A3 ARs all contribute to pharmacologic AR enhancement of LAD2 and BMMC degranulation; and (c) LAD2 cells depend on microenvironmental adenosine to trigger AR modulation.
doi:10.1007/s11302-014-9409-4
PMCID: PMC4152452  PMID: 24595664
FcεRI; C3a; A2A; A2B; A3; ATP release
6.  Rational Design of Sulfonated A3 Adenosine Receptor-Selective Nucleosides as Pharmacological Tools to Study Chronic Neuropathic Pain 
Journal of medicinal chemistry  2013;56(14):10.1021/jm4007966.
(N)-Methanocarba (bicyclo[3.1.0]hexane)-adenosine derivatives were probed for sites of charged sulfonate substitution, which precludes diffusion across biological membranes, e.g. blood brain barrier. Molecular modeling predicted that sulfonate groups on C2-phenylethynyl substituents would provide high affinity at both mouse (m) and human (h) A3 adenosine receptors (ARs), while a N6-p-sulfo-phenylethyl substituent would determine higher hA3AR vs. mA3AR affinity. These modeling predictions, based on steric fitting of the binding cavity and crucial interactions with key residues, were confirmed by binding/efficacy studies of synthesized sulfonates. N6-3-Chlorobenzyl-2-(3-sulfophenylethynyl) derivative 7 (MRS5841) bound selectively to h/m A3ARs (Ki hA3AR 1.9 nM) as agonist, while corresponding p-sulfo isomer 6 (MRS5701) displayed mixed A1/A3AR agonism. Both nucleosides administered i.p. reduced mouse chronic neuropathic pain that was ascribed to either A3 or A1/A3ARs using A3AR genetic deletion. Thus, rational design methods based on A3AR homology models successfully predicted sites for sulfonate incorporation, for delineating adenosine’s CNS vs. peripheral actions.
doi:10.1021/jm4007966
PMCID: PMC3858399  PMID: 23789857
Molecular modeling; G protein-coupled receptor; neuropathic pain; purines; radioligand binding; adenosine receptor
8.  Sarcolemmal ATP-sensitive potassium channels modulate skeletal muscle function under low-intensity workloads 
The Journal of General Physiology  2014;143(1):119-134.
ATP-sensitive potassium (KATP) channels have the unique ability to adjust membrane excitability and functions in accordance with the metabolic status of the cell. Skeletal muscles are primary sites of activity-related energy consumption and have KATP channels expressed in very high density. Previously, we demonstrated that transgenic mice with skeletal muscle–specific disruption of KATP channel function consume more energy than wild-type littermates. However, how KATP channel activation modulates skeletal muscle resting and action potentials under physiological conditions, particularly low-intensity workloads, and how this can be translated to muscle energy expenditure are yet to be determined. Here, we developed a technique that allows evaluation of skeletal muscle excitability in situ, with minimal disruption of the physiological environment. Isometric twitching of the tibialis anterior muscle at 1 Hz was used as a model of low-intensity physical activity in mice with normal and genetically disrupted KATP channel function. This workload was sufficient to induce KATP channel opening, resulting in membrane hyperpolarization as well as reduction in action potential overshoot and duration. Loss of KATP channel function resulted in increased calcium release and aggravated activity-induced heat production. Thus, this study identifies low-intensity workload as a trigger for opening skeletal muscle KATP channels and establishes that this coupling is important for regulation of myocyte function and thermogenesis. These mechanisms may provide a foundation for novel strategies to combat metabolic derangements when energy conservation or dissipation is required.
doi:10.1085/jgp.201311063
PMCID: PMC3874572  PMID: 24344248
9.  Expression of a Vacuole-Localized BURP-Domain Protein from Soybean (SALI3-2) Enhances Tolerance to Cadmium and Copper Stresses 
PLoS ONE  2014;9(6):e98830.
The plant-specific BURP family proteins play diverse roles in plant development and stress responses, but the function mechanism of these proteins is still poorly understood. Proteins in this family are characterized by a highly conserved BURP domain with four conserved Cys-His repeats and two other Cys, indicating that these proteins potentially interacts with metal ions. In this paper, an immobilized metal affinity chromatography (IMAC) assay showed that the soybean BURP protein SALI3-2 could bind soft transition metal ions (Cd2+, Co2+, Ni2+, Zn2+ and Cu2+) but not hard metal ions (Ca2+ and Mg2+) in vitro. A subcellular localization analysis by confocal laser scanning microscopy revealed that the SALI3-2-GFP fusion protein was localized to the vacuoles. Physiological indexes assay showed that Sali3-2-transgenic Arabidopsis thaliana seedlings were more tolerant to Cu2+ or Cd2+ stresses than the wild type. An inductively coupled plasma optical emission spectrometry (ICP-OES) analysis illustrated that, compared to the wild type seedlings the Sali3-2-transgenic seedlings accumulated more cadmium or copper in the roots but less in the upper ground tissues when the seedlings were exposed to excessive CuCl2 or CdCl2 stress. Therefore, our findings suggest that the SALI3-2 protein may confer cadmium (Cd2+) and copper (Cu2+) tolerance to plants by helping plants to sequester Cd2+ or Cu2+ in the root and reduce the amount of heavy metals transported to the shoots.
doi:10.1371/journal.pone.0098830
PMCID: PMC4047006  PMID: 24901737
10.  Quantitation and Composition of Cutaneous Microbiota in Diabetic and Nondiabetic Men 
The Journal of Infectious Diseases  2013;207(7):1105-1114.
Background. Diabetic foot infections are a leading cause of lower extremity amputations. Our study examines the microbiota of diabetic skin prior to ulcer development or infection.
Methods. In a case-control study, outpatient males were recruited at a veterans hospital. Subjects were swabbed at 4 cutaneous sites, 1 on the forearm and 3 on the foot. Quantitative polymerase chain reaction (qPCR) with primers and probes specific for bacteria, Staphylococcus species, Staphylococcus aureus, and fungi were performed on all samples. High-throughput 16S ribosomal RNA (rRNA) sequencing was performed on samples from the forearm and the plantar aspect of the foot.
Results. qPCR analysis of swab specimens from 30 diabetic subjects and 30 control subjects showed no differences in total numbers of bacteria or fungi at any sampled site. Increased log10 concentrations of Staphylococcus aureus, quantified by the number of nuc gene copies, were present in diabetic men on the plantar aspect of the foot. High-throughput 16S rRNA sequencing found that, on the foot, the microbiota in controls (n = 24) was dominated by Staphylococcus species, whereas the microbiota in diabetics (n = 23) was more diverse at the genus level. The forearm microbiota had similar diversity in diabetic and control groups.
Conclusions. The feet of diabetic men had decreased populations of Staphylococcus species, increased populations of S. aureus, and increased bacterial diversity, compared with the feet of controls. These ecologic changes may affect the risk for wound infections.
doi:10.1093/infdis/jit005
PMCID: PMC3583274  PMID: 23300163
microbiota; microbiome; diabetic foot; cutaneous; Staphylococcus; Staphylococcus aureus
11.  AMP-ACTIVATED PROTEIN KINASE AS REGULATOR OF P2Y6 RECEPTOR-INDUCED INSULIN SECRETION IN MOUSE PANCREATIC β-CELLS 
Biochemical pharmacology  2013;85(7):991-998.
5’-AMP-activated protein kinase (AMPK) and its pharmacological modulators have been targeted for treating type 2 diabetes. Extracellular uridine 5’-diphosphate (UDP) activates P2Y6 receptors (P2Y6Rs) in pancreatic β-cells to release insulin and reduce apoptosis, which would benefit diabetes. Here, we studied the role of P2Y6R in activation of AMPK in MIN6 mouse pancreatic β-cells and insulin secretion. Treatment with a potent P2Y6R dinucleotide agonist MRS2957 (500 nM) activated AMPK, which was blocked by P2Y6R-selective antagonist MRS2578. Also, MRS2957 induced phosphorylation of acetyl-coenzyme A carboxylase (ACC), a marker of AMPK activity. Calcium chelator BAPTA-AM, calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-069 and IP3 receptor antagonist 2-APB attenuated P2Y6R-mediated AMPK phosphorylation revealing involvement of intracellular Ca2+ pathways. P2Y6R agonist induced insulin secretion at high glucose, which was reduced by AMPK siRNA. Thus, P2Y6R has a crucial role in β-cell function, suggesting its potential as a therapeutic target in diabetes.
doi:10.1016/j.bcp.2012.11.029
PMCID: PMC3594329  PMID: 23333427
nucleotides; G protein-coupled receptor; insulin; AMPK; diabetes; P2Y6 receptor
12.  G Protein Inactivator RGS6 Mediates Myocardial Cell Apoptosis and Cardiomyopathy Caused by Doxorubicin 
Cancer research  2013;73(6):1662-1667.
Clinical use of the widely used chemotherapeutic agent doxorubicin is limited by life-threatening cardiotoxicity. The mechanisms underlying Dox-induced cardiomyopathy and heart failure remain unclear, but are thought to involve p53-mediated myocardial cell apoptosis. The tripartite G protein inactivating protein RGS6 has been implicated in reactive oxygen species (ROS) generation, ATM/p53 activation and apoptosis in Dox-treated cells. Thus, we hypothesized that RGS6, the expression of which is enriched in cardiac tissue, might also be responsible for the pathological effects of Dox treatment in heart. In this study, we show that RGS6 expression is induced strongly by Dox in the ventricles of mice and isolated ventricular myocytes (VCM) via a post-transcriptional mechanism. While Dox-treated wild type (WT) mice manifested severe left ventricular dysfunction, loss of heart and body mass, along with decreased survival five days after Dox administration, mice lacking RGS6 were completely protected against these pathogenic responses. Activation of ATM/p53-apoptosis signaling by Dox in ventricles of WT mice was also absent in their RGS6−/− counterparts. Dox-induced ROS generation was dramatically impaired in both the ventricles and VCM isolated from RGS6−/− mice, and the apoptotic response to Dox in VCM required RGS6-dependent ROS production. These results identify RGS6 as an essential mediator of the pathogenic responses to Dox in heart, and they argue that RGS6 inhibition offers a rational means to circumvent Dox cardiotoxicity in human cancer patients.
doi:10.1158/0008-5472.CAN-12-3453
PMCID: PMC3602152  PMID: 23338613
RGS6; doxorubicin cardiotoxicity; apoptosis; p53; reactive oxygen species
13.  Allosteric Modulation of A3 Adenosine Receptors by a Series of 3-(2-Pyridinyl)isoquinoline Derivatives 
Molecular pharmacology  2001;60(5):1057-1063.
Allosteric modulators of A1 and A2A adenosine receptors have been described; however, for the A3 adenosine receptor, neither an allosteric site nor a compound with allosteric effects has been described. In this study, the allosteric modulation of human A3 adenosine receptors by a series of 3-(2-pyridinyl)isoquinoline derivatives was investigated by examining their effects on the dissociation of the agonist radioligand, [125I]N6-(4-amino-3-iodobenzyl)-5′ -N-methylcarboxamidoadenosine (I-AB-MECA), from the receptor. Several 3-(2-pyridinyl)isoquinoline derivatives, including VUF5455, VUF8502, VUF8504, and VUF8507, slowed the dissociation of the agonist radioligand [125I]I-AB-MECA in a concentration-dependent manner, suggesting an allosteric interaction. These compounds had no effect on the dissociation of the radiolabeled antagonist [3H]PSB-11 from the A3 adenosine receptor, suggesting a selective enhancement of agonist binding. By comparison, compounds of similar structure (VUF8501, VUF8503, VUF8505), the classical adenosine receptor antagonist CGS15943 and the A1 receptor allosteric enhancer PD81723 did not significantly influence the dissociation rate of [125I]I-AB-MECA. The effect of agonist on forskolin-induced cAMP production was significantly enhanced by VUF5455. When the subtype-selectivity of the allosteric enhancement was tested the compounds had no effect on the dissociation of either [3H]N6-[(R)-phenylisopropyl]adenosine from the A1 adenosine receptor or [3H]CGS21680 from the A2A adenosine receptor. Probing of structure-activity relationships suggested that a carbonyl group is essential for allosterism but preferred only for competitive antagonism. The presence of a 7-methyl group decreased the competitive binding affinity without a major loss of the allosteric enhancing activity, suggesting that the structural requirements for allosteric enhancement might be distinct from those for competitive antagonism.
PMCID: PMC3953614  PMID: 11641434
14.  Selective Allosteric Enhancement of Agonist Binding and Function at Human A3 Adenosine Receptors by a Series of Imidazoquinoline Derivatives 
Molecular pharmacology  2002;62(1):81-89.
We have identified a series of 1H-imidazo-[4,5-c]quinolines as selective allosteric enhancers of human A3 adenosine receptors. Several of these compounds potentiated both the potency and maximal efficacy of agonist-induced responses and selectively decreased the dissociation of the agonist N6-(4-amino-3-[125I]iodobenzyl)-5′-N-methylcarboxamidoadenosine from human A3 adenosine receptors. There was no effect on the dissociation of the antagonist [3H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2.1-i]purin-5-one (PSB-11) from the A3 receptors, as well as [3H]N6-[(R)-phenylisopropy-l]adenosine from rat brain A1 receptors and [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamidoad-enosine from rat striatal A2A receptors, suggesting the selective enhancement of agonist binding at A3 receptors. The analogs were tested as antagonists of competitive binding at human A3 receptors, and Ki values ranging from 120 nM to 101 μM were observed; as for many allosteric modulators of G protein-coupled receptors, an orthosteric effect was also present. The most promising leads from the present set of analogs seem to be the 2-cyclopentyl-1H-imidazo[4,5-c]quinoline derivatives, of which the 4-phenylamino analog DU124183 had the most favorable degree of allosteric modulation versus receptor antagonism. The inhibition of forskolin-stimulated cyclic AMP accumulation in intact cells that express human A3 receptors was employed as a functional index of A3 receptor activation. The enhancer DU124183 caused a marked leftward shift of the concentration-response curve of the A3 receptor agonists in the presence of antagonist and, surprisingly, a potentiation of the maximum agonist efficacy by approximately 30%. Thus, we have identified a novel structural lead for developing allosteric enhancers of A3 adenosine receptors; such enhancers may be useful for treating brain ischemia and other hypoxic conditions.
PMCID: PMC3953617  PMID: 12065758
15.  CaMKII effects on inotropic but not lusitropic force frequency responses require phospholamban 
Increasing heart rate enhances cardiac contractility (force frequency relationship, FFR) and accelerates cardiac relaxation (frequency-dependent acceleration of relaxation, FDAR). The positive FFR together with FDAR promotes rapid filling and ejection of blood from the left ventricle (LV) at higher heart rates. Recent studies indicate that the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is involved in regulating FFR and FDAR. We used isolated perfused mouse hearts to study the mechanisms of FFR and FDAR in different genetic models, including transgenic myocardial CaMKII inhibition (AC3-I) and phosphalamban knockout (PLN−/−). When the rate was increased from 360 beats/min to 630 beats/min in wild type mouse hearts, the LV developed pressure (LVDP) and the maximum rate of increase in pressure (dP/dt max) increased by 37.6 ± 4.7% and 77.0 ± 8.1%, respectively. However, hearts from AC3-I littermates showed no increase of LVDP and a relatively modest (20.4 ± 3.9 %) increase in dP/dt max. PLN−/− hearts had a negative FFR, and myocardial AC3-I expression did not change the FFR in PLN−/− mice. PLN−/− mouse hearts did not exhibit FDAR, while PLN−/−mice with myocardial AC3-I expression showed further frequency dependent reductions in cardiac relaxation, suggesting CaMKII targets in addition to PLN were critical to myocardial relaxation. We incubated a constitutively active form of CaMKII with chemically-skinned myocardium and found that several myofilament proteins were phosphorylated by CaMKII. However, CaMKII did not affect myofilament calcium sensitivity. Our study shows that CaMKII plays an important role in modulating FFR and FDAR in murine hearts and suggest that PLN is a critical target for CaMKII effects on FFR, while CaMKII effects on FDAR partially require PLN-alternative targets.
doi:10.1016/j.yjmcc.2012.06.019
PMCID: PMC3936404  PMID: 22796260
CaM kinase II; force-frequency relation; frequency-dependent acceleration of relaxation; phospholamban
16.  Genetic Inhibition of Na+-Ca2+ Exchanger Current Disables Fight or Flight Sinoatrial Node Activity Without Affecting Resting Heart Rate 
Circulation research  2012;112(2):309-317.
Rationale
The sodium-calcium exchanger 1 (NCX1) is predominantly expressed in the heart and is implicated in controlling automaticity in isolated sinoatrial nodal (SAN) pacemaker cells, but the potential role of NCX1 in determining heart rate in vivo is unknown.
Objective
Determine the role of Ncx1 in heart rate.
Methods and Results
We employed global myocardial and SAN-targeted conditional Ncx1 knockout (Ncx1−/−) mice to measure the effect of the NCX current (INCX) in pacemaking activity in vivo, ex vivo and in isolated SAN cells. We induced conditional Ncx1−/− using a Cre/loxP system. Unexpectedly, in vivo and ex vivo hearts and isolated SAN cells showed that basal rates in Ncx1−/− (retaining ~20% of control level INCX) and control mice were similar, suggesting that physiological NCX1 expression is not required for determining resting heart rate. However, heart rate and SAN cell automaticity increases in response to isoproterenol or the dihydropyridine Ca2+ channel agonist BayK8644 were significantly blunted or eliminated in Ncx1−/− mice, indicating that NCX1 is important for fight or flight heart rate responses. In contrast the ‘pacemaker’ current (If) and L-type Ca2+ currents were equivalent in control and Ncx1−/− SAN cells under resting and isoproterenol-stimulated conditions. Ivabradine, an If antagonist with clinical efficacy, reduced basal SAN cell automaticity similarly in control and Ncx1−/− mice. However, ivabradine decreased automaticity in SAN cells isolated from Ncx1−/− mice more effectively than in control SAN cells after isoproterenol, suggesting that the importance of INCX in fight or flight rate increases is enhanced after If inhibition.
Conclusion
Physiological Ncx1 expression is required for increasing sinus rates in vivo, ex vivo and in isolated SAN cells but not for maintaining resting heart rate.
doi:10.1161/CIRCRESAHA.111.300193
PMCID: PMC3562595  PMID: 23192947
Na+-Ca2+ exchange; sinoatrial node; L-type Ca2+ channels; pacemaker current; ion channel
17.  Distinct cutaneous bacterial assemblages in a sampling of South American Amerindians and US residents 
The ISME Journal  2012;7(1):85-95.
The human skin harbors complex bacterial communities. Prior studies showing high inter-individual variation focused on subjects from developed countries. We therefore compared cutaneous bacterial communities of Amerindians in the Venezuelan Amazon with subjects in the United States. Forearm skin specimens were studied from healthy Amerindians in Platanillal village in Amazonas State, and from healthy persons in New York and Colorado. All skin sampling used similar swab/buffer techniques. Multiplexed V2-targeted 16S rRNA gene pyrosequencing yielded high quality sequences from 112 samples. The results show 20 phyla, with three (Proteobacteria, Firmicutes, Actinobacteria) predominating. US residents and Venezuelan Amerindians had significantly different forearm skin bacterial community compositions, with United States dominated by Propionibacterium. Among the Amerindians, there was a deep split based on bacterial community membership, with 30 and 42 samples, respectively, falling into each of the two groups, not associated with age, gender, or body mass index. One Amerindian group had diversity similar to the United States, but was dominated by Staphylococcus rather than Propionibacterium. The other Amerindian group was significantly more diverse and even than the US or the other Amerindian group, and featured a broad range of Proteobacteria. The results provide evidence that ethnicity, lifestyle and/or geography are associated with the structure of human cutaneous bacterial communities.
doi:10.1038/ismej.2012.81
PMCID: PMC3526177  PMID: 22895161
microbiome; cutaneous; microbial diversity; human; genetics
18.  Allosteric modulation and functional selectivity of G protein-coupled receptors 
Drug discovery today. Technologies  2012;10(2):e237-e243.
Agonists of a single G protein-coupled receptor (GPCR) may activate distinct signaling pathways. Functional selectivity, an emerging concept with therapeutic relevance for GPCRs, may be due to conformational selection or stabilization with respect to particular agonists, receptor dimerization, variable expression levels of GPCRs and downstream signaling molecules, and allosteric modulation. Allosteric modulators may have potential advantages over orthosteric ligands, including greater selectivity and safety. This review focuses on functional selectivity resulting from allosteric modulation.
doi:10.1016/j.ddtec.2012.08.004
PMCID: PMC3653340  PMID: 24050274
allosteric modulation; functional selectivity; GPCR; adenosine receptor; muscarinic receptor; metabotropic glutamate receptor; calcium sensing receptor; CCR5
19.  Community differentiation of the cutaneous microbiota in psoriasis 
Microbiome  2013;1:31.
Background
Psoriasis is a common chronic inflammatory disease of the skin. We sought to characterize and compare the cutaneous microbiota of psoriatic lesions (lesion group), unaffected contralateral skin from psoriatic patients (unaffected group), and similar skin loci in matched healthy controls (control group) in order to discern patterns that govern skin colonization and their relationship to clinical diagnosis.
Results
Using high-throughput 16S rRNA gene sequencing, we assayed the cutaneous bacterial communities of 51 matched triplets and characterized these samples using community data analysis techniques. Intragroup Unifrac β diversity revealed increasing diversity from control to unaffected to lesion specimens. Likewise, principal coordinates analysis (PCoA) revealed separation of the lesion samples from unaffected and control along the first axis, suggesting that psoriasis is a major contributor to the observed diversity. The taxonomic richness and evenness decreased in both lesion and unaffected communities compared to control. These differences are explained by the combined increased abundance of the four major skin-associated genera (Corynebacterium, Propionibacterium, Staphylococcus, and Streptococcus), which present a potentially useful predictor for clinical skin type. Psoriasis samples also showed significant univariate decreases in relative abundances and strong classification performance of Cupriavidus, Flavisolibacter, Methylobacterium, and Schlegelella genera versus controls. The cutaneous microbiota separated into two distinct clusters, which we call cutaneotypes: (1) Proteobacteria-associated microbiota, and (2) Firmicutes-associated and Actinobacteria-associated microbiota. Cutaneotype 2 is enriched in lesion specimens compared to control (odds ratio 3.52 (95% CI 1.44 to 8.98), P <0.01).
Conclusions
Our results indicate that psoriasis induces physiological changes both at the lesion site and at the systemic level, which select for specific differential microbiota among the assayed clinical skin types. These differences in microbial community structure in psoriasis patients are potentially of pathophysiologic and diagnostic significance.
doi:10.1186/2049-2618-1-31
PMCID: PMC4177411  PMID: 24451201
Cutaneous microbiota; Psoriasis markers; Microbiome analysis; Cutaneotypes
20.  Genome Wide Analysis of Narcolepsy in China Implicates Novel Immune Loci and Reveals Changes in Association Prior to Versus After the 2009 H1N1 Influenza Pandemic 
PLoS Genetics  2013;9(10):e1003880.
Previous studies in narcolepsy, an autoimmune disorder affecting hypocretin (orexin) neurons and recently associated with H1N1 influenza, have demonstrated significant associations with five loci. Using a well-characterized Chinese cohort, we refined known associations in TRA@ and P2RY11-DNMT1 and identified new associations in the TCR beta (TRB@; rs9648789 max P = 3.7×10−9 OR 0.77), ZNF365 (rs10995245 max P = 1.2×10−11 OR 1.23), and IL10RB-IFNAR1 loci (rs2252931 max P = 2.2×10−9 OR 0.75). Variants in the Human Leukocyte Antigen (HLA)- DQ region were associated with age of onset (rs7744020 P = 7.9×10−9 beta −1.9 years) and varied significantly among cases with onset after the 2009 H1N1 influenza pandemic compared to previous years (rs9271117 P = 7.8×10−10 OR 0.57). These reflected an association of DQB1*03:01 with earlier onset and decreased DQB1*06:02 homozygosity following 2009. Our results illustrate how genetic association can change in the presence of new environmental challenges and suggest that the monitoring of genetic architecture over time may help reveal the appearance of novel triggers for autoimmune diseases.
Author Summary
Narcolepsy-hypocretin deficiency results from a highly specific autoimmune attack on hypocretin cells. Recent studies have established antigen presentation by specific class II proteins encoded by (HLA DQB1*06:02 and DQA1*01:02) to the cognate T cell receptor as the main disease pathway, with a role for H1N1 influenza in the triggering process. Here, we have used a large and well-characterized cohort of Chinese narcolepsy cases to examine genetic architecture not observed in European samples. We confirmed previously implicated susceptibility genes (T cell receptor alpha, P2RY11), and identify new loci (ZNF365, IL10RB-IFNAR1), most notably, variants at the beta chain of the T cell receptor. We found that one HLA variant, (DQB1*03:01), is associated with dramatically earlier disease onset (nearly 2 years). We also identified differences in HLA haplotype frequencies among cases with onset following the 2009 H1N1 influenza pandemic as compared to before the outbreak, with fewer HLA DQB1*06:02 homozygotes. This may be the first demonstration of such an effect, and suggests that the study of changes in GWAS signals over time could help identify environmental factors in other autoimmune diseases.
doi:10.1371/journal.pgen.1003880
PMCID: PMC3814311  PMID: 24204295
21.  Functional characterization of protein 4.1 homolog in amphioxus: Defining a cryptic spectrin-actin-binding site 
Scientific Reports  2013;3:2873.
Vertebrate 4.1 proteins have a spectrin-actin-binding (SAB) domain, which is lacking in all the invertebrate 4.1 proteins indentified so far, and it was therefore proposed that the SAB domain emerged with the advent of vertebrates during evolution. Here we demonstrated for the first time that amphioxus (an invertebrate chordate) protein 4.1, though lacking a recognizable SAB, was able to bind both spectrin and actin, with a binding capacity comparable to that of human protein 4.1. Detailed structure-activity analyses revealed that the unique domain U2/3 was a newly identified SAB-like domain capable of interacting with spectrin and actin, suggesting the presence of a “cryptic” SAB domain in amphioxus 4.1 protein. We also showed that amphioxus 4.1 protein gene was the common ancestor of vertebrate 4.1 protein genes, from which 4.1R, 4.1N, 4.1G, and 4.1B genes originated. This work will encourage further study on the structure-activity of invertebrate 4.1 protein and its interacting proteins.
doi:10.1038/srep02873
PMCID: PMC3791450  PMID: 24096627
22.  Structural Sweet Spot for A1 Adenosine Receptor Activation by Truncated (N)- Methanocarba Nucleosides: Receptor Docking and Potent Anticonvulsant Activity 
Journal of medicinal chemistry  2012;55(18):8075-8090.
A1 adenosine receptor (AR) agonists display antiischemic and antiepileptic neuroprotective activity, but peripheral cardiovascular side effects impeded their development. SAR study of N6-cycloalkylmethyl 4′-truncated (N)-methanocarba-adenosines identified 10 (MRS5474, N6-dicyclopropylmethyl, Ki 47.9 nM) as a moderately A1AR-selective full agonist. Two stereochemically defined N6-methynyl group substituents displayed narrow SAR; larger than cyclobutyl greatly reduced AR affinity, and larger or smaller than cyclopropyl reduced A1AR selectivity. Nucleoside docking to A1AR homology model characterized distinct hydrophobic cyclopropyl subpockets, the larger “A” forming contacts with Thr270 (7.35), Tyr271 (7.36), Ile274 (7.39) and carbon chains of glutamates (EL2), and smaller subpocket “B” between TM6 and TM7. 10 suppressed minimal clonic seizures (6 Hz mouse model) without typical rotarod impairment of A1AR agonists. Truncated nucleosides, an appealing preclinical approach, have more drug-like physicochemical properties than other A1AR agonists. Thus, we identified highly restricted regions for substitution around N6 suitable for an A1AR agonist with anticonvulsant activity.
doi:10.1021/jm300965a
PMCID: PMC3463139  PMID: 22921089
G protein-coupled receptor; purines; molecular modeling; seizures; in vivo
23.  Structure-activity relationships and molecular modeling of 1,2,4-triazoles as adenosine receptor antagonists 
ACS medicinal chemistry letters  2012;3(9):715-720.
The structure-activity relationship (SAR) for a novel class of 1,2,4-triazole antagonists of the human A2A adenosine receptor (hA2AAR) was explored. Thirty-three analogs of a ligand that was discovered in a structure-based virtual screen against the hA2AAR were tested in hA1, A2A, and A3 radioligand binding assays and in functional assays for the A2BAR subtype. As a series of closely related analogs of the initial lead, 1, did not display improved binding affinity or selectivity, molecular docking was used to guide the selection of more distantly related molecules. This resulted in the discovery of 32, a hA2AAR antagonist (Ki 200 nM) with high ligand efficiency. In the light of the SAR for the 1,2,4-triazole scaffold, we also investigated the binding mode of these compounds based on docking to several A2AAR crystal structures.
doi:10.1021/ml300097g
PMCID: PMC3549271  PMID: 23342198
1,2,4-triazole; A2A adenosine receptor; antagonist; molecular docking; structure-activity relationship
24.  The Estimation of Prevalence, Incidence, and Residual Risk of Transfusion-Transmitted Human Hepatitis B Infection from Blood Donated at the Anhui Blood Center, China, from 2009 to 2011 
PLoS ONE  2013;8(9):e73472.
Background
The high prevalence of hepatitis B virus (HBV) among the Chinese population poses a threat to blood safety; however, few studies have examined epidemiological data regarding HBV infection of Chinese blood donors. The present study investigated the demographic characteristics of blood donors at the Anhui blood center in China, the prevalence, incidence, and residual risk (RR) associated with hepatitis B surface antigen (HBsAg) expression in terms of transfusion transmitted HBV (TTHBV) infections.
Methods
The demographic characteristics and HBV status of people who donated blood at the Anhui blood center between 2009 and 2011 were retrospectively analyzed. The incidence of HBV was estimated through HBsAg yield approach. The window period model was then used to estimate the RR of TTHBV infection.
Results
The typical donor at the Anhui blood center was a first-time volunteer, aged less than 25 years, unmarried, of Han ethnicity, and with an education below high school level. The prevalence of HBV infection among repeat donors, first-time donors, and all donors was 28.9, 127.2 and 82.1 per 100,000, respectively. The incidence estimate was 333.9 per 105 person-years. Using an infectious window period of 59 days, the RR for HBV was estimated to be 1 in 1853 between 2009 and 2011.
Conclusions
The incidence and RR of HBV in Chinese blood donors are much higher than those of donors in developed countries. This is because sensitive ELISAs and nucleic acid tests are not available in China. Further work is needed to improve both the safety and availability of blood products in China.
doi:10.1371/journal.pone.0073472
PMCID: PMC3772943  PMID: 24058476
25.  Structure–Activity Relationships and Molecular Modeling of 1,2,4-Triazoles as Adenosine Receptor Antagonists 
ACS Medicinal Chemistry Letters  2012;3(9):715-720.
The structure–activity relationship (SAR) for a novel class of 1,2,4-triazole antagonists of the human A2A adenosine receptor (hA2AAR) was explored. Thirty-three analogs of a ligand that was discovered in a structure-based virtual screen against the hA2AAR were tested in hA1, A2A, and A3 radioligand binding assays and in functional assays for the A2BAR subtype. As a series of closely related analogs of the initial lead, 1, did not display improved binding affinity or selectivity, molecular docking was used to guide the selection of more distantly related molecules. This resulted in the discovery of 32, a hA2AAR antagonist (Ki 200 nM) with high ligand efficiency. In light of the SAR for the 1,2,4-triazole scaffold, we also investigated the binding mode of these compounds based on docking to several A2AAR crystal structures.
doi:10.1021/ml300097g
PMCID: PMC3549271  PMID: 23342198
1,2,4-Triazole; A2A adenosine receptor; antagonist; molecular docking; structure−activity relationship

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