The present study sought to determine the signaling mechanisms by which inhibition of PKA activates β2-AR stimulation of cPLA2. We therefore determined the effects of zinterol  to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 μM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 μM GW5074), phospholipase C , PKC (4 μM chelerythrine) or IP3 receptor (IP3R) signaling (2 μM 2-APB) significantly inhibited zint-β2-AR stimulation of ICa,L in–PKA but not +PKA myocytes. Western blots showed that zint-β2-AR stimulation increased ERK1/2 phosphorylation in–PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-β2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-βgal. In +LMN myocytes, zint-β2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-βgal. In–PKA myocytes depletion of intracellular Ca2+ stores by 5 μM thapsigargin failed to inhibit zint-β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-β-cyclodextrin inhibited zint-β2-AR stimulation of ICa,L in–PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows β2-AR stimulation to act via PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of β-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates β Cell attachment to LMN decreases β1-AR signaling by inhibiting adenylate cyclase activity and diminishing cAMP levels via integrin-dependent activation of focal adhesion kinase (FAK)/phosphatidyinositol-3’ kinase (PI-3K)/protein kinase B (Akt) signaling . β2-AR activation of cPLA2 signaling is dependent on concomitant LMN-mediated inhibition of adenylate cyclase/cAMP-dependent kinase (PKA) [3]. In other words, cell attachment to LMN acts via inhibition of adenylate cyclase/PKA to both inhibit β1-AR signaling and enhance β2-AR signaling through activation of cPLA2. In embryonic chick ventricular myocytes [4] and rat ventricular myocytes [5] β2-AR stimulation also activates cPLA2/AA signaling. Moreover, these authors proposed that activation of β2-AR/cPLA2 signaling may compensate for depressed cAMP signaling [4]. Interestingly, in both of these studies by Pavoine et al. (1999) and Ait-Mamar et al., (2005) cardiomyocytes were cultured on LMN, supporting our findings that cell attachment to LMN may be responsible for inhibition of PKA and activation of β2-AR/cPLA2 signaling. However, the mechanism by which PKA inhibition activates β2-AR/cPLA2 signaling is not clear.We previously reported that attachment of atrial myocytes to the extracellular matrix protein laminin (LMN) acts via β2-AR activation of cPLA2 is Ca2+-dependent and mediated via ERK1/2 signaling [3]. This is consistent with studies in embryonic chick ventricular myocytes (cultured on LMN) in which β2-AR stimulation acts via ERK1/2 signaling to activate cPLA2 [6]. Moreover, in a variety of cell systems Raf-1 activates downstream ERK1/2 and PKA inhibits Raf-1 .Once fully anesthetized, a bilateral thoracotomy was performed, and the heart was rapidly excised and mounted on a Langendorff perfusion apparatus. After enzyme  digestion, atrial myocytes were isolated as previously reported [11]. L-type Ca2+ current  was activated by depolarizing pulses from a holding potential of -40 mV to 0 mV for 200 ms every 5 s and measured in relation to steady-state current. β2-AR stimulation was achieved by 0.1 μM zinterol (zint-β2-AR), a specific β2-AR agonist [12]. Agonist was applied for approximately 4 min and the effects on peak ICa,L amplitude were recorded at the steady-state response.Electrophysiological recordings from atrial myocytes were performed in the perforated (nystatin) patch whole-cell configuration at room temperature, as previously described [3]; atrial myocytes on uncoated glass cover-slips in the absence of PKA inhibitor (+PKA) and atrial myocytes on uncoated glass coverslips exposed to the specific PKA inhibitor (1 μM) KT5720 (-PKA). Because pharmacological inhibition of PKA elicits signaling mechanisms that are similar to those elicited by LMN-integrin signaling, we performed some experiments on atrial myocytes attached to glass cover-slips coated with laminin  for at least 2 hrs, as previously described , consistent with the relatively high endogenous PKA activity in cat atrial myocytes [11]. In addition, a variety of experimental results indicate that atrial cell attachment to LMN is not restoring LMN-mediated signaling somehow lost during the cell isolation procedure. For example, control experiments have shown that atrial myocytes plated on poly-L-lysine, a non-specific substrate for cell attachment, fail to exhibit changes in β-AR signaling similar to cells attached to LMN [1]. Moreover, freshly isolated cardiomyocytes not plated on LMN exhibit responses to β-AR stimulation which are similar to multicellular cardiac preparations, i.e. exhibit predominantly β1-AR over β2-AR signaling [1]. However, cell attachment to LMN decreases the β1-/β2-AR signaling ratio resulting in predominantly β2-AR over β1-AR signaling , kindly provided by Dr. Trevor Biden, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney Australia. An Adv expressing a dominant-negative (dn) mutant of rabbit PKCε  were generously provided by Dr. K.U. Malik, University of Tennessee Health Science Center, Memphis, TN. An Adv expressing a dn mutant of human Raf-1 [9] was kindly provided by Dr. Dan Kuppuswamy, Medical University of South Carolina, Charleston, SC. A control Adv expressing nuclear-encoded β-galactosidase  was used to control for nonspecific effects of adenoviral infection [16]. Adenoviruses were amplified and purified using HEK293 cells, and the multiplicity of infection (moi) for each virus was determined by dilution assay in HEK293 cells grown in 96 well clusters, as previously described [16]. Preliminary experiments using 5-bromo-4-chloro-3-indolyl-beta- D-galactopyranoside  staining of Adv-βgal infected cells determined that a concentration of 100 moi infected 93±3%  of cultured myocytes. The IP3 affinity trap consists of the ligand binding domain of the rat type 1 IP3R. The construction of this vector and subsequent production of adenovirus was previously described in detail [17]. Freshly isolated atrial myocytes were infected with Adv-IP3 affinity trap or the Adv-βgal (control) for 1 h, followed by 18 h short-term culture at 37°C.In some experiments, atrial myocytes were attached to laminin (2h) and then infected  with replication-defective adenovirus (Adv) prior to electrophysiological recording. PKCα was inhibited by infection with an Adv expressing kinase-inactive mouse PKCα  on zint-β2-AR stimulation of ICa,L in +PKA and–PKA atrial myocytes. In +PKA myocytes (A) 0.1 μM zint-β2-AR stimulation elicited a typical increase in ICa,L . In another group of +PKA myocytes from the same hearts (A), prior exposure to GW5074 for 30 min had no significant effects on basal ICa,L amplitude (open bar) or zint-β2-AR stimulation of ICa,L . In contrast, as shown in panel B, control zint-β2-AR stimulation of ICa,L in–PKA myocytes  was enhanced compared to +PKA myocytes  due to activation of cPLA2 signaling, as previously reported . In–PKA myocytes, GW5074 had no effects on basal ICa,L amplitudes (open bars) but in contrast to +PKA myocytes, GW5074 significantly inhibited zint-β2-AR stimulation of ICa,L . Additional experiments showed that zint-β2-AR stimulation of ICa,L in +LMN myocytes produced results similar to those found in–PKA myocytes  and  (see ).As shown in Fig 2A, in control +LMN myocytes expressing βgal, zint-β2-AR stimulation elicited a typically enhanced increase in ICa,L (202±19%). In +LMN myocytes expressing the dn-Raf-1 mutant, zint-β2-AR stimulation of ICa,L was significantly inhibited  compared to control. Together, these findings indicate that Raf-1 signaling plays no role in β2-AR stimulation of ICa,L in freshly isolated atrial myocytes not attached to LMN . However, when PKA is inhibited by either PKA inhibitor or cell attachment to LMN, β2-AR stimulation acts via Raf-1 to activate cPLA2 and stimulate ICa,L.To further establish the role of Raf-1, we infected atrial myocytes with an Adv that expresses a dn-Raf-1 mutant  and presence of PKA inhibitor (–PKA). Atrial myocytes were plated on poly-L-lysine, a biologically inactive substrate and therefore represent +PKA myocytes. As shown in Fig 3in control +PKA myocytes zint-β2-AR stimulation modestly increased ERK1/2 phosphorylation (solid bars). Exposure of +PKA myocytes to the PKA inhibitor (1 μM) KT5720, (–PKA myocytes) further increased ERK/1/2 phosphorylation, suggesting that inhibition of basal PKA activity removes PKA-induced inhibition of basal ERK1/2 phosphorylation. In–PKA myocytes, zint-β2-AR stimulation prominently stimulated ERK1/2 phosphorylation. Moreover, zint-β2-AR stimulation only modestly stimulated p38MAPK phosphorylation (open bars), and was not significantly affected by KT5720. The last lane on the Western blot is a positive control showing ERK1/2 and p38MAPK phosphorylation in A7r5 cells stimulated by 1μM angiotensin II (5 min). In conjunction with our previous findings , the present results indicate that inhibition of PKA preferentially activates β2-AR stimulation of ERK1/2 signaling.It is well established that Raf-1 activates downstream MEK/ERK1/2 signaling , 8. Moreover, our previous results indicated that inhibition of MEK/ERK1/2 signaling by U0126 inhibited zint-β2-AR stimulation of cPLA2 signaling . This raises the question of how β2-AR stimulation activates Raf-1. In a variety of cell systems [10], including cardiac muscle [9], protein kinase C (PKC) activates Raf-1/MEK/ERK1/2. Receptor-mediated activation of phospholipase C (PLC) and the hydrolysis of phospholipids results in the production of diacylglycerol and stimulation of PKC isoenzymes. Therefore, as shown in Fig 4we determined the effects of 0.5 μM edelfosine, a specific inhibitor of phospholipase C [19], on zint-β2-AR stimulation of ICa,L in +PKA and–PKA myocytes. In control cells (A), edelfosine had little effect on basal ICa,L amplitude (open bars) or zint-β2-AR stimulation of ICa,L . In–PKA myocytes (B) zint-β2-AR stimulation elicited a typically enhanced increase in ICa,L (215±5%) compared to control responses (A) due to activation of cPLA2 [3]. In contrast to +PKA myocytes, edelfosine now significantly inhibited zint-β2-AR stimulation of ICa,L . Additional experiments in +LMN myocytes showed results similar to those found in–PKA myocytes, i.e. zint, 204±2% vs edelfosine, 84±8%; P<0.03 (see ). In other words, inhibition of PKA allows β2-AR stimulation to act via PLC to stimulate cPLA2 signaling.It is well established that Raf-1 activates downstream MEK/ERK1/2 signaling 2+-dependent and Ca2+ -independent PKCs  on zint-β2-AR stimulation of ICa,L in +PKA and–PKA atrial myocytes. As shown in  in +PKA myocytes (panel A), chelerythrine had no significant effects on basal ICa,L amplitude (open bars) or zint-β2-AR stimulation of ICa,L . In–PKA myocytes (panel B) zint-β2-AR stimulation elicited a typically enhanced increase in ICa,L (278±13%) compared to control (panel A) due to activation of cPLA2. In contrast to +PKA myocytes, chelerythrine now significantly inhibited zint-β2-AR stimulation of ICa,L . These findings indicate PKC signaling plays no role in β2-AR stimulation of ICa,L in freshly isolated atrial myocytes. However when PKA is inhibited β2-AR stimulation acts via PKC to stimulate cPLA2, consistent with β2-AR stimulation of PLC.Because activation of PLC produces diacylglycerol and activation of PKC we next determined the effects of 4 μM chelerythrine, an inhibitor of Ca2+-dependent PKCα (dn-PKCα). Control cells were infected with Adv-βgal. Infected cells were cultured overnight on LMN and therefore represent +LMN myocytes. As shown in Fig 5C, in control +LMN myocytes  zint-β2-AR stimulation elicited a typically enhanced increase in ICa,L (208±8%) compared to +PKA myocytes (panel A). In +LMN myocytes expressing the dn-PKCα mutant, zint-β2-AR stimulation of ICa,L was significantly inhibited . Similar experiments performed on +LMN myocytes expressing a dn-PKCε mutant showed no differences with control +LMN  myocytes . These findings provide additional support for the idea that β2-AR stimulation acts via Ca2+-dependent PKCα to activate cPLA2.In another approach we infected atrial myocyte with an Adv expressing a dominant-negative mutant of Ca2 requires phosphorylation at serine sites S505 and S515 [23]. We therefore determined the effect of β2-AR stimulation in +LMN myocytes infected with Adv that either expressed a non-phosphorylatable dominant-negative cPLA2 (dn-cPLA2S505A/S515A) mutant, wild-type cPLA2 (wt-cPLA2) or βgal as control. Infected atrial myocytes were cultured on LMN overnight and therefore represent +LMN myocytes. Fig 6shows that in control +LMN myocytes expressing βgal, β2-AR stimulation elicited a typical increase in ICa,L (166±11%). In +LMN myocytes expressing the double dn-cPLA2S505A/S515A mutant β2-AR stimulation of ICa,L was significantly attenuated (84±8%) compared to control. In +LMN myocytes overexpressing wt-cPLA2, β2-AR stimulation of ICa,L was significantly enhanced (307±8%) compared to either control or dn-cPLA2S505A/S515A. These results confirm that cell attachment to LMN activates β2-AR/cPLA2 signaling and further indicates that β2-AR stimulation of cPLA2 requires serine phosphorylation at one or both sites.Activation of cPLA2-AR stimulation of cPLA2 signaling is dependent on intracellular Ca2+ [3]. The fact that β2-AR stimulation acts via PLC to activate cPLA2  suggests the potential involvement of IP3-mediated Ca2+ signaling. We therefore determined the effects of 2 μM 2-APB, a putative IP3 receptor (IP3R) blocking agent [24], on β2-AR stimulation of ICa,L in +PKA and–PKA myocytes. As shown in Fig 7A, in +PKA myocytes 2-APB had no significant effect on basal ICa,L (open bars) or zint-β2-AR stimulation of ICa,L . In–PKA myocytes (B) zint-β2-AR stimulation elicited a typically enhanced increase in ICa,L compared to control responses (panel A). In contrast to +PKA myocytes, 2-APB significantly inhibited zint-β2-AR stimulation of ICa,L . Similar results were obtained in +LMN myocytes where zint-β2-AR stimulation of ICa,L also was significantly blocked by 2-APB  (see ).Our previous work showed that β3 affinity trap which binds to IP3 in the cytosol and thereby inhibits IP3-mediated Ca2+ signaling by preventing IP3 from reaching and activating the IP3 receptor (IP3R) [17]. Cells were cultured overnight on LMN and therefore represent +LMN myocytes. As shown in  in control +LMN myocytes expressing βgal, zint-β2-AR stimulation elicited a typical increase in ICa,L (182±4%). However, in +LMN myocytes expressing the IP3 trap zint-β2-AR stimulation of ICa,L (111±17%) was significantly inhibited compared to controls (P<0.002). Together, these findings suggest that IP3R signaling plays no role in zint-β2-AR stimulation of ICa,L in freshly isolated atrial myocytes. However, when PKA is inhibited by either PKA inhibitor  or cell attachment to LMN , β2-AR stimulation of cPLA2 is dependent on IP3-mediated Ca2+ signaling.In another approach, we infected atrial myocytes with an adenovirus that expresses an IP3Rs are thought to be located primarily on the sarcoplasmic reticulum (SR) and nuclear envelope membranes. Because the SR and nuclear envelope membranes are highly interconnected, inhibition of SR Ca2+ uptake by thapsigargin depletes intracellular Ca2+ stores from both sites [25]. Therefore, in Fig 9we determined whether depletion of SR and nuclear Ca2+ by 5 μM thapsigargin  inhibits β2-AR stimulation of ICa,L in +PKA (A) and–PKA (B) myocytes. In +PKA myocytes (A) thapsigargin had no effect on basal ICa,L amplitude (open bars). Compared to control responses (107±8%), thapsigargin slightly enhanced zint-β2-AR stimulation of ICa,L (143±4%), although the change was not statistically significant. This modest increase in β2-AR stimulation of ICa,L is consistent with the effects of thapsigargin to inhibit SR Ca2+ release and thereby inhibit Ca2+-mediated inactivation of ICa,L. In fact, separate experiments showed that thapsigargin abolished SR Ca2+ transients (data not shown). In–PKA myocytes (B), zint-β2-AR stimulation of ICa,L (207±17%) was typically enhanced compared to control responses (107±8%) obtained in +PKA myocytes (A). Interestingly, in–PKA myocytes treated with thapsigargin β2-AR stimulation of ICa,L (238±12%) was still enhanced. In other words, depletion of Ca2+ from SR and nuclear membranes failed to prevent PKA inhibition from enhancing β2-AR signaling. Moreover, the last column in Fig 9B shows that in–PKA myocytes treated with thapsigargin, AACOCF3 (cPLA2 inhibitor) significantly inhibited zint-β2-AR stimulation of ICa,L (62±12%), indicating that the enhanced response to zint-β2-AR stimulation was in fact due to activation of cPLA2 signaling and that depletion of SR and nuclear Ca2+ stores failed to prevent β2-AR/cPLA2 signaling. Similar results were obtained when Ca2+ stores were depleted by treatment (10 min) with 10 μM ryanodine (data not shown). These results indicate that IP3-dependent Ca2+ signaling is not mediated via IP3Rs located on SR or nuclear envelope membranes.IP3R protein is present in caveolae , 27, which are abundant in atrial myocytes [28]. We therefore determined the effects of 10 mM methyl-β-cyclodextrin , an agent that disrupts caveolae formation [29], on zint-β2-AR stimulation of ICa,L in +PKA and–PKA myocytes. As shown in Fig 10A, in +PKA myocytes MCD caused a modest but significant inhibition of zint-β2-AR stimulation of ICa,L , representing a 24% decrease. This is consistent with the idea that β2-ARs are normally localized to caveolae [30]. However, in–PKA myocytes (B) MCD elicited a significantly larger inhibition of zint-β2-AR stimulation of ICa,L , representing a 77% decrease. The fact that MCD elicited a significantly larger inhibition of β2-AR signaling in–PKA compared to +PKA myocytes supports the idea that the IP3Rs that are essential for β2-AR/cPLA2 signaling are localized to the caveolae.Alternatively, IP2-AR/cPLA2 signaling , 3. The present study extends those findings by showing that when PKA is inhibited, β2-AR stimulation acts via PKCα/Raf-1/MEK/ERK1/2 and IP3-dependent Ca2+ signaling to activate cPLA2.We previously reported that in atrial myocytes inhibition of adenylate cyclase/PKA by either cell attachment to LMN or inhibition of PKA in cells not attached to LMN activates β2-AR-mediated phosphorylation of ERK1/2, consistent with β2-AR stimulation of ICa,L via MEK/ERK1/2 signaling . Moreover, inhibition of Raf-1 by either GW5074 or adenoviral expression of a dn-Raf-1 mutant significantly inhibited β2-AR stimulation of ICa,L in–PKA or +LMN myocytes but failed to affect β2-AR signaling in +PKA myocytes. These findings can be explained by the fact that PKA inhibits Raf-1 and Raf-1 activates MEK/ERK1/2 signaling , 31, 32. Because atrial myocytes normally exhibit relatively high endogenous levels of adenylate cyclase/PKA activity [11], Raf-1 is normally inhibited and therefore β2-AR stimulation is unable to activate Raf-1/MEK/ERK1/2 signaling in +PKA myocytes. However, when PKA is inhibited (–PKA or +LMN myocytes) β2-AR stimulation acts via Raf-1/MEK/ERK1/2 to activate cPLA2. The present experiments also show that adenoviral overexpression of wt-cPLA2 or expression of a dn-cPLA2S505A/S515A mutant in +LMN myocytes significantly enhanced or inhibited, respectively, β2-AR stimulation of ICa,L compared to control +LMN myocytes expressing βgal. In various cell systems including vascular smooth muscle and fibroblasts ERK1/2 phosphorylates cPLA2 at S505 and S515 , 21–23. In cardiac ventricular myocytes (cultured on LMN) β2-AR stimulation acts via ERK1/2 and p38MAPK to phosphorylate cPLA2 at S505 [6].In the present experiments, we showed that inhibition of PKA prominently stimulates β2-ARs are coupled to both Gs- and Gi-mediated signaling pathways [12]. We [3] and others [4] have reported that β2-ARs act via pertussis toxin-sensitive Gi to activate cPLA2. In various cell systems, including rat ventricular myocytes [33] activation of PKA phosphorylates β2-ARs and thereby switches β2-AR coupling from Gs-mediated adenylate cyclase to Gi-mediated ERK1/2 signaling . However, our findings are consistent with reports that PKA inhibits Raf-1 and its downstream MEK/ERK1/2 signaling pathway .The present results also indicate that inhibition of PLC (edelfosine) or PKC (chelerythrine or dn-PKCα) significantly inhibited β2+ by BAPTA prevented β2-AR stimulation via cPLA2. This is consistent with the fact that several of the signaling molecules involved in the proposed β2-AR/cPLA2 signaling cascade are Ca2+-dependent, including PLC, PKCα and cPLA2. In the present study, we investigated more specifically which source of intracellular Ca2+ is required for β2-AR/cPLA2 signaling. We found that 2-APB, an agent that inhibits IP3-mediated Ca2+ release in cat atrial myocytes [24] significantly inhibited β2-AR stimulation of ICa,L in both–PKA and +LMN myocytes but not in +PKA myocytes. Moreover, adenoviral expression of an IP3 affinity trap which inhibits IP3-mediated Ca2+ signaling [17] resulted in a similar inhibition of β2-AR signaling in +LMN myocytes. The fact that 2-APB had no effect on β2-AR signaling in control +PKA myocytes and that it exerted effects similar to those of the Adv-IP3 affinity trap suggests that 2-APB acted specifically to inhibit IP3-mediated Ca2+ signaling. Together, these results indicate that β2-AR stimulation of cPLA2 is dependent on IP3-mediated Ca2+ signaling. Although IP3Rs are typically located on SR and nuclear membranes, the present results showed that thapsigargin, an agent that depletes both SR and nuclear envelope Ca2+ stores [25] failed to prevent β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by methyl-β-cyclodextrin elicited a significantly larger inhibition of β2-AR stimulation in–PKA myocytes than control +PKA myocytes. We therefore conclude that IP3Rs on the SR and nuclear envelope membranes are not involved in the Ca2+ signaling required for β2-AR stimulation of cPLA2. Alternatively, β2-AR stimulation of ICa,L via cPLA2 is dependent on IP3-mediated Ca2+ signaling in caveolae. In endothelial and smooth muscle cells plasmalemmal caveolae contain IP3R protein that is speculated to mediate Ca2+ influx through the caveolar membrane . In fact, lipid rafts i.e. caveolae contain a wide variety of signaling components including β2-ARs, Gi, PKC, Raf-1, ERK1/2, IP3Rs [36], involved in β2-AR/cPLA2 signaling.Our previous findings showed that strong chelation of intracellular Casee Fig 11) that under normal conditions , β2-AR/Gs stimulation acts via adenylate cyclase (AC)/cAMP/PKA to stimulate ICa,L. The relatively high basal levels of endogenous PKA activity and β2-AR-stimulated PKA activity both inhibit Raf-1 signaling, thereby preventing β2-AR stimulation from normally activating cPLA2. However, inhibition of PKA by either PKA inhibitor  or by cell attachment to LMN , removes inhibition of Raf-1. Under these conditions, β2-AR/Gi stimulation of PLC activates PKCα and IP3R-mediated Ca2+ signaling in caveolae. With PKA inhibited, PKCα is now able to stimulate Raf-1/MEK/ERK1/2 signaling. Therefore, β2-AR/Gi-mediated activation of both ERK1/2 and IP3-dependent Ca2+ signaling stimulates cPLA2/AA. We previously reported that arachidonic acid (AA) stimulates ICa,L [3]. This scenario is consistent with the fact that cPLA2 activation requires both elevation of submicromolar intracellular [Ca2+] and phosphorylation by various kinases .We therefore propose K/Akt signaling to inhibit adenylate cyclase-mediated stimulation of ICa,L. Although, there is no direct evidence suggesting that aspirin or NSAIDs play a role in cardiomyocyte aging, we hypothesize that aspirin or NSAIDs may influence cPLA2/AA pathway and this requires further investigation.These findings may have important implications with respect to the aging and/or failing heart, both of which exhibit decreases in adenylate cyclase activity. In both animal models [S1 Fig2-AR stimulation of ICa,L was enhanced and GW5074 significantly inhibited zint-β2-AR stimulation of ICa,L. Numbers in parentheses indicate the number of myocytes studied. * = P<0.004.A; In +LMN myocytes, zint-β(TIF)Click here for additional data file.S2 Fig2-AR stimulation elicited a typically enhanced increase in ICa,L and edelfosine significantly inhibited zint-β2-AR stimulation of ICa,L. Numbers in parentheses indicate the number of myocytes studied. * = P<0.03.A; in +LMN myocytes zint-β(TIF)Click here for additional data file.S3 Fig2-AR stimulation elicited a typically enhanced increase in ICa,L and 2-APB significantly inhibited zint-β2-AR stimulation of ICa,L. Numbers in parentheses indicate the number of myocytes studied. * = P<0.01.A; +LMN myocytes, zint-β(TIF)Click here for additional data file.


It is well considered that reactive oxygen species (ROS) plays a prominent causative role in the development of allergic rhinitis (AR), and eosinophils cells as important allergic inflammatory cells contribute to elevating oxidative stress. Hydrogen, emerging as a novel antioxidant, has been proven effective in selectively reducing ROS in animals models of oxidative damage. We herein aim to verify protective effects of hydrogen on eosinophils cells in guinea pigs models of AR.Thirty two guinea pigs were random divided into four groups, and AR model was established through ovalbumin sensitization. The guinea pigs were injected with hydrogen-rich saline  or normal saline (control and AR group). The frequencies of sneezing and scratching were recorded. The IgE level, blood eosinophil count and eosinophil cationic protein (ECP) level in serum were measured. The serum malondialdehyde (MDA) and superoxide dismutase (SOD) assays were also measured to evaluate oxidative stress. The expression levels of eotaxin mRNA and protein in the nasal mucosa were also determined by real-time RT-PCR, Western blot and immunofluorescence.HRS reduced the ROS and MDA levels and increased SOD level in guinea pigs of AR-HRS group accompanied with decreased frequency of sneezing and scratches. Meanwhile, there was a decline of the number of eosinophils cells in blood and of thelevel of ECP in serum in the AR-HRS group. HRS also significantly decreased the expression of eotaxin in nasal mucosa.HRS may play a protective role in attenuating allergic inflammation, and suppressing the increase and activation of eosinophils in AR possibly through antioxidation effect of hydrogen. Rhinitis, especially allergic rhinitis (AR), is a major health problem. Although several treatments such as corticosteroid and anti-histamine drugs are available for dealing with it, their side effects have limited the use of them. Recent studies have shown that oxidative stress and the production of reactive oxygen species (ROS) contribute to allergic inflammation, such as asthma and AR. The main sources of ROS are NAD (P) H oxidase, xanthine oxidase, lipoxygenase, mitochondria, and the uncoupling nitric oxide synthase . It has Many observations suggest that oxidative stress plays an important role in the pathogenesis of airway allergic inflammation such as asthma and AR . As impoAirway and circulating inflammatory cells and especially eosinophils are the likely source of ROS increases. For instance, antigen challenge increases spontaneous ROS from airway eosinophils in patients with asthma , and som2S), hydrogen could selectively reduce the hydroxyl radical (∙OH) and peroxynitriteanion (ONOO-), the most cytotoxic chemicals of ROS, and effectively protect cells; in addition, hydrogen does not react with other ROS, which possesses physiological roles [Recently, it has been proved that hydrogen gas, a highly flammable gas, has potent antioxidant property. Compared with other antioxidants gas such as carbon monoxide (CO) and Hydrogen sulfide  and prepared as described previously . In shorMature healthy male guinea pigs weighing ±230 g were purchased from National Rodent Laboratory Animal Resources . All animal care and experimental procedures were approved by the Tongji University Institution Animal Care and Use Committee (2014-DW-009).n = 8 each group), namely, Control groups, normal-HRS, AR-NS and AR-HRS. The AR-NS and AR-HRS groups were both sensitized with ovalbumin . Each guinea pig was first sensitized intraperitoneally with 0.3 mg of OVA and 30 mg of Aluminum hydroxide (AL (OH) 3) every other day for a total of seven times. From day 15 on, the guinea pigs were treated with 0.5% OVA aerosol to stimulate AR symptoms for five times. Subsequently, each side of the nasal cavity was given 20 μL of 2% OVA solution intranasally once a day to maintain AR symptoms. The control group and normal-HRS groups were health guinea pigs who are given the same dose of saline treatment.The AR models were prepared as follows. 32 guinea pigs were randomly divided into four groups . These animals were sacrificed by rapid decapitation, and then blood and nasal mucosa were collected. Biopsies of the nasal mucosa were taken from the inferior turbinate and immediately placed in liquid nitrogen. Peripheral blood was collected and the separated serum was kept at − 20 °C. Serum total IgE  and ECP  were determined by enzyme-linked immunosorbent assay method (CAP system). The detectable range of the assay for total IgE is 2–200 ng/mL, and for serum ECP is 2 to 100 ng/L.A peripheral venous blood sample was collected and eosinophils were counted through automatic blood cell analyzer . The detectable range of blood eosinophil counted in the same way is 0 to 10,000/μL.The levels of serum ROS were measured with highly sensitive enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s recommendations . The absorbance (OD) was measured at 450 nm wavelength with the enzyme marker, and the ROS concentration in the sample was calculated by standard curve. The detectable range of the assay is 10 IU/ml -320 IU/ml.ROS levels in nasal mucosa were measured with 2,7-dichlorofuorescin diacetate (DCFH-DA). Collected nasal mucosa were incubated with DCFH-DA for 30 min at 37 °C. DCFH-DA forms a fluorescent product, DCF (dichloro fluorescein) upon oxidation with ROS. Fluorescence caused by DCF in each well was measured and recorded for 30 min at 500 nm (excitation) and 530 nm (emission).To further confirm that HRS-induced inhibition of inflammation was related to the anti-oxidative property of HRS, the oxidative stress markers were measured with MDA and SOD. SOD is endogenous antioxidants as free radical scavenger, and MDA is the lipid per oxidation end product.Serum MDA levels were measured with a commercial MDA assay kit . Briefly, hydroxytoluene combined with thiobarbituric acid was able to become red. The absorbance of condensation products was tested at a wavelength of 532 nm. The levels of MDA in nasal mucosa tissue were normalized against total protein (mg protein/mL). The activity of SOD was measured with a commercial assay kit , in accordance with the manufacturer’s instructions. Briefly, this assay kit uses a thiazole salt for detection of superoxide anions to produce a colored product; absorbance was tested at a wavelength of 450 nm.Fluorescent quantitative real time RT–PCR was performed to determine the expression levels of eotaxin mRNAs in nasal mucosa. The total RNA was extracted and cDNA was synthesized with a cDNA synthesis kit . To determine the expression of eotaxin, we performed a fluorescent quantitative real-time RT–PCR assay. Primer sequences used are listed in Table The guinea pig nasal mucosa (10 mg), which was frozen in liquid nitrogen, was homogenized in 1 mL of protein lysis buffer (PBS containing 0.1% Triton X-100) and centrifuged at 14,000 g for 10 min at 4 °C. Nasal mucosa lysates from each group were analyzed by Western blot. The blots were blocked with PBS-T containing 1% skim milk and then incubated with a 1:1000 dilution of anti-eotaxin antibody  at room temperature. After three additional washes, the blots were incubated with anti-mouse secondary antibody (1:5000)  and then conjugated to horseradish peroxidase for 1 h at room temperature. The second batch of images was quantified using Quantity One software . GAPDH was employed as an endogenous control for protein normalization. The experiments were performed in duplicate.Tissues from the specimens were fixed in 10% buffered formalin. Immunohistochemical stains were performed on formalin-fixed and paraffin-embedded 4 μm sections. The tissue sections were deparaffined, and permeabilized in precooled acetone at − 20 °C for 15 min. Slides were washed in PBS. Normal goat serum (10% in PBS) was used to block nonspecific antibody binding. The slides were incubated for 1 h at room temperature with anti–Eotaxin-1 rat monoclonal antibody . Slides were washed twice in PBS, and then incubated for 30 min at room temperature with anti-rat Cy3-conjugated IgG . Slides were washed twice in PBS and once in distilled water and allowed to dry. Fluorescence microscope was applied to observation and photograph.p < 0.05 was considered statistically significant.Statistical analyses were performed using SPSS version 17.0 . All data were expressed as mean ± S.D. Statistical analyses of data were performed using ANOVA for multiple comparison and Least Significant Difference (LSD) for comparison among groups, and Pearson Correlation for the two-variable correlation analysis. p < 0.05), and no significant changes were found in normal guinea pigs after HRS treatment (p > 0.05). In the AR-HRS group, the frequencies of scratching and sneezing decreased significantly compared with those of the AR-NS group (p < 0.05). It indicates that inflammatory symptom of AR was alleviated by HRS in guinea pigs.The frequencies of scratching and sneezing are shown in Fig. p < 0.01). After HRS treatment, the IgE content in the AR-HRS guinea pigs is significantly decreased . Otherwise, there is no significant difference between control and Normal-HRS group . All these results indicate that HRS could reduce the inflammatory response of AR accompanied with decreased IgE level.The level of IgE of groups is shown in Fig. p < 0.05). Moreover, decreased changes for SOD activity and increased MDA levels in serum were observed . Similarly, no significant difference between control and Normal-HRS group was found (p > 0.05). The correlation between the makers was also carried out, and there was a highly significant correlation between ROS and MDA levels    Fig. , suggest01) Fig. , suggest9/L vs.(0.26 ± 0.03) × 109/L, p < 0.05) and decreased significantly with HRS treatment ((0.31 ± 0.04) × 109/L, p < 0.05)  × 109/L, p > 0.05).Similarly, eosinophil count in AR group was significantly higher than in control group. ((0.41 ± 0.06) × 1005) Fig. . Otherwip < 0.05), while it was 554.48 ± 36.53 pg/mL in AR-HRS group and 512.39 ± 32.478 in normal-HRS group . Also, serum ECP was correlated with blood eosinophil count   significantly  Fig. , and sertly Fig. .Fig. 7Cop < 0.05). Similarly, no significant difference between control and Normal-HRS group was found (p > 0.05). However, the mRNA and protein expression of eotaxin in the HRS-treated group decreased significantly compared with that of the AR-NS group  and does not disrupt metabolic redox reactions or ROS concerned in cell signaling . It is dIn order to observe the antioxidation effect of HRS in AR, levels of MDA and SOD in serum were observed in this experiment. MDA can be used as a crosslinking agent to promote the cross-linking of nucleic acids, proteins and phospholipids, and to change the function of biological macromolecules. The content of MDA can reflect the degree of lipid per oxidation. SOD, as disproportionation of superoxide anion generation of H2O2, can scavenge ROS, prevent lipid per oxidation, and protect cells from damaging of toxic oxygen radicals. Therefore, SOD is an important enzyme in the defense of the superoxidion from the body or external environment. The increase of MDA levels and decrease of SOD activity can lead to oxidative stress reaction, which will result in cell damage and even cell death. It is showed that in the process of allergen-induced airway inflammation, the inflammatory mediators can stimulate eosinophils to produce ROS that can be inhibited by SOD . In thisIn order to investigate the influence of HRS on inflammation of AR, the function and counts of eosinophils were also studied, because abnormalities of quantity and activity of eosinophils are typical features of AR. The present observations indicate that the declined number of eosinophils may be associated with drug-induced eosinophil apoptosis of tissue. It has been widely assumed that apoptosis of eosinophils of airway tissue would be increased by treatment such as corticosteroid, and inducement of eosinophil apoptosis has thus been advocated as a major pharmacological mechanism to bring about resolution of allergic inflammation , 29. HowIt has been known that ECP is released by activated eosinophils, and ROS with activated eosinophils affect the ECP levels . Our stuMoreover, it is reported that eotaxin has a greater effect on priming eosinophil ROS, and eotaxin plays an important role in the pathogenesis of allergic inflammation through eosinophil activation by priming the eosinophil oxidative metabolism . In the 2S and H2, will be a new treatment strategy in the therapy of AR [Our study was the first to evaluate the molecular hydrogen protects cells and tissues against oxidative stress in AR models. It also raised the possibility of treating allergic respiratory diseases by using medical gas as antioxidant therapy. Antioxidants gas, such as CO,Hpy of AR .In conclusion, our data demonstrate that HRS attenuates airway inflammation and suppresses the function and counts of eosinophils in AR possibly through antioxidation effect of hydrogen. However, more investigations should be carried out to identify the underlying mechanisms in hydrogen-mediated anti-allergic inflammation in future. We also propose that molecular hydrogen could be widely used in medical applications as a safe and effective protective drug with minimal side effects.


Recently, flufenamic acid (FFA) was discovered among fenamates as a free radical scavenger and gap junction blocker; however, its effects have only been studied in cancer cells. Normal cells in the surroundings of a tumor also respond to radiation, although they are not hit by it directly. This phenomenon is known as the bystander effect, where response molecules pass from tumor cells to normal ones, through communication channels called gap junctions. The use of the enhanced permeability and retention effect, through which drug-loaded nanoparticles smaller than 200 nm may accumulate around a tumor, can prevent the local side effect upon controlled release of the drug. The present work, aimed at functionalizing MCM-41 (Mobil Composition of Matter No. 41) silica nanoparticles with FFA and determining its biocompatibility with human fibroblasts MRC-5 . MCM-41, was synthesized and characterized structurally and chemically, with multiple techniques. The biocompatibility assay was performed by Live/Dead technique, with calcein and propidium–iodide. MRC-5 cells were treated with FFA-grafted MCM-41 for 48 h, and 98% of cells remained viable, without signs of necrosis or morphological changes. The results show the feasibility of MCM-41 functionalization with FFA, and its potential protection of normal cells, in comparison to the role of FFA in cancerous ones. The International Agency for Cancer Research (IACR) and Globocan registered that approximately 17,100,000 new cases of cancer are expected worldwide by 2020. Besides surgery and chemotherapy, radiotherapy is often used in cancer patients to eliminate or control the growth of tumors. The radiation doses can reach up to 50 Gy . Technol2O2 is reduced by an enzyme known as glutathione reductase (GSH). In absence of oxidative stabilizing agents, such as divalent cations , the GSH tends to accumulate in the extracellular space, causing apoptosis of astrocytes. This effect was lowered after treating the cells with FFA at 100 µM, which decreased the GSH accumulation to approximately 65% [Recently, flufenamic acid (FFA) was discovered among fenamates as a free radical scavenger and gap junction blocker . Inside tely 65% ,8. The use of the enhanced permeability and retention (EPR) effect, through which drug-loaded nanoparticles smaller than 200 nm may accumulate around the tumor, can prevent the local side effect upon controlled release of the drug .Mesoporous silica nanoparticles can carry weakly water-soluble drugs, and protect most of them from degradation by extracellular enzymes . Some feNanostructured silica nanoparticles are hosts of a specific drug, which is released depending on certain parameters, such as pore size, pore shape, and the interaction between the drug and the mesoporous solid . Many noFunctionalization of aminated MCM-41 with folic acid (FA) has been successful after activation of a carboxyl group with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinamide (NHS) ,18. In aTaking all these considerations into account, and due to the lack of reports on the effects of FFA in normal cells, the present work is aimed primarily at the functionalization of MCM-41 with FFA, the characterization of this system, and determining its biocompatibility to MRC-5 fibroblasts, in order to verify the potential of FFA grafted onto silica MCM-41 surfaces to protect normal cells when exposed to radiation.The materials used for synthesis, functionalization, cell culture, and cell staining processes, and their respective manufacturers, are as follows: cetyltrimethylammonium bromide (CTAB), Tetraethylorthosilicate (TEOS), 3-Aminopropyltriethoxysilane (APTES), Dimethylaminopropyl)-N’-ethylcarbodiimide (EDC), N-Hydroxysuccinimide (NHS), flufenamic acid (FFA), Dulbecco’s Modified Eagles’s Medium- high glucose (DMEM), penicillin, streptomycin andamphotericin (PSA), and fetal bovine serum, which were all purchased from Sigma-Aldrich. Calcein-Orange and Hoescht 33258 were purchased from Invitrogen, ethanol from EMSURE, toluene from FMAIA, and Dimethyl Sulfoxide (DMSO) from ACRÓS ORGANICS. ® water. The temperature of the mixture was adjusted to 78 °C. TEOS (22.4 mmol) was added dropwise to the surfactant solution under vigorous stirring. The mixture was allowed to react for 2 h. After reaction, the mixture was filtered, washed with water and methanol, and dried at 60 °C for 24 h. The surfactant was removed by calcination, which was carried out by increasing the temperature to 550 °C under nitrogen flow for 2 h, followed by 3 h in air. MCM-41 silica was prepared in accordance to a published procedure , using c® H2O and ethanol (70%), followed by drying at 37 °C for two days. This sample was named MCM-41/AP. The first step in the functionalization process was to aminate the surface of MCM-41 to receive FFA moieties. Briefly, 27.5 mL of toluene was poured in a 50 mL round bottle. Then, 400 mg of MCM-41 was dispersed in the bottle, under constant stirring. Next, 156 µL of APTES was added, and this mixture was kept under constant magnetic stirring at 80 °C for 24 h. The suspension was filtered and washed thoroughly with Milli-QIn the next step, 22.5 mL of DMSO was poured in a 50 mL round bottle, then 60 mg of EDC, 60 mg of NHS, and 162 mg of FFA was added under magnetic stirring. After 1 h, 200 mg of MCM-41/AP was added, and the reaction occurred overnight at room temperature. The suspension was filtered, washed, and dried as mentioned previously, and the sample was named MCM-41/AP-FFA, following proportions from the standard protocol.2 adsorption was performed with the equipment Autosorb IQ , to obtain information on porosity and surface area of MCM-41 before and after addition of APTES and FFA. Samples of MCM-41 were treated at 300 °C for 4 h, whereas functionalized samples received treatment at 40 °C for 48 h. The values of the surface area, pore diameter, and volume were calculated by Brunnauer, Emmett, and Teller (BET) method, except for pore distribution data, which were obtained from Density Functional Theory (DFT) method instead. Small-angle X-ray spectroscopy  was used to confirm the diffraction pattern of the pores of MCM-41 before and after functionalization with APTES and FFA. The incident X-ray was set at a 1.54 Å wavelength, and the angle scattering (2θ) in the range of 0–6°. Fourier transform infrared spectroscopy  was performed as a qualitative technique to identify APTES and FFA after the process of functionalization of MCM-41. The samples were analyzed in KBr pellets, with 256 scans at the 4000 to 400 cm−1 range at a resolution of 4 cm−1. The ratio of MCM-4 to KBr was 1:100 (mg). Thermogravimetric analysis  was performed in nitrogen atmosphere at 50 mL/min from 25 to 700 °C at a rate of 10 °C/min. In order to determine the presence of C and N chemical elements, the electron energy loss spectroscopy  was performed with energy resolution of 1.5 eV and dispersion of 0.25 eV/pixel. Energy-filtered transmission electron microscopy (EFTEM) was performed, to increase contrast and retrieve a unique effect that later can be worked upon with ImageJ software, which can assign different colors to chemical elements to allow the merging of the two or more images. Photon correlation spectroscopy and Zeta Potential analysis  allowed to determine the mean diameter and surface charge of nanoparticles. The analytical procedure was conducted after the adequate dilution of the samples in ultra-pure MilliQ® water (0.05 mg/mL). The results were expressed as mean, ± standard deviation, for at least three different batches of each silica nanoparticles formulation. Morphological characterization was performed using the scanning electron microscope  operating at 30 kV, as well as a transmission electron microscope  at 200 kV. The NA suspension of nanoparticles functionalized with FFA (3 mg) in simulated body fluid (SBF)  was put into a dialysis tube (cut-off Mn = 3500-Max Molecular Weight) and then placed in a flask with 24 mL of SBF at 37 °C, under continuous stirring, at a rate of 50 rpm. At particular time intervals, the amount of FFA released from nanoparticles was calculated, by measuring the absorbance at 288 nm with a UV–Vis spectrometer . The in vitro experiments were carried out in triplicate.2.Human fibroblasts (MRC-5) were cultured in a T75 flask with 10 mL of Dulbecco’s Modified Eagles’s Medium- high glucose (DMEM), to which was added 10% fetal bovine serum and 10 mL of penicillin, streptomycin, and amphotericin (PSA). The cells were maintained in the incubator at 37 °C, 95% humidity, and 5% CO4 cells per well in a 24-well plate, in the absence or presence of MCM-41, MCM-41/AP, and MCM-41/AP-FFA  for 48 h. Viable and necrotic cells were assessed by calcein and propidium–iodide (PI) staining, respectively. Briefly, 7 µL of calcein AM (1 µM) and 50 µL of PI (1 mg/mL) were added in 7 mL of DMEM. Next, each well received 300 µL of the staining mixture, and was kept in the incubator at 37 °C for 10 min. The images were taken using an inverted fluorescence microscope . Calcein AM was detected at 288 nm, and PI at 570 nm. Analysis of the images was performed with ImageJ software. The MRC-5 cells were obtained upon informed consent from the patients, according to procedures approved by Ethics Committee of the Universidade Federal de Minas Gerais (no. ETIC 02887512.6.0000.5149), following the Brazilian laws statements on resolution CNS 196/96 published on 12 September 2011.MRC-5 human fibroblasts cells were seeded at 1.0 × 10The morphological characterization of silica nanoparticles was carried out by SEM and TEM techniques. Images of MCM-41 obtained through SEM showed spherical morphology of most nanoparticles, and low heterogeneity among them a. The paThe pore arrangement determined by TEM showed a well-defined hexagonal arrangement of uniform pores, observed when the electron beam was parallel to the main axis of the mesopores b-1, and 2 technique is characteristic of MCM-41, which matches type IV, and the hysteresis loop obtained from the adsorption fits type H1, according to classification by the International Union of Pure Applied Chemistry (IUPAC). This hysteresis indicates the amphiphilic feature of the mesoporous surface, where there is a capillary condensation of the adsorbate . Firstly, a monolayer forms on the walls of the material, followed by multiple layers that form, as suggested in the results from the graph, where the saturation pressure is reached before the saturation pressure of the gas . d parameter from 3.58 nm to 3.34 nm was expected, since APTES molecules were added in the mesoporous silica nanoparticles surface, including in the mesopores. The cell unit decreased for the (100) plane, from 4.13 nm to 3.85 nm, and the wall thickness h from 0.93 nm to 0.85 nm. This may indicate that the layer of APTES deposited on MCM-41 formed another layer that comprised a new and smaller cell unit, resulting in a decrease in pore diameter compared to the MCM-41 sample, according to BHJ calculations  Si-O-Si and its bending (δ) at 465 cm−1 [−1 and 3600 cm−1 refers to the stretching of (ν) H-O-H, due to the water adsorbed onto the surface of MCM-41 , showing less definition than in the MCM-41 spectrum, indicating a decrease on silanol groups, as expected after the amination process [−1, indicating the presence of a C-N bond at the aminated sample. This indicates that the adsorption of organic groups on the silica surface happens simultaneously with the disappearing of hydroxyl groups.In addition, after functionalization with APTES (MCM-41/AP), there is a modification in the shape of 950 cm process . The con−1 and 576 cm−1, indicating the presence of C(2)-N and benzene rings S(α)/S(β) characteristic of an FFA molecule, suggesting that the functionalization process was successful.In −1–1800 cm−1) was subjected to refinement through PeakFit v4  a change in band pattern could be observed between the samples of MCM-41/AP and MCM-41/AP-FFA. In the former sample, four peaks appeared at 1708 cm−1, 1639 cm−1, 1531 cm−1, 1465 cm−1, and 1412 cm−1, assigned to the bending of C-N-H bound, O-H vibration, N-H bending, and C-H stretching, respectively C=O of FFA molecule N-H2, S(α)/S(β), and N-C=O can be seen. The (δ)N-H2 is likely to be the secondary amine of FFA. S(α)/ S(β) and N-C=O peaks are another detectable vibration wavelength for the benzene rings of FFA, and there may be an amide group as a result of the reaction between carboxyl from FFA and amine groups from APTES. The presence of all the peaks described above supports the success of the grafting process in MCM-41 [In the refined spectra, obtained after further grafting with FFA, three peaks at 1679 cmmolecule b. Moreovn MCM-41 .2) structure of the MCM-41. Based on the data gathered from EELS, it was possible to determine the distribution of C and N in the MSNs. A homogeneous distribution of both elements can be seen in Analysis of the MCM-41/AP-FFA sample by electron energy loss spectroscopy (EELS) showed a strong and sharp peak assigned to carbon (C) at 284 eV (K edge), a second weak peak at 401 eV, which corresponds to nitrogen (N), and a third main sharp peak at 540 eV a. C and Thermogravimetric analysis (TGA) was carried out to determine the presence of organic compounds in the MCM-41, before and after chemical modification steps with APTES and FFA. The TGA graph shows the profile of mass loss curves, where the lines representing each sample register the increase in mass loss from MCM-41 towards the grafted samples MCM-41/AP and MCM-41/AP/FFA . Two temAfter functionalization with APTES, the mass loss between 150 and 700 °C increased to 10%, confirming the presence of APTES in the MCM-41. The MCM-41/AP-FFA sample lost 21% of its mass within the same temperature bracket. This result confirms the presence of FFA, counting for 10% variation (Δ) compared to MCM-41/AP ,34.The Derivative Thermogravimetric Analysis (DTG) provides the temperature brackets where mass loss occurred. All the analyzed samples presented a mass loss within 45.4 °C and 52.6 °C, related to the water adsorbed in the surface of nanoparticles (NPs) . Above 62 atmosphere [2, although the final mass loss content did not present a significant difference [Additionally, in a previous work, the present study’s authors have reported the loss of APTES by TGA in an Nmosphere , and anofference . Elemental analysis was carried out, in order to determine the presence of APTES and FFA in the grafted samples, and the data are presented in detail in The in vitro FFA release behavior was investigated as a function of time, and the result is shown in In order to access the biocompatibility of MCM-41 and its functionalized counterparts, human fibroblasts MRC-5 cells were cultured in the presence of each material  at 50 µg/mL for 48 h. Later, each group was stained with Calcein and PI to indicate live and dead cells, followed by fluorescence imaging. All treated groups showed a high biocompatibility compared to the control groups. Cell viability of MRC-5, in the presence of MCM-41, MCM-41/AP, and MCM-41/AP-AFF remained between 95% and 99% compared to control groups . This isMoreover, the contact of the cells with MCM-41, or MCM-41/AP and MCM-41/AP-AFF, did not lead to morphological changes, as seen in the bright-field images obtained by fluorescence microscopy. The positive control group was the only one that showed morphological changes, due to the treatment with cisplatin (5 µg/mL), where a large number of dead cells stained with PI could be seen. Results are shown in In closing, the characterization techniques showed that functionalization of MCM-41 with FFA is possible, and still allows for tailoring as to the amount of APTES and FFA that can be grafted onto the surface of MCM-41. The treatment of MRC-5 cells with MCM-41, MCM-41/AP, and MCM-41/AP-FFA showed high biocompatibility (up to 99%), to the point where necrotic cells could hardly be seen. Such behavior is essential for the potential use of silica mesoporous grafted with FFA for the protection of normal cells from damaging molecules released by cancer cells. The EPR effect would lead to accumulation of those silica nanoparticles around the tumor, where it indicates a beneficial effect on normal cells while increasing cell death on cancerous ones.


A decrease in ECD1 expression induced by RNAi resulted in seedlings with albino cotyledons but normal true leaves. The aberrant morphology and under-developed thylakoid membrane system in cotyledons of RNAi seedlings suggests a role of ECD1 specifically in chloroplast development in seedlings. In cotyledons of ECD1-RNAi plants, RNA-editing of rps14-149  was seriously impaired. In addition, dramatically decreased plastid-encoded RNA polymerase-dependent gene expression and abnormal chloroplast rRNA processing were also observed. Taken together, our results indicate that ECD1 is indispensable for chloroplast development at the seedling stage in Arabidopsis.Chloroplast development is a highly complex process and the regulatory mechanisms have not yet been fully characterized. In this study, we identified Early Chloroplast Development 1 (ECD1), a chloroplast-localized pentatricopeptide repeat protein (PPR) belonging to the PLS subfamily. Inactivation of  Chloroplasts are not only the exclusive organelles that perform photosynthesis but they are also responsible for many other biosynthetic processes, such as the synthesis of amino acids, hormones, and metabolites . During  Derived through endosymbiosis from cyanobacteria, chloroplasts are semi-autonomous organelles that have their own genome. However, there are only about 100–150 genes in the plastid genome, the products of which are mainly involved in photosynthesis and plastid gene expression , and almatpI-atpH or psaf-rpl33 intercistronic region  in plastids were detected in cotyledons of the ECD1-RNAi transgenic plants. Mutation of ECD1 also resulted in decreased expression of PEP-dependent genes and abnormal rRNA processing. Our results indicate that ECD1 plays a vital role in chloroplast development in seedlings.Despite the presence of numerous PPR proteins in higher plants, their functions in the regulation of chloroplast development has not yet been elucidated. Here, we report a novel chloroplast factor, Early Chloroplast Development 1 (ECD1), that is involved in early chloroplast development in seedlings and which belongs to the PLS subgroup of the PPR family. Disruption of https://abrc.osu.edu/; last accessed 27 April 2018). This line is the same as the one used in previous studies by –2 s–1 at 22 °C. For lincomycin and spectinomycin treatment, the wild-type seeds were surface-sterilized and placed on MS media with the addition of 500 μM lincomycin or 50 mg l–1 spectinomycin. The seedlings were harvested after 7 d.We obtained the T-DNA insertion line CS16045 (ecotype Columbia) from the Arabidopsis Biological Resource Center  was amplified and inserted into the PFGC5941 vector. The forward restriction endonucleases were NcoI and SwaI, and the reverse enzymes were XbaI and BamHI. The constructs were transformed into Agrobacterium tumefaciens strain GV3101 and introduced into the wild-type plants by the floral dip method  fusion vector pUC18-35S-sGFP with GFP as a reporter. The controls with mitochondrial-, chloroplast-, and nuclear-localization signals were FROSTBITE1 (FRO1), ribulose bisphosphate carboxylase small subunit (RbcS), and PTM-N (ECD1-RNAi-1 line was separated on 1.3% (w/v) agarose-formaldehyde gels, blotted to a nylon membrane, and subsequently hybridized with a probe labeled with 32P. The probes were prepared by PCR amplification and labeled using the Prime-a-Gene Labeling System (SGMB01-Promega-U1100).Total leaf RNA was extracted from 7-d-old seedlings, and from 14-d-old cotyledons and true leaves using an RNeasy Plant Mini kit (Qiagen). RNA concentration was determined using thermo NanoDrop 2000. Total RNA from seedlings of the wild-type and the ACTIN control.RNA was used to generate first-strand cDNA in a 20-μl reaction using the Superscript III cDNA synthesis system (Invitrogen). The resulting cDNA samples were used as templates for RT-PCR analysis. Quantitative RT-PCR was performed using the SYBR Premix ExTaq Kit (Takara) following the manufacturer’s instructions with a Light Cycler 480 system. The expression level was normalized to that of an Tissues were incubated in cold 90% (v/v) acetone and placed in a staining buffer . After vacuum-infiltration for 15–30 min, samples were incubated for 16–24 h at 37 °C. β-Glucuronidase (GUS)-stained tissues were cleared with an ethanol series of 20% (v/v), 30% (v/v), and 50% (v/v) for 30 min in turn, and finally incubated in a solution of 70% (v/v) ethanol and 30% (v/v) acetic acid for at least 30 min until the tissues became transparent enough to observe under a dissecting Olympus SZX16 microscope.www.ab-mart.com/; last accessed 27 April 2018).Total proteins were prepared as previously described (JXB online). The levels of RNA editing were estimated by the relative heights of the peaks of the nucleotide in the sequence analysed. Plasmids prepared from approximately 90 independent colonies of each sample were sequenced to determine the RNA-editing efficiency of rps14-80 and rps14-149.A series of specific primers were used to amplify the regions of the genes containing the editing sites in Arabidopsis .For TEM processing, wild-type and ecd1+/ siliques at different developmental stages and cleared in Hoyer’s solution  as described by Embryos were excised from wild-type and ECD1 was cloned into pSAT4A-nEYFP-N1, and full-length cDNAs of MORF2 and MORF9 (multiple organellar RNA-editing factor) were cloned into pSAT4A-cEYFP-N1. The plasmids were co-transformed into protoplasts. Yellow fluorescent protein (YFP) was imaged using a confocal laser scanning microscope .BiFC assays were performed as previously described  according to the manufacturer’s instructions. Interactions were determined by growing diploid yeast colonies on synthetic dropout (SD) medium containing 40 μg ml–1 X-α-Gal  without tryptophan, leucine, and histidine.The CDS of 35S::ECD1-FLAG transgenic seedlings. Anti-FLAG M2 magnetic beads were obtained from Sigma (M8823). Protein A/G Sepharose incubated with pre-immune serum was used as the control. RNA was isolated by phenol-chloroform isoamyl alcohol extraction and then analysed by qRT-PCR.RIP assays were performed as described previously . After incubation for 20 min at 25 °C, the samples were resolved on a 6% Tris-borate gel in 0.5× TBE buffer, transferred to a nylon membrane, and subsequently processed using a chemiluminescent detection kit (Thermo 89880). The 5′-end biotin-labeled oligoribonucleotides ETMALc-H . RecombiAT3G49170, designated as ecd1 , attracted our attention for further investigation. Failure to identify any progeny homozygous for the ecd1 mutant allele suggested that the mutation causes embryonic lethality. We dissected the developing siliques and assessed the seeds under a dissecting microscope. In wild-type siliques, all the ovules developed normally, while in the heterozygous ecd1/+ siliques, some ovules were white  (data not shown). In older siliques, the white ovules became shrunken and aborted.To study the detailed mechanisms of chloroplast development, we obtained a series of T-DNA insertion lines from the Arabidopsis Biological Resource Center, the products of which are predicted to be candidates for chloroplast biogenesis factors. An embryo-lethal line CS16045 of the gene re white . In 33 sECD1 mutant arrested development, developing seeds at various stages from self-pollinated heterozygous plants were cleared and observed using differential interference contrast microscopy. The normal wild-type embryos underwent typical developmental stages, ranging from pre-globular, globular, heart-shaped, torpedo-shaped, through to cotyledon and maturity. However, in the heterozygous ecd1/+ siliques, although the homozygous mutant embryos consistently initiated cotyledons and showed continued growth and cell division, beyond the heart stage the development of the embryos was significantly slower than that of the wild-type . In further studies, three RNAi transgenic lines with a range of stable phenotypes with respect to white cotyledons and stunted plant growth were selected  to constitute a chloroplast transit peptide. Sequence analysis revealed that the ECD1 protein contains 17 PPR or PPR-like  motifs, together with one E motif, one E+ motif, and one DYW motif in the C-terminal part , eggplant (Solanum), Zea mays, and rice (Oryza) (The part see . It belo (Oryza) .To determine the subcellular localization of the ECD1 protein, the 218 N-terminal amino acids were fused to the N terminus of synthetic GFP (sGFP). The ECD1-GFP fusion protein was transiently expressed in Arabidopsis protoplasts under the control of the cauliflower mosaic virus 35S promoter. We observed that the GFP fluorescence merged with the chlorophyll autofluorescence , indicatECD1 gene in Arabidopsis, we made transgenic plants expressing the GUS protein under the control of the ECD1. The highest expression levels were observed in the cotyledons of seedlings. In addition, GUS activity was also detected in rosette leaves, flower buds, flowers, and siliques, with minimal expression also observed in roots , PSII (D1 and LHCII), Cytb6/f (Cytb6 and Cytf), and ATPase (CF0II) of cotyledons in ECD1-RNAi-1 were dramatically decreased compared to those of the wild type  including MORF2, MORF3, MORF6, MORF8, and MORF9 . We alsorps14 transcripts, rps14-80 and rps14-149. In cotyledons of the ECD1-RNAi transgenic plants, editing of rps14-149 was decreased dramatically, while that of rps14-80 remained normal. If the mutation of ECD1 specifically affects RNA-editing of rps14-149, then ECD1 should bind to a cis-element surrounding this editing site. To analyse the ability of ECD1 to interact with such a cis-element, electrophoretic mobility shift assays (EMSAs) were performed. The recombinant ECD1 protein with an N-terminal MBP tag was expressed  were significantly reduced in ild-type . In cont greatly . These rECD1-RNAi transgenic plants using ethidium bromide-stained agarose gel assays  was severely impaired, leading to the formation of white cotyledons. In recent years, several mutants with a phenotype of cotyledon-specific impairment have been isolated. wco mutants have a specific defect in 16S rRNA maturation in a cotyledon-specific manner (sco1 mutants are defective in the chloroplast elongation factor G, which not only affects chloroplast mRNA translation during chloroplast formation in cotyledons, but also other developmental processes such as germination and flowering (sco2/cyo1 are required for protein folding and both of them have DnaJ-like zinc finger domains (ECD1 is not limited to cotyledons only; however, the higher amount in cotyledons and its specific role in RNA-editing of rps14-149 in cotyledons but not in true leaves suggest that ECD1 is more important in cotyledons than in leaves and other organs. Since ECD1-RNAi-1 is not lethal, the editing of rps14-149 is not completely abolished. It is expected that more severe editing defects occur in the ecd1 homozygotes, leading to an embryo-lethal phenotype.In the rps14-149 RNA-editing site, and it is required for early chloroplast development in seedlings. A decrease in ECD1 expression leads to editing defects of rps14-149 in cotyledons, which result in decreased accumulation of the RPS14 protein; this in turn leads to lower levels of plastid ribosomes in cotyledons, and thus to defects in chloroplast development. Decreased expression of PEP-dependent genes and defective plastid rRNA processing were also observed in ECD1-RNAi transgenic plants, which can largely be accounted for by limiting amounts of plastid ribosomes.In conclusion, the results of our study indicate that the PPR protein ECD1 is a site-specific factor for the JXB online.Supplementary data are available at Fig. S1. Motifs and sequence analysis of ECD1.ECD1 within seedlings.Fig. S2. Expression patterns of ECD1-RNAi-1 and wild-type seedlings.Fig. S3. Ultrastructure of chloroplasts in ECD1-RNAi-1 and wild-type seedlings, and in seedlings treated with lincomycin or spectinomycin.Fig. S4. RNA-editing efficiency of various target sites in plastids of cotyledons in rps14 transcripts in seedlings treated with lincomycin or spectinomycin.Fig. S5. RNA editing efficiency of ECD1-RNAi-1 and wild-type seedlings.Fig. S6. Plastid RNA editing in true leaves of Fig. S7. Purification of MBP-ECD1.Fig. S8. Transcript levels of chloroplast genes for which the transcribing RNA polymerase is unknown.ECD1-RNAi-1 seedlings.Fig. S9. RNA gel blot analysis of chloroplast RNAs in cotyledons and true leaves of 14-d-old wild-type and ECD1-RNAi-1 seedlings.Fig. S10. RNA gel blot analysis of rRNAs in cotyledons and true leaves of 14-d-old wild-type and trnI and trnA in cotyledons and true leaves of wild-type and ECD1-RNAi-1 seedlings.Fig. S11. RNA gel blot analysis of Table S1. List of primers used in this study.Supplementary Figures S1-S11 and Table S1Click here for additional data file.


Recent studies provide strong evidence that the androgen receptor (AR) signaling pathway remains active in castration-resistant prostate cancer (CRPC). However, the underlying mechanisms are not well understood. In this study, we demonstrate that plant homeo domain finger protein 8 (PHF8 )interacts with and functions as an essential histone demethylase activity-dependent AR coactivator. Furthermore, we demonstrate that the expression of PHF8 is induced by hypoxia in various prostate cancer cell lines. Knockdown of either hypoxia-inducible factor HIF2α or HIF1α almost completely abolished hypoxia-induced PHF8 expression. Importantly, we observed that PHF8 is highly expressed in clinical androgen deprived prostate cancer samples and expression of PHF8 correlates with increased levels of HIF1α and HIF2α. Moreover, elevated PHF8 is associated with higher grade prostate cancers and unfavorable outcomes. Our findings support a working model in which hypoxia in castrated prostate cancer activates HIF transcription factors which then induces PHF8 expression. The elevated PHF8 in turn promotes the AR signaling pathway and prostate cancer progression. Therefore, the HIF/PHF8/AR axis could serve as a potential biomarker for CRPC and is also a promising therapeutic target in combating CRPC. Unfortunately, although the majority of patients are initially responsive to androgen-deprivation therapy, most tumors eventually progress from hormone-dependent prostate cancer to castration-resistant prostate cancer (CRPC).2 Importantly, recent studies indicate that the androgen receptor (AR) still has a pivotal role even in CRPC.3 Multiple mechanisms have been proposed to explain the role of AR in androgen-deprivation conditions, including enhanced local synthesis of androgens, increased levels of AR due to upregulated transcription and/or translation, AR mutations and alterations in regulatory factors such as coactivators and corepressors.4 Understanding the functions of the AR signaling pathway in CRPC has led to the development of next-generation AR antagonists for CRPC therapy.5 Despite these advancements, CRPC is still the major cause of prostate cancer-related death in men.Prostate cancer is the most frequently diagnosed cancer and the second leading cause of cancer-related death in men in the United States.6 Previous studies have also shown increased hypoxia-inducible factor 1α (HIF1α) gene expression in prostate cancer tissues.7 Furthermore, hypoxia has been shown to increase AR transcriptional activity in prostate cancer cells.9 These observations could partially explain why inhibiting HIF1α attenuates AR signaling pathways and represses tumor progression in CRPC.10 Castration induced local prostate hypoxia was initially observed in animal models11 and recent studies have provided evidence that both chemical and surgical castration treatments for patients with hormone-dependent prostate cancer are also associated with local hypoxia and subsequent activation of the HIF pathway.11 Thus, it is of critical significance to elucidate the underlying mechanisms by which castration-induced hypoxia promotes AR activation and the development of CRPC.It has been reported that the degree of tumor hypoxia positively correlates with prostate cancer progression and poor clinical outcomes.16 Consistent with its histone demethylase activity, PHF8 has been demonstrated to promote transcriptional activation of various Pol II-transcribed genes and ribosomal DNA transcription by RNA polymerase I.18 Consistent with the finding that PHF8 mutations are loosely linked with X-linked mental retardation, PHF8 was also shown to function as a coactivator for retinoic acid receptor and has a role in neural differentiation.19 Furthermore, PHF8 was observed to be highly expressed in cancers, including non-small cell lung cancer, esophageal squamous cell carcinoma, acute promyelocytic leukemia, cervical cancer and prostate cancer.25 A recent study reported that PHF8 promotes prostate cancer cell growth by activating miR-125b.26 However, the underlying mechanism for enhanced PHF8 expression in prostate cancer is unknown. Furthermore, the functional relationship between PHF8 and the AR signaling pathway and prostate cancer progression following castration treatment remain poorly understood.Plant homeo domain finger protein 8 (PHF8), also known as Jumonji domain-containing histone demethylase, is a member of the histone demethylase family. Numerous studies collectively show that PHF8 is capable of demethylating mono- and di-methylated histone H3 lysine 9 (H3K9me1/2), di-methylated histone H3 lysine 27 (H3K 27me2), mono-methylated histone H4 lysine 20 (H4K20me1) and possibly di-methylated histone H3 lysine 36 (H3K36me2).In this study, we demonstrate that PHF8 interacts with and functions as a coactivator for the AR. Furthermore, we demonstrate that the expression of PHF8 is induced by hypoxia in prostate cancer cell lines and this induction requires HIF1α and HIF2α. Finally, we provide evidence that the levels of PHF8 in prostate cancer clinical samples correlate with increased Gleason grade, poor prognosis and lower overall survival of prostate cancer patients. We propose that a novel regulatory axis, HIFs/PHF8/AR, exists in prostate cancer and targeting this axis could be a potential therapeutic strategy in combating castration-induced CRPC.27 Given the reported increased PHF8 expression in prostate cancer clinical samples,24 we tested if PHF8 also interacts with the AR. We co-expressed a green fluorescent protein (GFP)-tagged AR and Flag-tagged PHF8 in 293FT cells and treated the transfected cells with or without the AR agonist dihydrotestosterone (DHT) for 24 h. Co-immunoprecipitation was then conducted with antibodies against Flag or GFP. As shown in In previous studies, PHF8 has been shown to interact with and enhance transcriptional activation of the retinoic acid receptor.To test if endogenous PHF8 and AR interact with each other, we prepared whole-cell extracts from the AR positive prostate cancer cell line, LNCaP. We detected endogenous PHF8 when AR was immunoprecipitated with anti-AR antibody in DHT-treated LNCaP cells . In addiHaving demonstrated that PHF8 interacts with AR, we next examined if PHF8 regulates AR transcriptional activity. Toward this end, a luciferase reporter assay was employed. We transfected a 4xUAS-TK-luc reporter, a plasmid expressing a fusion protein of Gal4 DNA-binding domain and AR , and various concentrations (as indicated) of plasmids encoding the wild-type or H247A mutant PHF8 into 293FT cells . The celWe next analyzed the role of PHF8 in AR transcriptional activation in LNCaP cells. We first tested if PHF8 were recruited to AR target genes upon DHT-induced AR transcriptional activation. To this end, a chromatin immunoprecipitation (ChIP) assay was performed to examine the association of PHF8 with two well characterized AR target genes, PSA and NKX3.1. As shown in We then examined the role of PHF8 in DHT-induced transcriptional activation of AR target genes. We first established conditions to knockdown PHF8 in LNCaP cells using short hairpin RNA (shRNA) against PHF8 . Quantit29 Since increased PHF8 expression has been reported to occur broadly in prostate cancer, we hypothesized that increased PHF8 expression in prostate cancer could be at least partly induced by hypoxia. To test this hypothesis, we cultured three prostate cancer cell lines, LNCaP, 22RV1 and DU145 under hypoxic conditions for 0, 12 and 24 h and then examined the levels of PHF8 protein by western blot analysis and mRNA by RT–PCR. As shown in 30 were induced by hypoxia, thus verifying our hypoxic conditions. RT–PCR analysis demonstrated that hypoxia treatment elevated the levels of PHF8 mRNA  and hypoxic (1% O2) conditions by immunofluorescent staining. The results showed that PHF8 overexpression in two cell lines (indicated by arrows) correlated with reduced levels of H3K9me1, H3K9me2 and H4K20me1, but not H3K9me3 under both conditions . However, compared with the weak-PHF8 group, the strong-PHF8 group contained more poorly differentiated patients with Gleason scores 8–10 (P<0.001). Moreover, PHF8 displayed more cytoplasmic staining in tumors with lower Gleason grades and tended to exhibit a stronger nuclear staining in tumors with higher Gleason grades . The general characteristics of the patients, including age, Gleason scores, PHF8 IHC staining scores and the survival status are presented in n grades .P=0.008) and overall survival (P=0.016) were significantly lower in patients with strong PHF8 expression  than those with weak PHF8 expression . Comparing mortality among PHF8-strong patients to that of PHF8-weak patients as the reference, the hazard ratio was 2.5 . In addition, the levels of PHF8 in benign prostatic hyperplasia, castration-sensitive and castration-resistant specimens are highly correlated with differentiation grade and the highest level of PHF8 was observed in castration-resistant cancers  and elevated levels of HIF1α and HIF2α  according to the manufacturer's protocol.Primary antibodies: Anti-β-Actin , Anti-GLUT-1 , Anti-HIF1α , Anti-HIF2α, Anti-PHF8 , Anti-AR, Anti-H3 , Anti-H4 , Anti-H3K9me1 , Anti-H3K9me2 , Anti-H3K9me3 , Anti-H4K20me1 , Anti-Flag-tag , Anti-GFP-tag , Anti-ER , and anti-PSA . Anti-rabbit secondary antibodies were purchased from Proteintech (SA00001-2) and ComWin Biotech . Anti-mouse secondary antibodies were purchased from ComWin Biotech and Proteintech (SA00003-1). Constructs: empty vector, pcDNA3.1-Flag-HIF1α, pcDNA3.1-Flag-HIF2α, pSG5-Flag-PHF8 wild type (WT), pSG5-Flag-PHF8 H247A mutant (MU), pPYCAGIP-GFP-AR, UAS-TK-Luc and Gal-AR were as described.24 Formalin-fixed, paraffin-embedded tissue specimens were obtained and handled by standard surgical oncology procedure. After deparaffinization and rehydration, the sections were heated in EDTA buffered solution  for 20–40 min in 100 °C boiling water for antigen retrieval. Flanking sections were stained with monoclonal antibodies again HIF1α, HIF2α and PHF8. The secondary antibody conjugated with streptavidin–biotin-horseradish peroxidase complex (Biotinylated Anti-Rabbit IgG (H+L), SP-9001; Biotinylated Anti-Mouse IgG (H+L), SP-9002 . Horseradish peroxidase conjugate used with DAB  produced a brown stain . Nuclei were lightly counterstained with hematoxylin. All antibodies have been validated for IHC. Negative controls were performed using phosphate-buffered saline without primary antibody. Tissue microarray was scanned by Axio Scan.Z1  and analyzed by Zen 2012 (Blue Edition). The HIF1α, HIF2α and PHF8 staining score were assessed by two certified clinical pathologists  as described previously.42 The intensity and extent of scoring of the PHF8 staining were scaled between 0–3. Final scores were computed using a composite of intensity scores multiplied by the extent of staining score. The score of 1~2 was labeled as ‘+', 3~4 as ‘++', and 6~9 as ‘+++'. The score of 1~4 was assessed as weak expression and 6~9 as strong expression. IHC pictures were photographed under light microscope , and analyzed by Image J software version 1.44p .All clinical samples were collected from the Department of Pathology with approval from the Research Ethics Committee of Daping Hospital, Third Military Medical University and written informed consent was obtained from each patient. The sample size was chosen to ensure adequate power based on previous report.P-values were calculated by a log-rank test. We used Cox proportional hazard regression to compare mortality of PHF8-strong patients to that of PHF8-weak patients.Patients were categorized into weak or strong PHF8 groups based on PHF8 levels. Kaplan–Meier survival curves were plotted for survival until death or censored from the study. The 5-year survival and overall survival were shown. Transrectal ultrasound examinations were performed using a Sequoia 512 ultrasound scanner  equipped with a 7-or 10-MHz endorectal probe. The lesions were primarily detected based on gray-scale sonography followed by color Doppler imaging using a low velocity, high-sensitivity color setting for evaluating vascularity signals. A standardized approach was used for each patient with the same color Doppler settings: the velocity scale was set at±0.011 m/s, the wall filter at 1, and the color frequency at 6 MHz. Ultrasound images of prostate cancer tissue were observed and analyzed by professional ultrasound specialist (Yanli Guo) based on blood flow and vascularity.2 . Hypoxic conditions  were created in a Forma Series II Water Jacket CO2 incubator . For DHT  treatment, cells were preconditioned in hormone-free culture overnight and the final concentration of DHT was 10 nM.We analyzed five human prostate cancer cell lines, LNCaP, VCaP, 22RV1, DU145 and PC-3 cells and 293FT cells. LNCaP, VCaP, 22RV1 and PC-3 were obtained from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All cell lines were authenticated by short tandem repeat (STR) profiling and tested negative for mycoplasma contamination. Cell lines were grown in RPMI medium or high-glucose Dulbecco's Modified Eagle Medium supplemented with 10% heat-inactivated FBS, 10 IU/ml penicillin, and 10 μg/ml streptomycin. Cells were cultured and stored according to the suppliers' instructions. All cells for this study were used within 6 months of resuscitation and cultured at 37 °C in 5% COControl shRNA or shRNAs specific for human HIF1α (SH001530), HIF2α (SH001430) and PHF8 (SH184896) mRNAs were purchased from Yingrun Biotechnology Inc.  . LNCaP cProtein concentrations were determined by RCDC assay  according to the manufacturer's instructions. Proteins on polyvinylidene difluoride Membranes  were detected with primary and secondary antibodies as indicated. Detection was performed using ECL western blotting detection reagents  and signals were visualized and quantified using the Quantity One application . The gray analysis was based on Image J software version 1.44p .Total RNA was isolated using the RNA Extraction Kit  according to the manufacturer's instructions, and complementary DNA was made using First Strand complementary DNA Synthesis Kit  and HiScript II Q RT SuperMix for quantitative PCR. Semi-quantitative RT–PCR was performed with Taq DNA Polymerase (Invitrogen). Quantitative real-time polymerase chain reaction (RT–PCR) was performed based on iCycle System (Bio-Rad) and The SensiMix SYBR Master Mix . PCR data were analyzed using Graph Pad Prism . Information about the primers and probes used in quantitative PCR is in the To verify that PHF8 is upregulated by HIFs, 293FT cells were co-transfected with plasmids containing either a control promoter or full PHF8 promoter  and plasg for 15 min. A sample of the lysate was set aside for analysis by western blotting to assess expression of transfected DNA proteins. The remainder of the cleared lysates was divided into two equal portions. Antibodies against either Flag  or GFP  was incubated with cell lysate overnight at 4 °C and precipitated with protein A/G-Sepharose  for 2 h at 4 °C. The precipitates were washed four times with phosphate-buffered saline at 4 °C. The proteins were eluted from the beads in 50 μl 2 × SDS sample buffer by heating for 5 min at 80 °C and analyzed by western blotting. To determine whether AR can interact with endogenous PHF8, lysate from LNCaP cells as well as 22RV1 cells were precipitated with antibodies against either AR  or PHF8  followed by western blotting with antibodies against PHF8 and AR.Flag-tagged PHF8 and GFP-tagged AR were co-expressed in HEK293FT cells in the presence or absence of DHT. Antibodies against Flag or GFP were used for immunoprecipitation and western blot assays. Briefly, after co-transfection of plasmids with flag-tagged PHF8 and GFP-tagged AR for 48 h, HEK293FT cells were lysed in RIPA buffer with a protease inhibitor tablet. Lysates were rotated at 4 °C for 20 min and then homogenized by passage through a 21-gauge syringe needle five times, followed by another 20 min rotation at 4 °C. Insoluble material was cleared from the lysates by centrifugation at 12 000 LNCaP or PC-3 cells transfected with plasmid expressing Flag-tagged PHF8 Wt were incubated for 40 h and further treated by 1% hypoxia for 12 h at 37 ºC, followed by fixation with 4% paraformaldehyde, cells were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline at room temperature for 20 min, and blocked in 10% goat serum for 30 min at 37 ºC. Flag-tag was stained with mouse anti-Flag monoclonal antibody  at 4 °C and goat anti-mouse antibodies conjugated to fluorescein isothiocyanate  for 1 h at 37 ºC. To observe the methylation level, rabbit anti-H3K9me1, H3K9me2 and H4k20me1 primary antibodies  and goat anti-rabbit secondary antibodies conjugated to cyanine 3  were used. The nuclei were stained for 15 min with 40, 60-diamidino-2-phenylindole  at 37 ºC, and images were obtained with confocal microscopy .Cells were cross-linked with 1% formaldehyde (Sigma) and chromatin DNA was extracted using the chromatin extraction kit . The DNA was sheared to 200–1000 bp by sonicating 4x10s at 40% of maximum force. IgG or antibody against PHF8 or AR were added to the lysates and incubated for 2 h at room temperate on an orbital shaker (100 r.p.m.). After washing four times, the cross-linked protein–DNA complex was reversed by proteinase K at 65 °C for 15–20 min, followed by incubation at 95 °C for 5–10 min. Purified DNA was used for PCR with specific primer pairs targeting the enhancer regions of the PSA and NKX3.1 promoter. Information of the primers was shown in the VCaP cells were transfected with chemical systhesis siRNA against human PHF8 (Invitrogen). RT–PCR were used to detect PHF8 knockdown in VCaP cells. Information of the primers was shown in the t-test. Experiments with more than two groups were compared by one-way ANOVA with Tukey's multiple comparison for equal variances or by Kruskal–Wallis test followed by Dunn's Multiple Comparison if equal variances were not assumed. Statistical analyses were performed using Graph Pad Prism version 5 . A value of P<0.05 was considered to be statistically significant. Kaplan–Meier plots with log-rank statistical testing and Cox regression analysis was used in patient survival analysis. Tests were undertaken using SPSS, version 19.0, computer software .All data are expressed as means±standard error. Comparisons between two groups were conducted using unpaired Student's


Bemisia tabaci infesting cassava in Africa. Isofemale B. tabaci colonies of sub-Saharan Africa 1-subgroup 3 (SSA1-SG3), infected with two secondary endosymbiotic bacteria Arsenophonus and Rickettsia (AR+) and those free of AR infections (AR−), were compared for fitness parameters on healthy and East African cassava mosaic virus-Uganda variant (EACMV-UG)-infected cassava plants. The whitefly fecundity and nymph development was not affected by bacterial infections or the infection of cassava by the virus. However, emergence of adults from nymphs was 50 and 17% higher by AR− on healthy and virus-infected plants, respectively, than AR+ flies. Development time of adults also was 10 days longer in AR+ than AR−. The whiteflies were further compared for acquisition and retention of EACMV-UG. Higher proportion of AR− acquired (91.8%) and retained (87.6%) the virus than AR+ . Similarly, the AR− flies retained higher quantities of virus (~ninefold more) than AR+. These results indicated that bacteria-free whiteflies were superior and better transmitters of EACMV-UG, as they had higher adult emergence, quicker life cycle and better virus retention abilities than those infected with bacteria.We investigated the dual effects of bacterial infections and diseased cassava plants on the fitness and biology of the The online version of this article (doi:10.1007/s10340-017-0910-8) contains supplementary material, which is available to authorized users. Cassava is a staple crop of millions of poor in SSA and its production is constrained by two whitefly-borne virus epidemics.Unusually high populations of whitefly are driving these epidemics.High numbers (~40%) of these populations were free of secondary bacterial symbionts.Here, we provide experimental evidence that bacterial symbionts negatively impact the biology of whiteflies by reducing adult emergence and delayed development.Whitefly population free of symbionts also retained higher titres of cassava mosaic virus.Bemisia tabaci harbour many endosymbiotic bacteria including an obligate primary symbiont (P-symbiont), Portiera aleyrodidarum (γ-proteobacteria) and seven other facultative secondary symbionts (S-symbiont); Arsenophonus, Hamiltonella (γ-proteobacteria), Cardinium (bacteriodetes), Fritschea (Chlamydiae), Rickettsia, Wolbachia and Hemipteriphilus  in the USA, for example, provided fitness benefits with higher adult progenies, female bias and faster development time  and cassava brown streak disease (CBSD)  technique. An understanding of the role of bacterial infections is essential to better understand cassava whitefly population dynamics in SSA and the viral diseases they spread.We therefore set out to investigate the effects of the two most predominant S-endosymbiotic bacterial infections B. tabaci infesting cassava were generated by random isolation of individual male and female with specific bacterial infections, and allowing sibling mating for developing into colonies. Whiteflies used in this study for generating isofemale lines have been maintained and inbreeding in cassava plants in the NRI insectary for 20 years   (family Geminiviridae) were grown in the NRI quarantine glass house. Plants were grown in 10 × 10 cm plastic pots containing John Innes no. 2 compost and soil in equal mixture at 28 ± 5 °C and 50–60% relative humidity. Two-month-old plants were used in experiments to compare fitness parameters (see below) of the two whitefly populations.Cassava plants of the var. Ebwanateraka infected with the begomovirus 2 for 5 s. Insects from each colony were sexed separately under a stereobinocular microscope, and one female and two males from the same colony were transferred to 25-mm-diameter clip cages. The cages were then attached to a leaf of 2-month-old cassava plant var. Ebwanateraka for oviposition. The experiment was set up on 10 EACMV-UG-infected and healthy plants each for AR+ and AR− whiteflies for measuring whitefly fecundity and biology, and the experiment was repeated twice. The clip cages were removed after 12 days, and the whitefly eggs and nymphs were counted using a stereobinocular microscope. Only clip cages that had live females after 12 days were used for the analysis of fitness assays. Fecundity was measured on the 12th day immediately after removing clip cages.Two-day-old adult whiteflies were collected from SSA1-SG3 AR+ and AR− colonies using an aspirator and anesthetised using COThe leaves with eggs and nymphs were enclosed in perforated bread bags to prevent contamination from other insects, and the number of nymphs developed and adults emerged was recorded every 3 days. The average number of eggs hatching was estimated based on the total number of nymphs emerged. The mean adult emergence  and mean adult development time  was also recorded for both whitefly colonies.All statistical analyses were done using the R software (R Development Core Team Adult SSA1-SG3 AR+ and AR− whiteflies were given acquisition access period (AAP) of 48 h on a 3-month-old EACMV-UG-infected plant var. Ebwanateraka in a large Perspex cage (50 × 50 × 100 cm). Twenty-five AR+ or AR− potentially viruliferous whiteflies were then transferred on to 2-month-old healthy cassava plants var. Ebwanateraka for 48-h virus inoculation access period (IAP). The experiment had three replications, and a total of 30 healthy plants were inoculated using each whitefly colony. Potentially viruliferous whiteflies were collected from all the experimental plants . Statistical significance of the transmission efficiency and frequency of AR+ and AR− whiteflies carrying EACMV-UG after 48 h each of AAP and IAP was estimated in contingency tables using Fisher’s exact test at P < 0.05. All statistical analyses were done using the R software  in twenty replicates using TRIzolArsenophonus and Rickettsia were localised in the cassava whitefly nymphs and adults by fluorescent in situ hybridisation (FISH) using specific probes labelled with cy3 or cy5 at the Volcani centre, Israel . Twenty-five nymphs and adults from each isofemale lines were tested for the location of symbionts in whitefly bodies.The S-symbionts P = 0.04) on virus-infected plants  on virus-infected plants compared to healthy plants 23.0 ± 1.84 eggs). AR− whiteflies laid almost identical number of eggs both on virus-infected (22.4 ± 1.84 eggs) and healthy plants  than AR+ on healthy (0.32 ± 0.01) and virus-infected (0.42 ± 0.02) plants  and virus-infected plants (0.59 ± 0.02) was significantly higher (P < 0.001) for AR− whiteflies on both healthy (35.5 ± 0.7 days) and virus-infected (34.2 ± 0.9 days) plants compared to AR+ whiteflies .About 91.8% (45/49) of SSA1-SG3 AR− whiteflies acquired the virus after 48 h AAP, compared to 71.8% (28/39) of AR+ flies (P = 0.0002) for AR−  than AR+  whiteflies  due to inconsistent transmission rates across replications.SSA1-SG3 AR−  were more efficient in transmitting EACMV-UG to healthy cassava plants than AR+ whiteflies . The differences in transmission were, however, not statistically significant , Knottin2  and Knottin3  were overexpressed by minimum of twofold in AR+ compared to AR− populations  was also upregulated in AR+ populations . Arsenophonus and Rickettsia could not be detected in SSA1-SG3 AR− whiteflies .lts Fig. , both coies Fig. , but PorArsenophonus and Rickettsia was investigated to understand their implications on host biology and virus transmission abilities. In our conditions, AR+ infections had no effect on whitefly fecundity or nymph development. However, they had significant negative effect on adult development and generation time. AR+ infections caused 50% reduction in the number of nymphs developing into adults, and the adult development time was delayed by 10 days compared to AR− whiteflies. Both these parameters, if operational in cassava fields, will have significant negative impact on the whitefly population development and thus its pest status. The SSA1-SG3 used in this study has been found only in coastal areas of eastern Africa  harbouring both B. tabaci and thus enhance their population development  to Wolbachia caused significant reductions in the acquisition and transmission of mosquito-transmitted dengue virus . Our results thus support the hypothesis of using bacteria as potential biocontrol agents to reduce the whitefly population development as well as the spread of CMD and CBSD in Africa.Further investigations on virus acquisition and retention showed that a higher proportion of SSA1-SG3 AR− flies (91.8%) acquired and retained the virus than AR+ (71.8%). The AR− also retained higher quantities of virus. These results are also similar to those seen with mosquitoes in which infection by B. tabaci  such as Knottins are expressed constitutively in B. tabaci increased begomovirus load and transmission efficiency indicating its role in the regulating virus quantities inside the whitefly body Below is the link to the electronic supplementary material.