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1.  The Phosphodiesterase-4 (PDE4) Inhibitor Rolipram Decreases Ethanol Seeking and Consumption in Alcohol-preferring Fawn-Hooded Rats 
Alcohol dependence is a complex psychiatric disorder demanding development of novel pharmacotherapies. Since the cyclic AMP (cAMP) signaling cascade has been implicated in mediating behavioral responses to alcohol, key components in this cascade may serve as potential treatment targets. Phosphodiesterase 4 (PDE4), an enzyme that specifically catalyzes the hydrolysis of cAMP, represents as a key point in regulating intracellular cAMP levels. Thus, it was of interest to determine whether PDE4 was involved in the regulation of alcohol use and abuse.
Male Fawn-Hooded (FH/Wjd) rats were tested for 5% (v/v) ethanol and 10% (w/v) sucrose operant oral self-administration following treatment with the selective PDE4 inhibitor rolipram (0.0125, 0.025, or 0.05 mg/kg, s.c.); rolipram at higher doses (0.05, 0.1, and 0.2 mg/kg, s.c.) was tested to determine its impact on the intake of ethanol, sucrose, or water using the two-bottle choice drinking paradigm. Subsequent open-field testing was performed to evaluate the influence of higher doses of rolipram on locomotor activity.
Acute administration of rolipram dose-dependently reduced operant self-administration of 5% ethanol, but had no effect on 10% sucrose responding. Time-course assessment revealed significant decreases in ethanol consumption after rolipram (0.1, 0.2 mg/kg) treatment in continuous- and intermittent-access to ethanol at 5% or 10%, respectively. Moreover, chronic rolipram treatment time-dependently decreased 5% ethanol consumption and preference during treatment days and after the termination of rolipram administration. Rolipram at the highest doses (0.1 and 0.2 mg/kg) did decrease locomotor activity, but the effect lasted only 10 and 20 min, respectively, which did not likely alter long-term ethanol drinking.
These results suggest that PDE4 plays a role in alcohol seeking and consumption behavior. Drugs interfering with PDE4 may be a potential pharmacotherapy for alcohol dependence.
PMCID: PMC4335658  PMID: 22671516
Cyclic AMP Signaling; Phosphodiesterase-4 (PDE4); Rolipram; FH/Wjd Rat; Ethanol Intake
2.  Benzodiazepines and Z-Drug Use among HIV-Infected Patients in Taiwan: A 13-Year Nationwide Cohort Study 
BioMed Research International  2015;2015:465726.
Introduction. Benzodiazepines (BZDs) and zolpidem, zopiclone, and zaleplon (Z-drugs) are commonly prescribed to HIV-infected patients. We hypothesized that frequent BZD and Z-drug use among these patients may be associated with psychiatric illnesses, particularly in long-term users. Methods. We included 1,081 patients with HIV between 1998 and 2011 from the Taiwan National Health Insurance Research Database and matched them according to age, sex, and comorbidity with uninfected controls to investigate the psychiatric diagnoses and prescriptions of BZDs and Z-drugs. Cumulative defined daily dose (cDDD) was assessed as the indicator of the duration of medication exposure. Patients exhibiting a cDDD exceeding 180 were defined as long-term users. Results. The patients with HIV had an increased risk of any use (odds ratio (OR): 8.70, 95% confidence interval (CI): 6.82–10.97) and long-term use (OR: 5.06, 95% CI: 3.63–7.04) of BZD and Z-drugs compared with those without HIV during the follow-up after demographic data and psychiatric comorbidities were adjusted. Conclusion. A large proportion of the HIV-infected patients received prescriptions of BZDs and Z-drugs. Mood disorders, insomnia, anxiety disorders, HIV infection, and substance use disorder were substantial predictors among the BZD and Z-drug users. These findings suggest that providing psychiatric services for HIV-infected patients is vital.
PMCID: PMC4309025  PMID: 25654104
3.  Role of chemokines and their receptors in chronic lymphocytic leukemia 
Cancer Biology & Therapy  2013;15(1):3-9.
Chemokines produced in distinct tissue microenvironments sustain migration of mature lymphocytes in lymphoglandula. Chemokine receptors expressed on chronic lymphocytic leukemia (CLL) cells regulate the migration of the leukemia cells within the bone marrow (BM), lymphoid organs in collaboration with chemokines. Chemokines form a pro-survival circuitry by regulating leukocyte trafficking, maintaining extended lymphocyte survival. Therefore, chemokines in tumor cell–microenvironment interactions represent a target for treatment of CLL. AMD3100 disrupts the CLL/microenvironment interactions and influences CXCL12/CXCR4 survival signaling. Fostamatinib, ibrutinib, and GS-1101 as B-cell receptor (BCR)-related kinase inhibitors inhibit BCR- and chemokine-receptor-signal-regulated kinase and have a good clinical response in CLL. Lenalidomide, sorafenib, and dasatinib are other additional drugs associated with chemokine in microenvironment. Inhibiting signaling through chemokine and microenvironment associated signaling are emerging as innovative therapeutic targets in CLL. In this article, we reviewed the role of chemokines in CLL microenvironment and novel therapeutics targeting CLL microenvironment.
PMCID: PMC3938521  PMID: 24149438
chronic leukemia lymphoma; microenvironment; chemokines; chemokines receptors; targeted therapy
4.  Inhibition of phosphodiesterase-4 decreases ethanol intake in mice 
Psychopharmacology  2011;218(2):331-339.
Cyclic AMP (cAMP)-protein kinase A (PKA) signaling has been implicated in the regulation of ethanol consumption. Phosphodiesterase-4 (PDE4) specifically hydrolyzes cAMP and plays a critical role in controlling intracellular cAMP levels in the brain. However, the role of PDE4 in ethanol consumption remains unknown.
To examine whether PDE4 was involved in regulating ethanol intake.
The two-bottle choice paradigm was used to assess intake of ethanol, sucrose, and quinine in C57BL/6J mice treated with the selective PDE4 inhibitor rolipram or Ro 20-1724; locomotor activity was also monitored using the open-field test in mice treated with rolipram.
Administration (i.p.) of either rolipram (0.25 and 0.5 mg/kg) or Ro 20-1724 (10 mg/kg) reduced ethanol intake and preference by 60-80%, but did not alter total fluid intake. In contrast, rolipram even at the higher dose of 0.5 mg/kg was not able to affect intake of sucrose or quinine, alcohol-induced sedation, or blood ethanol elimination. At 0.5 mg/kg, rolipram did decrease locomotor activity, but the effect only lasted for approximately 40 min, which did not likely affect behavior of ethanol drinking.
These results suggest that PDE4 is a novel target for drugs that reduce ethanol intake; PDE4 inhibitors may be used for treatment of alcohol dependence.
PMCID: PMC4210373  PMID: 21509503
Ethanol intake; phosphodiesterase-4 (PDE4); two-bottle choice; rolipram; locomotor activity; sucrose; quinine; cyclic AMP; mice
5.  PDE4 as a target for cognition enhancement 
The second messengers cAMP and cGMP mediate fundamental aspects of brain function relevant to memory, learning and cognitive functions. Consequently, cyclic nucleotide phosphodiesterases (PDEs), the enzymes that inactivate the cyclic nucleotides, are promising targets for the development of cognition-enhancing drugs.
Areas covered
PDE4 is the largest of the eleven mammalian PDE families. This review covers the properties and functions of the PDE4 family, highlighting procognitive and memory-enhancing effects associated with their inactivation.
Expert opinion
PAN-selective PDE4 inhibitors exert a number of memory- and cognition-enhancing effects and have neuroprotective and neuroregenerative properties in preclinical models. The major hurdle for their clinical application is to target inhibitors to specific PDE4 isoforms relevant to particular cognitive disorders to realize the therapeutic potential while avoiding side effects, in particular emesis and nausea. The PDE4 family comprises four genes, PDE4A-D, each expressed as multiple variants. Progress to date stems from characterization of rodent models with selective ablation of individual PDE4 subtypes, revealing that individual subtypes exert unique and non-redundant functions in the brain. Thus, targeting specific PDE4 subtypes, as well as splicing variants or conformational states, represents a promising strategy to separate the therapeutic benefits from the side effects of PAN-PDE4 inhibitors.
PMCID: PMC4066988  PMID: 23883342
Alzheimer’s disease; schizophrenia; cognition; memory; cyclic nucleotide; cAMP; phosphodiesterase; PDE4; Rolipram
6.  Senescent-induced dysregulation of cAMP/CREB signaling and correlations with cognitive decline 
Brain research  2013;1516:93-109.
It is well known that alongside senescence there is a gradual decline in cognitive ability, most noticeably certain kinds of memory such as working, episodic, spatial, and long term memory. However, until recently, not much has been known regarding the specific mechanisms responsible for the decline in cognitive ability with age. Over the past decades, researchers have become more interested in cAMP signaling, and its downstream transcription factor cAMP response element binding protein (CREB) in the context of senescence. However, there is still a lack of understanding on what ultimately causes the cognitive deficits observed with senescence. This review will focus on the changes in intracellular signaling in the brain, more specifically, alterations in cAMP/CREB signaling in aging. In addition, the downstream effects of altered cAMP signaling on cognitive ability with age will be further discussed. Overall, understanding the senescent-related changes that occur in cAMP/CREB signaling could be important for the development of novel drug targets for both healthy aging, and pathological aging such as Alzheimer's disease.
PMCID: PMC3760033  PMID: 23623816
Aging; Memory; Camp; CREB; Phosphodiesterase; Protein kinase A
7.  Molecular Characterization of PauR and Its Role in Control of Putrescine and Cadaverine Catabolism through the γ-Glutamylation Pathway in Pseudomonas aeruginosa PAO1 
Journal of Bacteriology  2013;195(17):3906-3913.
Pseudomonas aeruginosa PAO1 grows on a variety of polyamines as the sole source of carbon and nitrogen. Catabolism of polyamines is mediated by the γ-glutamylation pathway, which is complicated by the existence of multiple homologous enzymes with redundant specificities toward different polyamines for a more diverse metabolic capacity in this organism. Through a series of markerless gene knockout mutants and complementation tests, specific combinations of pauABCD (polyamine utilization) genes were deciphered for catabolism of different polyamines. Among six pauA genes, expression of pauA1, pauA2, pauA4, and pauA5 was found to be inducible by diamines putrescine (PUT) and cadaverine (CAD) but not by diaminopropane. Activation of these promoters was regulated by the PauR repressor, as evidenced by constitutively active promoters in the pauR mutant. The activities of these promoters were further enhanced by exogenous PUT or CAD in the mutant devoid of all six pauA genes. The recombinant PauR protein with a hexahistidine tag at its N terminus was purified, and specific bindings of PauR to the promoter regions of most pau operons were demonstrated by electromobility shift assays. Potential interactions of PUT and CAD with PauR were also suggested by chemical cross-linkage analysis with glutaraldehyde. In comparison, growth on PUT was more proficient than that on CAD, and this observed growth phenotype was reflected in a strong catabolite repression of pauA promoter activation by CAD but was completely absent as reflected by activation by PUT. In summary, this study clearly establishes the function of PauR in control of pau promoters in response to PUT and CAD for their catabolism through the γ-glutamylation pathway.
PMCID: PMC3754595  PMID: 23794626
8.  Hair Metabolomics: Identification of Fetal Compromise Provides Proof of Concept for Biomarker Discovery 
Theranostics  2014;4(9):953-959.
Analysis of the human metabolome has yielded valuable insights into health, disease and toxicity. However, the metabolic profile of complex biological fluids such as blood is highly dynamic and this has limited the discovery of robust biomarkers. Hair grows relatively slowly, and both endogenous compounds and environmental exposures are incorporated from blood into hair during growth, which reflects the average chemical composition over several months.
We used hair samples to study the metabolite profiles of women with pregnancies complicated by fetal growth restriction (FGR) and healthy matched controls. We report the use of GC-MS metabolite profiling of hair samples for biomarker discovery. Unsupervised statistical analysis showed complete discrimination of FGR from controls based on hair composition alone. A predictive model combining 5 metabolites produced an area under the receiver-operating curve of 0.998.
This is the first study of the metabolome of human hair and demonstrates that this biological material contains robust biomarkers, which may lead to the development of a sensitive diagnostic tool for FGR, and perhaps more importantly, to stable biomarkers for a range of other diseases.
PMCID: PMC4107295  PMID: 25057319
metabolite profiling; hair; biomarker; fetal growth restriction; GC-MS.
9.  Effect of Xiaoyaosan Decoction on Learning and Memory Deficit in Rats Induced by Chronic Immobilization Stress 
Xiaoyaosan (XYS) decoction is a famous prescription which can protect nervous system from stress and treat liver stagnation and spleen deficiency syndrome (LSSDS). In this experiment, we observed the effect of XYS decoction on chronic immobilization stress (CIS) induced learning and memory deficit in rats from behaviors and changes of proteins in hippocampus. We used XYS decoction to treat CIS induced learning and memory deficit in rats with rolipram as positive control, used change of body weight and behavioral tests to determine whether the rats have LSSDS and have learning and memory deficit or not. We used Western blotting to determine the content of postsynaptic density protein 95 (PSD-95) and synaptophysin (SYP) in hippocampus. Results showed that XYS could improve the situation of slow weight gain induced by CIS, improve the ability of learning and memory, reverse the symptom of liver stagnation and spleen deficiency syndrome (LSSDS) in rats, and increase the levels of PSD-95 and SYP on the hippocampal nerve synapses. These findings suggested that XYS decoction may be helpful in reversing CIS induced learning and memory deficit by increasing the levels of PSD-95 and SYP on the hippocampal nerve synapses and improving synaptic plasticity.
PMCID: PMC3891437  PMID: 24459529
10.  Effect of whey protein on plasma amino acids in diabetic mice 
The aim of this study was to investigate the effect of whey protein on plasma amino acid levels in a mouse model of type II diabetes, using high-performance liquid chromatography (HPLC). The composition and content of amino acids in the whey proteins were analyzed using HPLC. Type I and type II diabetic mouse models were prepared using streptozotocin (STZ) and normal mice were used as a control. The ICR mice in each group were then randomly divided into four subgroups, to which 0, 10, 20 and 40% whey protein, respectively, was administered for four weeks. Changes in the plasma amino acid levels were observed in each group. The proportions of leucine, isoleucine and valine in the whey proteins were 14.40, 5.93 and 5.32% of the total amino acids, respectively, that is, the branched-chain amino acid content was 25.65%. The levels of branched-chain amino acids increased in the plasma of the normal and model mice following the administration of whey proteins by gavage and the amino acid levels increased as the concentration of the administered protein increased. In addition, the branched-chain amino acid levels in the blood of the model mice were higher than those in the normal mice. The levels of plasma amino acids in diabetic mice increased following gavage with whey protein, which is rich in branched-chain amino acids.
PMCID: PMC3829761  PMID: 24255674
HPLC; diabetic mice; branched-chain amino acids; whey protein
11.  Enhancing Cell Nucleus Accumulation and DNA Cleavage Activity of Anti-Cancer Drug via Graphene Quantum Dots 
Scientific Reports  2013;3:2852.
Graphene quantum dots (GQDs) maintain the intrinsic layered structural motif of graphene but with smaller lateral size and abundant periphery carboxylic groups, and are more compatible with biological system, thus are promising nanomaterials for therapeutic applications. Here we show that GQDs have a superb ability in drug delivery and anti-cancer activity boost without any pre-modification due to their unique structural properties. They could efficiently deliver doxorubicin (DOX) to the nucleus through DOX/GQD conjugates, because the conjugates assume different cellular and nuclear internalization pathways comparing to free DOX. Also, the conjugates could enhance DNA cleavage activity of DOX markedly. This enhancement combining with efficient nuclear delivery improved cytotoxicity of DOX dramatically. Furthermore, the DOX/GQD conjugates could also increase the nuclear uptake and cytotoxicity of DOX to drug-resistant cancer cells indicating that the conjugates may be capable to increase chemotherapy efficacy of anti-cancer drugs that are suboptimal due to the drug resistance.
PMCID: PMC3790198  PMID: 24092333
12.  Correction: Metabolic Response of Candida albicans to Phenylethyl Alcohol under Hyphae-Inducing Conditions 
PLoS ONE  2013;8(8):10.1371/annotation/d395f7a5-ac0a-41e2-abd8-fa947717f6a3.
PMCID: PMC3782339
13.  Metabolic Response of Candida albicans to Phenylethyl Alcohol under Hyphae-Inducing Conditions 
PLoS ONE  2013;8(8):e71364.
Phenylethyl alcohol was one of the first quorum sensing molecules (QSMs) identified in C. albicans. This extracellular signalling molecule inhibits the hyphal formation of C. albicans at high cell density. Little is known, however, about the underlying mechanisms by which this QSM regulates the morphological switches of C. albicans. Therefore, we have applied metabolomics and isotope labelling experiments to investigate the metabolic changes that occur in C. albicans in response to phenylethyl alcohol under defined hyphae-inducing conditions. Our results showed a global upregulation of central carbon metabolism when hyphal development was suppressed by phenylethyl alcohol. By comparing the metabolic changes in response to phenylethyl alcohol to our previous metabolomic studies, we were able to short-list 7 metabolic pathways from central carbon metabolism that appear to be associated with C. albicans morphogenesis. Furthermore, isotope-labelling data showed that phenylethyl alcohol is indeed taken up and catabolised by yeast cells. Isotope-labelled carbon atoms were found in the majority of amino acids as well as in lactate and glyoxylate. However, isotope-labelled carbon atoms from phenylethyl alcohol accumulated mainly in the pyridine ring of NAD+/NADH and NADP−/NADPH molecules, showing that these nucleotides were the main products of phenylethyl alcohol catabolism. Interestingly, two metabolic pathways where these nucleotides play an important role, nitrogen metabolism and nicotinate/nicotinamide metabolism, were also short-listed through our previous metabolomics works as metabolic pathways likely to be closely associated with C. albicans morphogenesis.
PMCID: PMC3741116  PMID: 23951145
14.  Effects of rocuronium and vecuronium on initial rundown of endplate potentials in the isolated phrenic nerve diaphragm preparation of rats 
SpringerPlus  2013;2:155.
Rocuronium and vecuronium, two non-depolarizing neuromuscular blockers, have been widely used in surgery procedures. However, their electrophysiological properties need to be more widely explored. We examined the effects of rocuronium and vecuronium on initial rundown of endplate potential amplitudes in the non-uniform stretched muscle preparation of the rat isolated phrenic nerve diaphragm. More specifically, the endplate potentials were recorded with one microelectrode from a single endplate. The effects of rocuronium or vecuronium each at 4 concentrations (0.5 ×, l ×, 2 ×, 4 × EC95; EC95 = concentration of the drug required to produce the inhibitory effect by 95%) on the amplitude of endplate potentials and its rundown were observed. Treatment of the isolated rat phrenic nerve-diaphragm preparation with rocuronium (2.5–20 μg/ml) or vecuronium (0.5–4 μg/ml) decreased the amplitude of endplate potentials and inhibited its rundown in a concentration-dependent manner. At the concentration (2.5 μg/ml for rocuronium and 0.5 μg/ml for vecuronium) that did not alter the endplate potential amplitude, the onset of reduced endplate potential rundown was 3 and 5 min after administration of rocuronium or vecuronium, respectively. The results suggest that rocuronium and vecuronium block the neuromuscular junction presynaptically and that rocuronium does it faster than vecuronium.
PMCID: PMC3639353  PMID: 23641322
Rocuronium; Vecuronium; Motor endplate; Neuromuscular junction; Diaphragm
15.  Guidelines for the use and interpretation of assays for monitoring autophagy 
Klionsky, Daniel J. | Abdalla, Fabio C. | Abeliovich, Hagai | Abraham, Robert T. | Acevedo-Arozena, Abraham | Adeli, Khosrow | Agholme, Lotta | Agnello, Maria | Agostinis, Patrizia | Aguirre-Ghiso, Julio A. | Ahn, Hyung Jun | Ait-Mohamed, Ouardia | Ait-Si-Ali, Slimane | Akematsu, Takahiko | Akira, Shizuo | Al-Younes, Hesham M. | Al-Zeer, Munir A. | Albert, Matthew L. | Albin, Roger L. | Alegre-Abarrategui, Javier | Aleo, Maria Francesca | Alirezaei, Mehrdad | Almasan, Alexandru | Almonte-Becerril, Maylin | Amano, Atsuo | Amaravadi, Ravi K. | Amarnath, Shoba | Amer, Amal O. | Andrieu-Abadie, Nathalie | Anantharam, Vellareddy | Ann, David K. | Anoopkumar-Dukie, Shailendra | Aoki, Hiroshi | Apostolova, Nadezda | Arancia, Giuseppe | Aris, John P. | Asanuma, Katsuhiko | Asare, Nana Y.O. | Ashida, Hisashi | Askanas, Valerie | Askew, David S. | Auberger, Patrick | Baba, Misuzu | Backues, Steven K. | Baehrecke, Eric H. | Bahr, Ben A. | Bai, Xue-Yuan | Bailly, Yannick | Baiocchi, Robert | Baldini, Giulia | Balduini, Walter | Ballabio, Andrea | Bamber, Bruce A. | Bampton, Edward T.W. | Juhász, Gábor | Bartholomew, Clinton R. | Bassham, Diane C. | Bast, Robert C. | Batoko, Henri | Bay, Boon-Huat | Beau, Isabelle | Béchet, Daniel M. | Begley, Thomas J. | Behl, Christian | Behrends, Christian | Bekri, Soumeya | Bellaire, Bryan | Bendall, Linda J. | Benetti, Luca | Berliocchi, Laura | Bernardi, Henri | Bernassola, Francesca | Besteiro, Sébastien | Bhatia-Kissova, Ingrid | Bi, Xiaoning | Biard-Piechaczyk, Martine | Blum, Janice S. | Boise, Lawrence H. | Bonaldo, Paolo | Boone, David L. | Bornhauser, Beat C. | Bortoluci, Karina R. | Bossis, Ioannis | Bost, Frédéric | Bourquin, Jean-Pierre | Boya, Patricia | Boyer-Guittaut, Michaël | Bozhkov, Peter V. | Brady, Nathan R | Brancolini, Claudio | Brech, Andreas | Brenman, Jay E. | Brennand, Ana | Bresnick, Emery H. | Brest, Patrick | Bridges, Dave | Bristol, Molly L. | Brookes, Paul S. | Brown, Eric J. | Brumell, John H. | Brunetti-Pierri, Nicola | Brunk, Ulf T. | Bulman, Dennis E. | Bultman, Scott J. | Bultynck, Geert | Burbulla, Lena F. | Bursch, Wilfried | Butchar, Jonathan P. | Buzgariu, Wanda | Bydlowski, Sergio P. | Cadwell, Ken | Cahová, Monika | Cai, Dongsheng | Cai, Jiyang | Cai, Qian | Calabretta, Bruno | Calvo-Garrido, Javier | Camougrand, Nadine | Campanella, Michelangelo | Campos-Salinas, Jenny | Candi, Eleonora | Cao, Lizhi | Caplan, Allan B. | Carding, Simon R. | Cardoso, Sandra M. | Carew, Jennifer S. | Carlin, Cathleen R. | Carmignac, Virginie | Carneiro, Leticia A.M. | Carra, Serena | Caruso, Rosario A. | Casari, Giorgio | Casas, Caty | Castino, Roberta | Cebollero, Eduardo | Cecconi, Francesco | Celli, Jean | Chaachouay, Hassan | Chae, Han-Jung | Chai, Chee-Yin | Chan, David C. | Chan, Edmond Y. | Chang, Raymond Chuen-Chung | Che, Chi-Ming | Chen, Ching-Chow | Chen, Guang-Chao | Chen, Guo-Qiang | Chen, Min | Chen, Quan | Chen, Steve S.-L. | Chen, WenLi | Chen, Xi | Chen, Xiangmei | Chen, Xiequn | Chen, Ye-Guang | Chen, Yingyu | Chen, Yongqiang | Chen, Yu-Jen | Chen, Zhixiang | Cheng, Alan | Cheng, Christopher H.K. | Cheng, Yan | Cheong, Heesun | Cheong, Jae-Ho | Cherry, Sara | Chess-Williams, Russ | Cheung, Zelda H. | Chevet, Eric | Chiang, Hui-Ling | Chiarelli, Roberto | Chiba, Tomoki | Chin, Lih-Shen | Chiou, Shih-Hwa | Chisari, Francis V. | Cho, Chi Hin | Cho, Dong-Hyung | Choi, Augustine M.K. | Choi, DooSeok | Choi, Kyeong Sook | Choi, Mary E. | Chouaib, Salem | Choubey, Divaker | Choubey, Vinay | Chu, Charleen T. | Chuang, Tsung-Hsien | Chueh, Sheau-Huei | Chun, Taehoon | Chwae, Yong-Joon | Chye, Mee-Len | Ciarcia, Roberto | Ciriolo, Maria R. | Clague, Michael J. | Clark, Robert S.B. | Clarke, Peter G.H. | Clarke, Robert | Codogno, Patrice | Coller, Hilary A. | Colombo, María I. | Comincini, Sergio | Condello, Maria | Condorelli, Fabrizio | Cookson, Mark R. | Coombs, Graham H. | Coppens, Isabelle | Corbalan, Ramon | Cossart, Pascale | Costelli, Paola | Costes, Safia | Coto-Montes, Ana | Couve, Eduardo | Coxon, Fraser P. | Cregg, James M. | Crespo, José L. | Cronjé, Marianne J. | Cuervo, Ana Maria | Cullen, Joseph J. | Czaja, Mark J. | D'Amelio, Marcello | Darfeuille-Michaud, Arlette | Davids, Lester M. | Davies, Faith E. | De Felici, Massimo | de Groot, John F. | de Haan, Cornelis A.M. | De Martino, Luisa | De Milito, Angelo | De Tata, Vincenzo | Debnath, Jayanta | Degterev, Alexei | Dehay, Benjamin | Delbridge, Lea M.D. | Demarchi, Francesca | Deng, Yi Zhen | Dengjel, Jörn | Dent, Paul | Denton, Donna | Deretic, Vojo | Desai, Shyamal D. | Devenish, Rodney J. | Di Gioacchino, Mario | Di Paolo, Gilbert | Di Pietro, Chiara | Díaz-Araya, Guillermo | Díaz-Laviada, Inés | Diaz-Meco, Maria T. | Diaz-Nido, Javier | Dikic, Ivan | Dinesh-Kumar, Savithramma P. | Ding, Wen-Xing | Distelhorst, Clark W. | Diwan, Abhinav | Djavaheri-Mergny, Mojgan | Dokudovskaya, Svetlana | Dong, Zheng | Dorsey, Frank C. | Dosenko, Victor | Dowling, James J. | Doxsey, Stephen | Dreux, Marlène | Drew, Mark E. | Duan, Qiuhong | Duchosal, Michel A. | Duff, Karen E. | Dugail, Isabelle | Durbeej, Madeleine | Duszenko, Michael | Edelstein, Charles L. | Edinger, Aimee L. | Egea, Gustavo | Eichinger, Ludwig | Eissa, N. Tony | Ekmekcioglu, Suhendan | El-Deiry, Wafik S. | Elazar, Zvulun | Elgendy, Mohamed | Ellerby, Lisa M. | Eng, Kai Er | Engelbrecht, Anna-Mart | Engelender, Simone | Erenpreisa, Jekaterina | Escalante, Ricardo | Esclatine, Audrey | Eskelinen, Eeva-Liisa | Espert, Lucile | Espina, Virginia | Fan, Huizhou | Fan, Jia | Fan, Qi-Wen | Fan, Zhen | Fang, Shengyun | Fang, Yongqi | Fanto, Manolis | Fanzani, Alessandro | Farkas, Thomas | Farre, Jean-Claude | Faure, Mathias | Fechheimer, Marcus | Feng, Carl G. | Feng, Jian | Feng, Qili | Feng, Youji | Fésüs, László | Feuer, Ralph | Figueiredo-Pereira, Maria E. | Fimia, Gian Maria | Fingar, Diane C. | Finkbeiner, Steven | Finkel, Toren | Finley, Kim D. | Fiorito, Filomena | Fisher, Edward A. | Fisher, Paul B. | Flajolet, Marc | Florez-McClure, Maria L. | Florio, Salvatore | Fon, Edward A. | Fornai, Francesco | Fortunato, Franco | Fotedar, Rati | Fowler, Daniel H. | Fox, Howard S. | Franco, Rodrigo | Frankel, Lisa B. | Fransen, Marc | Fuentes, José M. | Fueyo, Juan | Fujii, Jun | Fujisaki, Kozo | Fujita, Eriko | Fukuda, Mitsunori | Furukawa, Ruth H. | Gaestel, Matthias | Gailly, Philippe | Gajewska, Malgorzata | Galliot, Brigitte | Galy, Vincent | Ganesh, Subramaniam | Ganetzky, Barry | Ganley, Ian G. | Gao, Fen-Biao | Gao, George F. | Gao, Jinming | Garcia, Lorena | Garcia-Manero, Guillermo | Garcia-Marcos, Mikel | Garmyn, Marjan | Gartel, Andrei L. | Gatti, Evelina | Gautel, Mathias | Gawriluk, Thomas R. | Gegg, Matthew E. | Geng, Jiefei | Germain, Marc | Gestwicki, Jason E. | Gewirtz, David A. | Ghavami, Saeid | Ghosh, Pradipta | Giammarioli, Anna M. | Giatromanolaki, Alexandra N. | Gibson, Spencer B. | Gilkerson, Robert W. | Ginger, Michael L. | Ginsberg, Henry N. | Golab, Jakub | Goligorsky, Michael S. | Golstein, Pierre | Gomez-Manzano, Candelaria | Goncu, Ebru | Gongora, Céline | Gonzalez, Claudio D. | Gonzalez, Ramon | González-Estévez, Cristina | González-Polo, Rosa Ana | Gonzalez-Rey, Elena | Gorbunov, Nikolai V. | Gorski, Sharon | Goruppi, Sandro | Gottlieb, Roberta A. | Gozuacik, Devrim | Granato, Giovanna Elvira | Grant, Gary D. | Green, Kim N. | Gregorc, Ales | Gros, Frédéric | Grose, Charles | Grunt, Thomas W. | Gual, Philippe | Guan, Jun-Lin | Guan, Kun-Liang | Guichard, Sylvie M. | Gukovskaya, Anna S. | Gukovsky, Ilya | Gunst, Jan | Gustafsson, Åsa B. | Halayko, Andrew J. | Hale, Amber N. | Halonen, Sandra K. | Hamasaki, Maho | Han, Feng | Han, Ting | Hancock, Michael K. | Hansen, Malene | Harada, Hisashi | Harada, Masaru | Hardt, Stefan E. | Harper, J. 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Peter | Rodríguez de Córdoba, Santiago | Rohrer, Bärbel | Roninson, Igor B. | Rosen, Kirill | Rost-Roszkowska, Magdalena M. | Rouis, Mustapha | Rouschop, Kasper M.A. | Rovetta, Francesca | Rubin, Brian P. | Rubinsztein, David C. | Ruckdeschel, Klaus | Rucker, Edmund B. | Rudich, Assaf | Rudolf, Emil | Ruiz-Opazo, Nelson | Russo, Rossella | Rusten, Tor Erik | Ryan, Kevin M. | Ryter, Stefan W. | Sabatini, David M. | Sadoshima, Junichi | Saha, Tapas | Saitoh, Tatsuya | Sakagami, Hiroshi | Sakai, Yasuyoshi | Salekdeh, Ghasem Hoseini | Salomoni, Paolo | Salvaterra, Paul M. | Salvesen, Guy | Salvioli, Rosa | Sanchez, Anthony M.J. | Sánchez-Alcázar, José A. | Sánchez-Prieto, Ricardo | Sandri, Marco | Sankar, Uma | Sansanwal, Poonam | Santambrogio, Laura | Saran, Shweta | Sarkar, Sovan | Sarwal, Minnie | Sasakawa, Chihiro | Sasnauskiene, Ausra | Sass, Miklós | Sato, Ken | Sato, Miyuki | Schapira, Anthony H.V. | Scharl, Michael | Schätzl, Hermann M. | Scheper, Wiep | Schiaffino, Stefano | Schneider, Claudio | Schneider, Marion E. | Schneider-Stock, Regine | Schoenlein, Patricia V. | Schorderet, Daniel F. | Schüller, Christoph | Schwartz, Gary K. | Scorrano, Luca | Sealy, Linda | Seglen, Per O. | Segura-Aguilar, Juan | Seiliez, Iban | Seleverstov, Oleksandr | Sell, Christian | Seo, Jong Bok | Separovic, Duska | Setaluri, Vijayasaradhi | Setoguchi, Takao | Settembre, Carmine | Shacka, John J. | Shanmugam, Mala | Shapiro, Irving M. | Shaulian, Eitan | Shaw, Reuben J. | Shelhamer, James H. | Shen, Han-Ming | Shen, Wei-Chiang
Autophagy  2012;8(4):445-544.
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
PMCID: PMC3404883  PMID: 22966490
LC3; autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole
16.  Genome-Wide Microarrray Analysis Reveals Roles for the REF-1 Family Member HLH-29 in Ferritin Synthesis and Peroxide Stress Response 
PLoS ONE  2013;8(3):e59719.
In Caenorhabditis elegans, the six proteins that make up the REF-1 family have been identified as functional homologs of the Hairy/Enhancer of Split (HES) proteins. These transcription factors act in both Notch dependent and Notch-independent pathways to regulate embryonic events during development; however, their post-embryonic functions are not well defined. As a first step toward understanding how the REF-1 family works together to coordinate post-embryonic events, we used gene expression microarray analysis to identify transcriptional targets of HLH-29 in L4/young adult stage animals. Here we show that HLH-29 targets are genes needed for the regulation of growth and lifespan, including genes required for oxidative stress response and fatty acid metabolism, and the ferritin genes, ftn-1 and ftn-2. We show that HLH-29 regulates ftn-1 expression via promoter sequences upstream of the iron-dependent element that is recognized by the hypoxia inducible factor, HIF-1. Additionally, hlh-29 mutants are more resistant to peroxide stress than wild-type animals and ftn-1(RNAi) animals, even in the presence of excess iron. Finally we show that HLH-29 acts parallel to DAF-16 but upstream of the microphthalmia transcription factor ortholog, HLH-30, to regulate ftn-1 expression under normal growth conditions.
PMCID: PMC3606163  PMID: 23533643
17.  Effect of glutamine on apoptosis of intestinal epithelial cells of severe acute pancreatitis rats receiving nutritional support in different ways 
Objective: To investigate the effect of glutamine (Gln) on pro-inflammatory cytokines (TNF-α, IL-2 and IL-10) and the balance between pro-inflammatory cytokines and anti-inflammatory cytokines in severe acute pancreatitis (SAP) rats receiving nutritional support in different ways. Methods: Male SD rats (n=80) were randomly assigned into 5 groups: sham group, SAP+ parenteral nutrition (PN) group, SAP+ enteral nutrition (EN) group, SAP+EN+Gln group and SAP+PN+Gln group. At the same time, rats in 5 groups were sacrificed at 4 and 7 days after nutritional support. ELISA was employed to detect the pro-inflammatory cytokines including TNF-α, IL-2 and IL-10. Results: The serum TNF-α in the EN+Gln group after 7-day treatment was significantly lower than that in the EN, PN and PN+Gln groups at corresponding time point (P<0.05). The serum IL-2 in the EN+Gln group after 7-day treatment was markedly higher than that in the EN, PN and PN+Gln groups at corresponding time point (P<0.01). After 7-day treatment, the serum IL-2 in the EN+Gln and EN groups were markedly higher than that after 4-day treatment (P<0.01), but the serum IL-2 in the PN group was significantly lower than that after 4-day treatment (P<0.01). The serum IL-10 after 7-day treatment was markedly lower than that after 4-day treatment in all groups (P<0.01), and PN group had the lowest serum IL-10. Serum IL-10 in the EN+Gln group was significantly higher than that in the PN and PN+Gln groups at both time points (P<0.01). The serum IL-10 in the EN group was significantly higher than that in the PN group after 4-day treatment (P<0.01), but the serum IL-10 in the EN group was comparable to that in the PN group after 7-day treatment. The serum IL-10/TNF-α in the EN+Gln group was only slightly higher than that in the control group at both time points. The serum IL-10/TNF-α in the EN group was significantly lower than that in the EN+Gln group at both time points (P<0.05). The serum IL-10/TNF-α in the PN group was markedly lower than that in the EN group and EN+Gln group (P<0.05 and P<0.01, respectively).Conclusion: EN in combination with Gln are superior to EN alone, PN alone and PN in combination with Gln in regulating inflammation in SAP rats, and the EN has more potent capability to regulate the balance between pro-inflammation and anti-inflammation than PN.
PMCID: PMC3563192  PMID: 23412711
Pancreatitis; glutamine; inflammatory cytokines; rat
18.  Pervasive and dynamic protein binding sites of the mRNA transcriptome in Saccharomyces cerevisiae 
Genome Biology  2013;14(2):R13.
Protein-RNA interactions are integral components of nearly every aspect of biology, including regulation of gene expression, assembly of cellular architectures, and pathogenesis of human diseases. However, studies in the past few decades have only uncovered a small fraction of the vast landscape of the protein-RNA interactome in any organism, and even less is known about the dynamics of protein-RNA interactions under changing developmental and environmental conditions.
Here, we describe the gPAR-CLIP (global photoactivatable-ribonucleoside-enhanced crosslinking and immunopurification) approach for capturing regions of the untranslated, polyadenylated transcriptome bound by RNA-binding proteins (RBPs) in budding yeast. We report over 13,000 RBP crosslinking sites in untranslated regions (UTRs) covering 72% of protein-coding transcripts encoded in the genome, confirming 3' UTRs as major sites for RBP interaction. Comparative genomic analyses reveal that RBP crosslinking sites are highly conserved, and RNA folding predictions indicate that secondary structural elements are constrained by protein binding and may serve as generalizable modes of RNA recognition. Finally, 38% of 3' UTR crosslinking sites show changes in RBP occupancy upon glucose or nitrogen deprivation, with major impacts on metabolic pathways as well as mitochondrial and ribosomal gene expression.
Our study offers an unprecedented view of the pervasiveness and dynamics of protein-RNA interactions in vivo.
PMCID: PMC4053964  PMID: 23409723
19.  Akt1 Mediates Neuronal Differentiation in Zebrafish via a Reciprocal Interaction with Notch Signaling 
PLoS ONE  2013;8(1):e54262.
Akt1 is well known for its role in regulating cell proliferation, differentiation, and apoptosis and is implicated in tumors and several neurological disorders. However, the role of Akt1 in neural development has not been well defined. We have isolated zebrafish akt1 and shown that this gene is primarily transcribed in the developing nervous system, and its spatiotemporal expression pattern suggests a role in neural differentiation. Injection of akt1 morpholinos resulted in loss of neuronal precursors with a concomitant increase in post-mitotic neurons, indicating that knockdown of Akt1 is sufficient to cause premature differentiation of neurons. A similar phenotype was observed in embryos deficient for Notch signaling. Both the ligand (deltaA) and the downstream target of Notch (her8a) were downregulated in akt1 morphants, indicating that Akt1 is required for Delta-Notch signaling. Furthermore, akt1 expression was downregulated in Delta-Notch signaling-deficient embryos and could be induced by constitutive activation of Notch signaling. In addition, knockdown of Akt1 was able to nullify the inhibition of neuronal differentiation caused by constitutive activation of Notch signaling. Taken together, these results provide in vivo evidence that Akt1 interacts with Notch signaling reciprocally and provide an explanation of why Akt1 is essential for the inhibition of neuronal differentiation.
PMCID: PMC3544797  PMID: 23342113
20.  Comparative Effects of Er-Xian Decoction, Epimedium Herbs, and Icariin with Estrogen on Bone and Reproductive Tissue in Ovariectomized Rats 
Er-Xian Decoction (EXD), Epimedium herbs (herbs of Epimedium brevicornum Maxim, EBH), and icariin (ICA) have been proven to have estrogen-like and antiosteoporotic activity and are used for the treatment of osteoporosis, menopausal syndrome, and age-associated diseases. The present study found that EXD, EBH, and ICA treatments, emulating estrogen, significantly contributed to bone density and architecture in OVX rats and that EXD is similar to estrogen and exerts a concomitant effect on bone formation and bone resorption at the tissue level, while EBH and ICA produced bone-protective effects mainly by inhibiting bone resorption. Nevertheless, EXD, EBH, and ICA treatments manifested a fewer adverse effects on the uterus, mammary gland, and vagina compared to estrogen administrations. Among the EXD, EBH, and ICA, EXD was found to have superior efficacy and safety profile.
PMCID: PMC3511842  PMID: 23227099
21.  Effects and Interaction of Icariin, Curculigoside, and Berberine in Er-Xian Decoction, a Traditional Chinese Medicinal Formula, on Osteoclastic Bone Resorption 
Er-Xian decoction (EXD), a traditional Chinese medicine, has been reported to have a protective effect against bone loss in ovariectomized osteoporotic rats, and the inclusion of icariin (I), curculigoside (C), and berberine (B) in EXD displays inhibitory effects on osteoclastic bone resorption. In the present paper, we investigated the interaction and effects of I, C, B, and their combination on bone resorption activity in vitro on osteoclasts derived from rat bone marrow cells. ICB synergistically decreased the formation of bone resorption pits, the number of multinucleated osteoclasts, and the activity of tartrate-resistant acid phosphatase (TRAP) and showed antagonistic or additive effects on cathepsin K activity in the coculture system of osteoblasts and bone marrow cells in the presence of 1, 25-dihydroxyvitamin D3 and dexamethasone. The combination of ICB also enhanced the inhibitory effects on the formation of F-actin ring, a cytoskeleton structure of osteoclasts induced from bone marrow cells with macrophage colony stimulation factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). In addition, ICB synergistically improved the ratio of protein expression of osteoprotegerin (OPG) and RANKL in osteoblasts and interfered with the mitogen-activated protein kinases (MAPKs) pathway in osteoclast. These results clearly show that I, C, B, and their combination in EXD exert effects of mutual reinforcement. However, IBC does not show an intensified adverse effect in the ovariectomized murine model, as revealed by change in body and uterine weight, confirming the safety of EXD. These observations are in agreement with the rationality of the formula used in this paper.
PMCID: PMC3519276  PMID: 23243450
22.  Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation 
ASN NEURO  2012;4(6):e00098.
The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.
PMCID: PMC3449306  PMID: 22909302
Akt (protein kinase B); dopamine D2S receptor; glycogen synthase kinase 3; nucleus accumbens; receptor internalization; CNS, central nervous system; ConA, concanavalin A; CREB, cAMP-response-element-binding protein; Cy3, indocarbocyanine; DA, dopamine; DAPI, 4′,6-diamidino-2-phenylindole; EGF, epidermal growth factor; ERK, extracellular-signal-regulated kinase; GSK, glycogen synthase kinase; HEK, human embryonic kidney; HRP, horseradish peroxidase; MAPK, mitogen-activated protein kinase; MDC, monodansylcadaverine; MEK, MAPK/ERK kinase; MEM, minimum essential medium; METH, methamphetamine; Nrf2, nuclear factor-erythroid 2-related factor 2; PDGF, platelet-derived growth factor; PI3K, phosphoinositide 3-kinase
23.  The association between online gaming, social phobia, and depression: an internet survey 
BMC Psychiatry  2012;12:92.
Online gaming technology has developed rapidly within the past decade, and its related problems have received increasing attention. However, there are few studies on the psychiatric symptoms associated with excessive use of online games. The aim of this study is to investigate the characteristics of online gamers, and the association between online gaming hours, social phobia, and depression using an internet survey.
An online questionnaire was designed and posted on a popular online game websites, inviting the online gamers to participate the survey. The content of the questionnaire included demographic data, profiles of internet usage and online gaming, and self-rating scales of Depression and Somatic Symptoms Scale (DSSS), Social Phobia Inventory (SPIN), and Chen Internet Addiction Scale (CIAS).
A total of 722 online gamers with a mean age of 21.8 ± 4.9 years completed the online survey within one month. 601 (83.2%) participants were male, and 121 (16.8%) were female. The mean weekly online gaming time was 28.2 ± 19.7 hours, which positively associated with history of online gaming (r = 0.245, p < 0.001), total DSSS (r = 0.210, p < 0.001), SPIN (r = 0.150, p < 0.001), and CIAS (r = 0.290, p < 0.001) scores. The female players had a shorter history of online gaming (6.0 ± 3.1 vs. 7.2 ± 3.6 years, p = 0.001) and shorter weekly online gaming hours (23.2 ± 17.0 vs. 29.2 ± 20.2 hours, p = 0.002), but had higher DSSS (13.0 ± 9.3 vs. 10.9 ± 9.7, p = 0.032) and SPIN (22.8 ± 14.3 vs. 19.6 ± 13.5, p = 0.019) scores than the male players. The linear regression model showed that higher DSSS scores were associated with female gender, higher SPIN scores, higher CIAS scores, and longer weekly online gaming hours, with controlling for age and years of education.
The online gamers with longer weekly gaming hours tended to have a longer history of online gaming, and more severe depressive, social phobic, and internet addiction symptoms. Female online gamers had fewer weekly online gaming hours and a shorter previous online gaming history, but tended to have more severe somatic, pain, and social phobic symptoms. The predictors for depression were higher social phobic symptom, higher internet addiction symptoms, longer online gaming hours, and female gender.
PMCID: PMC3545926  PMID: 22839747
24.  The Caenorhabditis elegans HEN1 Ortholog, HENN-1, Methylates and Stabilizes Select Subclasses of Germline Small RNAs 
PLoS Genetics  2012;8(4):e1002617.
Small RNAs regulate diverse biological processes by directing effector proteins called Argonautes to silence complementary mRNAs. Maturation of some classes of small RNAs involves terminal 2′-O-methylation to prevent degradation. This modification is catalyzed by members of the conserved HEN1 RNA methyltransferase family. In animals, Piwi-interacting RNAs (piRNAs) and some endogenous and exogenous small interfering RNAs (siRNAs) are methylated, whereas microRNAs are not. However, the mechanisms that determine animal HEN1 substrate specificity have yet to be fully resolved. In Caenorhabditis elegans, a HEN1 ortholog has not been studied, but there is evidence for methylation of piRNAs and some endogenous siRNAs. Here, we report that the worm HEN1 ortholog, HENN-1 (HEN of Nematode), is required for methylation of C. elegans small RNAs. Our results indicate that piRNAs are universally methylated by HENN-1. In contrast, 26G RNAs, a class of primary endogenous siRNAs, are methylated in female germline and embryo, but not in male germline. Intriguingly, the methylation pattern of 26G RNAs correlates with the expression of distinct male and female germline Argonautes. Moreover, loss of the female germline Argonaute results in loss of 26G RNA methylation altogether. These findings support a model wherein methylation status of a metazoan small RNA is dictated by the Argonaute to which it binds. Loss of henn-1 results in phenotypes that reflect destabilization of substrate small RNAs: dysregulation of target mRNAs, impaired fertility, and enhanced somatic RNAi. Additionally, the henn-1 mutant shows a weakened response to RNAi knockdown of germline genes, suggesting that HENN-1 may also function in canonical RNAi. Together, our results indicate a broad role for HENN-1 in both endogenous and exogenous gene silencing pathways and provide further insight into the mechanisms of HEN1 substrate discrimination and the diversity within the Argonaute family.
Author Summary
Small RNAs serve as sentinels of the genome, policing activity of selfish genetic elements, modulating chromatin dynamics, and fine-tuning gene expression. Nowhere is this more important than in the germline, where endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) promote formation of functional gametes and ensure viable, fertile progeny. Small RNAs act primarily by associating with effector proteins called Argonautes to direct repression of complementary mRNAs. HEN1 methyltransferases, which methylate small RNAs, play a critical role in accumulation of these silencing signals. In this study, we report that the 26G RNAs, a class of C. elegans endo-siRNAs, are differentially methylated in male and female germlines. 26G RNAs derived from the two germlines are virtually indistinguishable, except that they associate with evolutionarily divergent Argonautes. Our data support a model wherein the methylation status and, consequently, stability of a small RNA are determined by the associated Argonaute. Therefore, selective expression of Argonautes that permit or prohibit methylation may represent a new mechanism for regulating small RNA turnover. As we observe this phenomenon in the germline, it may be particularly pertinent for directing inheritance of small RNAs, which can carry information not encoded in progeny DNA that is essential for continued transgenerational genome surveillance.
PMCID: PMC3330095  PMID: 22548001
25.  Mechanical pain sensitivity of deep tissues in children - possible development of myofascial trigger points in children 
It is still unclear when latent myofascial trigger points (MTrPs) develop during early life. This study is designed to investigate the mechanical pain sensitivity of deep tissues in children in order to see the possible timing of the development of latent MTrPs and attachment trigger points (A-TrPs) in school children.
Five hundreds and five healthy school children (age 4- 11 years) were investigated. A pressure algometer was used to measure the pressure pain threshold (PPT) at three different sites in the brachioradialis muscle: the lateral epicondyle at elbow (site A, assumed to be the A-TrP site), the mid-point of the muscle belly (site B, assumed to be the MTrP site), and the muscle-tendon junction as a control site (site C).
The results showed that, for all children in this study, the mean PPT values was significantly lower (p < 0.05) at the assumed A-TrP site (site A) than at the other two sites, and was significantly lower (p < 0.05) at the assumed MTrP site (site B) than at the control site (site C). These findings are consistent if the data is analyzed for different genders, different dominant sides, and different activity levels.
It is concluded that a child had increased sensitivity at the tendon attachment site and the muscle belly (endplate zone) after age of 4 years. Therefore, it is likely that a child may develop an A-Trp and a latent MTrP at the brachioradialis muscle after the age of 4 years. The changes in sensitivity, or the development for these trigger points, may not be related to the activity level of children aged 7-11 years. Further investigation is still required to indentify the exact timing of the initial occurrence of a-Trps and latent MTrPs.
PMCID: PMC3298468  PMID: 22316064
myofascial trigger points; children; pressure pain threshold; algometry

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