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NMR Innovation Offers Insights into Protein Interaction

By MedImaging International staff writers
Posted on 17 Jul 2013

Image: A permissive captor A computer rendering depicts a GroEL protein hunting down an Aβ protein. Four complementary styles of nuclear magnetic resonance spectroscopy contributed to an understanding of the protein-to-protein interaction (Photo courtesy of Fawzi lab at Brown University).

By taking a novel approach to nuclear magnetic resonance spectroscopy--a fusion of four techniques--scientists have been able to resolve a key interaction between two proteins that could never be seen before.

The findings were published the week of June 24, 2013, in the Proceedings of the National Academy of Sciences of the United States of America (PNAS). The interaction the researchers became the first ones to describe is nearly universal across all of life. A protein unit called a chaperone takes hold of a disordered smaller protein to help it find its correct folded conformation. The scientists, in this instance, initiated test-tube experiments where they hoped to visualize the capsule-shaped bacterial chaperone GroEL capture a disordered amyloid beta (A-beta) protein, a molecule that in humans is central in Alzheimer's disease.

The two proteins are well researched, but the motions they go through when they first meet, i.e., when the open GroEL capsule captures its target, have been invisible to scientists. Electron microscopy and X-ray crystallography are only good for taking snapshots of easily frozen moments in time. NMR is capable of sensing the interactions and kinetics of protein interactions as they occur, but in some cases, any single technique can provide only clues into what is actually going on.

Brown University (Providence, RI, USA) biologist Dr. Nicolas Fawzi, who was a post-doc in the group of Marius Clore’s at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) within the US National Institutes of Health (NIH; Bethesda, MD, USA), worked with coauthors and NIDDK researchers Drs. David Libich, Jinfa Yang, and Marius Clore assembling the interactions of the proteins by combining four different NMR techniques. They determined what each one could inform them about the interaction and built the case presented in PNAS.

“None of the four techniques alone gave us sufficient information,” said Dr. Fawzi, now an assistant professor in Brown’s department of molecular pharmacology, physiology, and biotechnology. “Only by using them all together would we be able to figure out the structure and motions of A-beta; when it was bound to GroEL. By having four indirect measurements together, that was able to give us a complete picture.”

The NMR techniques they used were lifetime line broadening, Carr-Purcell-Meinboom-Gill (CPMG) relaxation dispersion spectroscopy, and exchange-induced chemical shifts. “The fourth technique we employed was dark-state exchange saturation transfer [DEST] spectroscopy, which we had developed in my lab at the NIH in 2011,” said Dr. Clore, also the article’s corresponding author. “We were able to more effectively conduct our research by using that tool to corroborate and extend the information afforded by the other three measurements.”

The elusive process debated among molecular biologists was about what the GroEL chaperone requires of its captives at the moment they engage. Does it force them into a specific conformation? Does it hold on tightly while it closes its capsule lid around the smaller protein, or does the captive stay in motion at all?

What the investigators observed is that the GroEL is a permissive captor. It bound A-beta; at just two hydrophobic sites, leaving the smaller protein to otherwise swing in a range of conformations. It also did not keep it bound the entire time, letting it instead detach and re-bind. Basically, A-beta would jump off and on within GroEL’s binding cavity.

“By using these four techniques together we were able to extract information about the structure of the protein while it binds as well as how fast it comes on and off and what it's doing at each position,” Dr. Fawzi said. “Instead of forming more particular structure upon binding it appears to retain great conformational heterogeneity.”

The lifetime line-broadening technique, for example, informed them that the A-beta; was interacting with something big (GroEL), while the CPMG and chemical shift observations combined to show the length of time A-beta; spent on GroEL before unbinding, as well as the structural characteristics of A-beta; when it was bound to GroEL. DEST provided data that could validate much of the story of the other techniques.

Dr. Fawzi noted that GroEL’s relaxed strategy could be a matter of being able to bind many different proteins in disordered conformations, but also of saving energy. Forcing proteins into a specific conformation just to make and sustain the initial capture would require more energy than it is worth. Eventually, in moments after those the team resolved in this study, GroEL closes its lid and encapsulates its target proteins fully, according to Dr. Fawzi said. Thatis when it capitalizes on in compelling them to fold in the correct manner.

For molecular and structural biologists, the newly proven combination of NMR techniques could create a number of other cold cases of elusive interactions. “We can now look at how these big machines can do their job while they are working,” Dr. Fawzi said. “This is not just limited to this GroEL machine.”

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51 (68.187713623047): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

52 (142.09747314453): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

53 (74.863433837891): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

54 (693.08280944824): SELECT name FROM jos_deeppockets WHERE id=294747273 LIMIT 1

55 (225.06713867188): SELECT name FROM jos_deeppockets_archive WHERE id=294747273 LIMIT 1

56 (63.180923461914): SELECT a.* FROM jos_components AS a WHERE ( a.admin_menu_link = 'option=com_syndicate' OR a.admin_menu_link = 'option=com_syndicate&hidemainmenu=1' ) AND a.option = 'com_syndicate'

57 (106.0962677002): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.cat=10 AND dp.id=294747273 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

58 (571.01249694824): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets_archive as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.cat=10 AND dp.id=294747273 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

59 (!!!!! 1318.9315795898): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

60 (69.141387939453): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

61 (152.8263092041): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

62 (72.002410888672): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

63 (122.0703125): select cover from jos_number where journalid in (5) and cover is not null order by left(issue,2)+right(issue,2)*12 desc limit 1

64 (580.78765869141): SELECT fid FROM jos_deeppockets WHERE id=294747273 AND published='1' union SELECT fid FROM jos_deeppockets_archive WHERE id=294747273 AND published='1' LIMIT 1

65 (49.114227294922): SELECT id,name,sponsor_text FROM jos_deeppockets dp left outer join jos_channel_sponsor cs on dp.id=cs.fid WHERE cat=10 AND TYPE='file_folder' AND published=1 AND id=213 ORDER BY `ordering`

66 (60.081481933594): select jos_twitters.link from jos_twitters where cat=10

67 (30.994415283203): select jos_facebook.link from jos_facebook where cat=10

68 (31.948089599609): select jos_google_plus.link from jos_google_plus where cat=10

69 (30.040740966797): select jos_linkedin.link from jos_linkedin where cat=10

70 (54.121017456055): SELECT fid FROM jos_deeppockets WHERE id=294747273 AND published='1' union SELECT fid FROM jos_deeppockets_archive WHERE id=294747273 AND published='1' LIMIT 1

71 (61.988830566406): SELECT id,name,sponsor_text FROM jos_deeppockets dp left outer join jos_channel_sponsor cs on dp.id=cs.fid WHERE cat=10 AND TYPE='file_folder' AND published=1 AND id=213 ORDER BY `ordering`

72 (324.96452331543): SELECT fid FROM jos_deeppockets WHERE cat=10 AND id=294747273 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

73 (229.1202545166): SELECT fid FROM jos_deeppockets_archive WHERE cat=10 AND id=294747273 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

74 (46.014785766602): SELECT id,name,title FROM jos_deeppockets WHERE cat=10 AND TYPE='file_folder' AND published=1 AND id != 294743300 AND id != 294743301 AND id != 294743302 AND id != 294743303 AND id != 294743304 AND id != 294743305 AND id != 294743306 AND id != 294743307 AND id != 294743308 AND id != 294743309 AND id != 294743310 AND metakey != 'aacc' ORDER BY `ordering`

75 (77.962875366211): SELECT show_live FROM jos_com_show_setting LIMIT 1

76 (45.061111450195): SELECT show_live FROM jos_com_show_setting LIMIT 1

77 (31.948089599609): SELECT show_live FROM jos_com_show_setting LIMIT 1

78 (27.894973754883): SELECT show_live FROM jos_com_show_setting LIMIT 1

79 (26.226043701172): SELECT show_live FROM jos_com_show_setting LIMIT 1

80 (25.98762512207): SELECT show_live FROM jos_com_show_setting LIMIT 1

81 (25.98762512207): SELECT show_live FROM jos_com_show_setting LIMIT 1

82 (26.941299438477): SELECT show_live FROM jos_com_show_setting LIMIT 1

83 (25.98762512207): SELECT show_live FROM jos_com_show_setting LIMIT 1

84 (26.941299438477): SELECT show_live FROM jos_com_show_setting LIMIT 1

85 (25.98762512207): SELECT show_live FROM jos_com_show_setting LIMIT 1

86 (26.226043701172): SELECT show_live FROM jos_com_show_setting LIMIT 1

87 (25.98762512207): SELECT show_live FROM jos_com_show_setting LIMIT 1

88 (25.98762512207): SELECT show_live FROM jos_com_show_setting LIMIT 1

89 (815.86837768555): SELECT fid FROM jos_deeppockets WHERE cat=10 AND id=294747273 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

90 (46.014785766602): SELECT fid FROM jos_deeppockets_archive WHERE cat=10 AND id=294747273 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

91 (38.14697265625): SELECT id,name,title FROM jos_deeppockets WHERE cat=10 AND TYPE='file_folder' AND published=1 AND id != 294743300 AND id != 294743301 AND id != 294743302 AND id != 294743303 AND id != 294743304 AND id != 294743305 AND id != 294743306 AND id != 294743307 AND id != 294743308 AND id != 294743309 AND id != 294743310 AND metakey != 'aacc' ORDER BY `ordering`

92 (!!!!! 2153.8734436035): SELECT show_live FROM jos_com_show_setting LIMIT 1

93 (82.015991210938): SELECT show_live FROM jos_com_show_setting LIMIT 1

94 (41.007995605469): SELECT show_live FROM jos_com_show_setting LIMIT 1

95 (31.948089599609): SELECT show_live FROM jos_com_show_setting LIMIT 1

96 (45.061111450195): SELECT show_live FROM jos_com_show_setting LIMIT 1

97 (32.901763916016): SELECT show_live FROM jos_com_show_setting LIMIT 1

98 (77.962875366211): SELECT show_live FROM jos_com_show_setting LIMIT 1

99 (40.054321289062): SELECT show_live FROM jos_com_show_setting LIMIT 1

100 (32.186508178711): SELECT show_live FROM jos_com_show_setting LIMIT 1

101 (!!!!! 1752.1381378174): SELECT show_live FROM jos_com_show_setting LIMIT 1

102 (56.028366088867): SELECT show_live FROM jos_com_show_setting LIMIT 1

103 (31.948089599609): SELECT show_live FROM jos_com_show_setting LIMIT 1

104 (43.153762817383): SELECT show_live FROM jos_com_show_setting LIMIT 1

105 (40.054321289062): SELECT show_live FROM jos_com_show_setting LIMIT 1

106 (!!!!! 5213.9759063721): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

107 (167.1314239502): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

108 (133.99124145508): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.cat=10 AND dp.id=294747273 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

109 (74.863433837891): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets_archive as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.cat=10 AND dp.id=294747273 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

110 (!!!!! 4041.9101715088): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

111 (141.14379882812): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

112 (386.95335388184): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.id=294747273 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

113 (350.9521484375): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets_archive as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.id=294747273 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

114 (83.92333984375): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.id=294747272 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

115 (376.93977355957): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets_archive as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.id=294747272 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

116 (900.9838104248): SELECT dp.name, dp.id, dp2.name as channel, ex.name as author_name, ex.specialization, ex.images FROM jos_deeppockets as dp LEFT JOIN jos_experts as ex ON ex.id = dp.author LEFT JOIN jos_deeppockets as dp2 ON dp2.id = dp.fid WHERE dp.author != 0 AND dp.cat = 10 AND dp.published = 1 AND ( (dp.fid = 294743300) OR (dp.fid = 294743301) OR (dp.fid = 294743302) OR (dp.fid = 294743303) OR (dp.fid = 294743304) OR (dp.fid = 294743305) OR (dp.fid = 294743306) OR (dp.fid = 294743307) OR (dp.fid = 294743308) OR (dp.fid = 294743309) OR (dp.fid = 294743310) ) ORDER BY RAND() LIMIT 1

117 (727.89192199707): SELECT dp.name, dp.id, dp2.name as channel, ex.name as author_name, ex.specialization, ex.images FROM jos_deeppockets as dp LEFT JOIN jos_experts as ex ON ex.id = dp.author LEFT JOIN jos_deeppockets as dp2 ON dp2.id = dp.fid WHERE dp.author != 0 AND dp.cat = 10 AND dp.published = 1 AND ( (dp.fid = 294743300) OR (dp.fid = 294743301) OR (dp.fid = 294743302) OR (dp.fid = 294743303) OR (dp.fid = 294743304) OR (dp.fid = 294743305) OR (dp.fid = 294743306) OR (dp.fid = 294743307) OR (dp.fid = 294743308) OR (dp.fid = 294743309) OR (dp.fid = 294743310) ) ORDER BY RAND() LIMIT 1

118 (!!!!! 5036.8309020996): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

119 (123.02398681641): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

120 (144.00482177734): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

121 (66.995620727539): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

122 (101.08947753906): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

123 (63.896179199219): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

124 (83.92333984375): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

125 (63.896179199219): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

126 (524.99771118164): SELECT * FROM trending_articles WHERE `journal_id` = 10 AND `timestamp` <= NOW() + INTERVAL 7 DAY ORDER BY `order`

127 (!!!!! 57790.040969849): SELECT * FROM jos_deeppockets WHERE jos_deeppockets.cat = 10 AND jos_deeppockets.type = 'deeppockets_item' AND jos_deeppockets.published = 1 AND jos_deeppockets.created + INTERVAL 14 DAY >= NOW() ORDER BY jos_deeppockets.hits DESC LIMIT 7

128 (594.13909912109): SELECT fid FROM jos_deeppockets WHERE cat=10 AND id=294747273 AND type='deeppockets_item' AND published='1' union SELECT fid FROM jos_deeppockets_archive WHERE cat=10 AND id=294747273 AND type='deeppockets_item' AND published='1' LIMIT 1

129 (!!!!! 20519.971847534): ( SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.video != '' AND dp.video_source != '' AND dp.cat = 10 AND dp.published = 1 AND dp.type != 'deeppockets_item_link' ORDER BY created DESC LIMIT 3 ) UNION ( SELECT dpa.id, dpa.name, dpa.title, dpa.video, dpa.video_source, dpa.created FROM jos_deeppockets_archive AS dpa WHERE dpa.video != '' AND dpa.video_source != '' AND dpa.cat = 10 AND dpa.published = 1 AND dpa.type != 'deeppockets_item_link' ORDER BY created DESC LIMIT 3 ) ORDER BY created DESC LIMIT 3

130 (76.055526733398): ( SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.video != '' AND dp.video_source != '' AND dp.cat = 10 AND dp.published = 1 AND dp.type != 'deeppockets_item_link' ORDER BY created DESC LIMIT 3 ) UNION ( SELECT dpa.id, dpa.name, dpa.title, dpa.video, dpa.video_source, dpa.created FROM jos_deeppockets_archive AS dpa WHERE dpa.video != '' AND dpa.video_source != '' AND dpa.cat = 10 AND dpa.published = 1 AND dpa.type != 'deeppockets_item_link' ORDER BY created DESC LIMIT 3 ) ORDER BY created DESC LIMIT 3

131 (61.988830566406): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.id = 294803337 LIMIT 1

132 (44.107437133789): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.id = 294803337 LIMIT 1

133 (94.890594482422): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.id = 294803276 LIMIT 1

134 (91.075897216797): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.id = 294803276 LIMIT 1

135 (427.0076751709): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets_archive AS dp WHERE dp.id = 294803276 LIMIT 1

136 (56.028366088867): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets_archive AS dp WHERE dp.id = 294803276 LIMIT 1

137 (42.915344238281): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.id = 294802958 LIMIT 1

138 (34.093856811523): SELECT dp.id, dp.name, dp.title, dp.video, dp.video_source, dp.created FROM jos_deeppockets AS dp WHERE dp.id = 294802958 LIMIT 1

139 (!!!!! 1092.1955108643): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

140 (572.91984558105): SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

141 (!!!!! 3304.9583435059): SELECT name FROM jos_deeppockets WHERE id=213

142 (127.07710266113): SELECT name FROM jos_deeppockets WHERE id=213

143 (!!!!! 1039.0281677246): SELECT *, DATE_FORMAT(publish_up,'%d %b %Y') AS date_publish_up, DATE_FORMAT(publish_down,'%d %b %Y') AS date_publish_down FROM jos_events WHERE state=1 AND publish_up > NOW() and extra_info REGEXP '((;)|(^))213((;)|($))' AND catid IN (0,110) ORDER BY publish_up ASC LIMIT 3

144 (792.02651977539): SELECT *, DATE_FORMAT(publish_up,'%d %b %Y') AS date_publish_up, DATE_FORMAT(publish_down,'%d %b %Y') AS date_publish_down FROM jos_events WHERE state=1 AND publish_up > NOW() and extra_info REGEXP '((;)|(^))213((;)|($))' AND catid IN (0,110) ORDER BY publish_up ASC LIMIT 3

145 (122.0703125): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

146 (75.817108154297): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

147 (!!!!! 21389.007568359): TRUNCATE TABLE jos_trademed_module

148 (!!!!! 2534.1510772705): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.cat=10 AND dp.id=294747273 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

149 (987.05291748047): SELECT dp.*, ch.title as channel, exp.name as author_name, exp.specialization as author_specialization, exp.images as author_photo FROM jos_deeppockets_archive as dp LEFT JOIN jos_experts as exp ON exp.id = dp.author LEFT JOIN jos_deeppockets as ch ON dp.fid = ch.id WHERE 1=1 AND dp.cat=10 AND dp.id=294747273 AND (dp.type='deeppockets_item' OR dp.type='deeppockets_item_link') AND dp.published='1' LIMIT 1

150 (!!!!! 1372.0989227295): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

151 (119.92454528809): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND (type='deeppockets_item' OR type='deeppockets_item_link') AND published='1' LIMIT 1

152 (!!!!! 3540.0390625): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

153 (164.03198242188): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

154 (!!!!! 1977.9205322266): SELECT jos_item.* FROM jos_item INNER JOIN jos_number ON jos_item.numberID=jos_number.id WHERE 1=1 AND (jos_item.imgMini<>'') AND jos_item.type='L' AND jos_item.numberID= ( SELECT jos_number.id FROM jos_number WHERE jos_number.journalID IN (10) and jos_number.id in (select distinct jos_number.id from jos_number join jos_item on jos_item.numberid=jos_number.id where jos_item.type='P') ORDER BY (CONVERT(SUBSTRING(issue,1,2),SIGNED) + (CONVERT(SUBSTRING(issue,3,2),SIGNED)*100)) DESC LIMIT 0,1 ) ORDER BY RAND() DESC LIMIT 6

155 (118.01719665527): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

156 (80.82389831543): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

157 (102.99682617188): SELECT fid FROM jos_deeppockets WHERE cat=10 AND id=294747273 AND type='deeppockets_item' AND published='1' union SELECT fid FROM jos_deeppockets_archive WHERE cat=10 AND id=294747273 AND type='deeppockets_item' AND published='1' LIMIT 1

158 (64.13459777832): select distinct links.* from jos_textlinks links join jos_textlinks_placement placement on links.id=placement.link_id where journal_id=(10) and (channel_id=0 or channel_id is null)

159 (51.975250244141): select distinct links.* from jos_textlinks links join jos_textlinks_placement placement on links.id=placement.link_id where journal_id=(10) and (channel_id=0 or channel_id is null)

160 (56.982040405273): SELECT livesite FROM jos_dp_categories WHERE id=10 AND published='1' LIMIT 1

161 (!!!!! 1961.9464874268): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

162 (164.03198242188): SELECT * FROM jos_deeppockets WHERE 1=1 AND id=294747273 AND published='1' union SELECT * FROM jos_deeppockets_archive WHERE 1=1 AND id=294747273 AND published='1' LIMIT 1

163 (89.883804321289): select jos_twitters.link from jos_twitters where cat=10

164 (45.061111450195): select jos_facebook.link from jos_facebook where cat=10

165 (45.061111450195): select jos_google_plus.link from jos_google_plus where cat=10

166 (41.961669921875): select jos_linkedin.link from jos_linkedin where cat=10

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