Drosophila in vivo quantitative approach, we have analysed the impact of AMPK and autophagy on tau-mediated toxicity, recapitulating the AMPK-mediated tauopathy condition: increased tau phosphorylation, without corresponding autophagy activation. We have demonstrated that AMPK binding to and phosphorylating tau at Ser-262, a site reported to facilitate soluble tau accumulation, affects its degradation. This phosphorylation results in exacerbation of tau toxicity and is ameliorated via rapamycin-induced autophagy stimulation. Our findings support the development of combinatorial therapies effective at reducing tau toxicity targeting tau phosphorylation and AMPK-independent autophagic induction. The proposed in vivo tool represents an ideal readout to perform preliminary screening for drugs promoting this process.Neurodegenerative tauopathies are characterised by accumulation of hyperphosphorylated tau aggregates primarily degraded by autophagy. The 5′AMP-activated protein kinase (AMPK) is expressed in most cells, including neurons. Alongside its metabolic functions, it is also known to be activated in Alzheimer's brains, phosphorylate tau, and be a critical autophagy activator. Whether it plays a neurotoxic or neuroprotective role remains unclear. In tauopathies stress conditions can result in AMPK activation, enhancing tau-mediated toxicity. Paradoxically, in these cases AMPK activation does not always lead to protective autophagic responses. Using a  Summary: Dissection of the impact of AMPK and autophagy on tau-mediated toxicity by using an in vivo Drosophila tool as readout to perform preliminary drug screening supported by quantitative analyses. The microtubule (MT)-associated protein tau regulates assembly and stability of MTs . In physThe tau MT-binding ability is strongly dependent on tau phosphorylation status . Severalin vitro and in vivo models of familial amyotrophic lateral sclerosis, as well as in an in vivo mutant TDP-43 model of motor neuron disease (in vivo model (mouse primary neurons) where they reported an increase of tau phosphorylation at multiple sites, whereas AMPK inhibition led to a rapid decrease of tau phosphorylation. Furthermore, in a mouse model of AD it has been demonstrated that AMPK mediates the synaptotoxic effects of Aβ42 oligomers via phosphorylating tau at Ser-262  mutation, we exacerbate tau toxicity by increasing tau phosphorylation without corresponding autophagy activation. Interestingly, treatment with the autophagy activator rapamycin was able to rescue the additional toxicity.Here we report an Our findings support the development of new therapies targeting tau clearance via AMPK-independent autophagic induction.It has been demonstrated that activated AMPK phosphorylated at Thr-172 (p-AMPK) is abnormally accumulated in all the major tauopathies, including AD . To detein vitro assay .It has previously been demonstrated that AMPK can phosphorylate tau at Ser 262 in a cell-free ro assay , as wellro assay . We now ro assay . As reporo assay , transfeKnockdown of endogenous AMPK via AMPKα1-KD overexpression caused dramatic reductions in total tau soluble fraction levels  under the control of the retina-specific GMR driver. The presence of the hemizygous GMR driver  partially rescued the tau-induced eye phenotype as determined by scanning electron microscopy , for the assessment of ommatidial distortion in terms of deviation from ommatidial roundness caused by transgenic manipulations of the eye . The disin vivo Drosophila model, we co-expressed human tau with the relevant human AMPKα1 mutants previously characterised in vitro and assayed the resultant toxicity. Several insertion lines for both transgenes were used for the initial experiments; the results for two lines of the human AMPKα1-CA transgene (hereafter AMPKα1-CA) and three lines of the human AMPKα1-KD transgene (hereafter AMPKα1-KD) were used for initial SEM analyses and similar results were obtained for each transgene (data not shown). The overexpression of the AMPKα1 transgenes under the control of the GMR-GAL4 driver in the resulting AMPKα1-CA and AMPKα1-KD lines were validated by western blot . The overexpression of those transgenes caused no appreciable external eye phenotype .In order to recapitulate the human AMPK/tau pathway in our n=10; Co-expression of tau and AMPKα1-CA  caused an exacerbation of the toxic tau eye phenotype A. By usin=12) and the rescue resulted in a shift to the left of the DC distribution compared with tau alone  ameliorated the basal tau phenotype. Quantitative analysis using QED validated the SEM analyses . Previous work has shown that pathologically hyperphosphorylated (including pSer-262) human tau band runs at ∼60 kDa, whereas the normally phosphorylated tau runs in the range of 55–60 kDa . ConsistTo address whether the reduced tau toxicity associated with AMPK downregulation B coincidProbing our samples with an antibody against tau phosphorylated on Ser-262 (p262), we validated that the pathologically hyperphosphorylated band consists of tau hyperphosphorylated at Ser-262 and the immunoreactivity for that band is reduced by AMPK downregulation F,G. ThisIt has been demonstrated that Ref(2)P, the homologue of mammalian p62, is a major component of protein aggregates formed during normal aging in the adult fly brain. It also accumulates in protein aggregates in flies that are defective in autophagy and in models of human neurodegenerative diseases . AccordiGiven AMPK's known roles in autophagy activation, we sought to test the autophagy status in our transgenic fly models. We therefore assayed Ref(2)P accumulation in tau, tau/AMPKα1-CA and tau/AMPKα1-KD flies head extracts.In our model, we detected no changes in Ref(2)P accumulation in tau/AMPKα1-CA compared to tau alone B, demonsn=9 for DMSO control flies versus 0.51±0.11, n=7 for rapamycin-treated flies) and the rescue resulted in a clear shift to the left of the DC distribution compared with DMSO-treated flies  and analysed the eye phenotypes. Whereas 0.1 µM rapamycin had no effect (data not shown), 1 µM treatment ameliorated the tau AMPKα1-CA mediated exacerbation of the toxic tau eye phenotype, compared with the same genotype fed with DMSO C. QED quDrosophila have strengthened this notion demonstrating that AMPK deficiency causes neurodegeneration .We have also demonstrated that downregulating AMPK activity affects tau in both  species B. In thein vitro via expression of AMPKα1-KD altered the phosphorylation profile of tau and led to compromised soluble tau stability in cells P, the umulates . This apumulates . This isumulates . From thConversely, the increase in tau toxicity provoked by AMPKα1-CA is due to the increased phosphorylation and possible reduced degradation of tau, concomitant with the inability of AMPKα1-CA to effectively induce autophagy. Indeed, the lack of increased autophagic induction by AMPKα1-CA has been previously demonstrated in HT-29 and HeLa mammalian cells .Eventually, the reduction in tau toxicity found in AMPKα1-CA-tau flies treated with rapamycin C,D, is cDrosophila model presented in this paper demonstrates an ability to affect tau toxicity via AMPK-modulation in vivo.The In human tauopathies, stress conditions result in AMPK overactivation and increased kinase activity, responsible for tau phosphorylation. Additionally, AD patients are known to be defective in autophagy activation . Our AMPin vivo for AMPK-CA, we propose that this results on balance in toxic accumulation of phospho-tau. Accordingly, our results indicate that pharmacological induction of autophagy downstream of AMPK may be beneficial in tau-dependent neurodegeneration.While AMPK is potentially able to offer protective effects through regulation of autophagy , given tDrosophila model presented here offers an opportunity to test the development of such drugs.Taken together our findings raise the possibility that new therapies targeted at AMPK-mediated tau phosphorylation might be effective at reducing tau toxicity. The potential for such treatments to compromise autophagy are likely to be outweighed by the benefits associated with reducing phospho-tau toxicity. This opens the possibility of combinatorial therapy with treatments targeted at AMPK-independent autophagic induction to promote tau clearance. The 2. Transfections were performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol with empty pCMV-FLAG, pCMV-FLAG-tau , pCMV-FLAG-S262A tau or pCMV-FLAG-T231A tau plasmids in combination with empty pcDNA3 (Invitrogen), pcDNA3 N-terminal-Myc-AMPKα1-CA . pCMV-FLAG-tau is an N-terminal Flag-tagged human microtubule-associated protein tau isoform 2 (Genbank NP_005901.2), cloned into the pCMV FLAG-vector. pCMV-Myc Tag3b AMPKα1-KD and pCMV-FLAG-S262A tau were obtained by site-directed mutagenesis respectively on pCMV-Myc Tag3b AMPKα1-wt and pCMV-FLAG-tau, via site-directed mutagenesis using a QuikChange site-directed mutagenesis kit .HEK293T cells were maintained in DMEM of high glucose, supplemented with 10% FBS and grown at 37°C in a humidified atmosphere with 5% CODrosophila expression vector, pUAST. Transgenic flies were produced by BestGene Inc. using standard procedures. UAS-AMPK RNAi flies were obtained from the VDRC . All other fly stocks were obtained from the Bloomington Drosophila Stock Center . Crosses were maintained on standard cornmeal-based Drosophila medium at 25°C.The transgenic fly line carrying the human 0N4R tau, with the four tubulin-binding domains (R) at the C-terminal region and no N-terminal insert (N), was a kind gift from Dr Mel Feany  . UAS-AMPg for 5 min in a microfuge and resuspended in 0.5 ml lysis buffer . After sonication for 15 min and incubation on ice for 10 min, samples were centrifuged at 15700 g for 30 min. The supernatant was recovered and the protein concentration determined. Proteins were either directly analysed after electrophoresis, by western blotting (see below), or immunoprecipitated. In the second case, extracts were incubated under rotation for 2 h at 4°C with ANTI-FLAG M2 Affinity Gel  according to manufacturer's instructions, or with the relevant antibody. In the latter case, Protein A-Sepharose  was then added and the mixture incubated for a further 2 h at 4°C. Sepharose beads were quickly centrifuged in a microfuge (2′ at 8000 rpm) and washed three times with lysis buffer. After the final wash, the beads were resuspended in 30 µl Laemmli sample buffer, boiled 10 min at 90°C and electrophoresed through a 10% acrylamide gel. Adult flies were collected, heads dissected and homogenised in a lysis buffer containing 50 mM Tris-HCl pH 7.5, 0.1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM Sodium Orthovanadate, 50 mM Sodium Fluoride, 5 mM Sodium Pyrophosphate, 0.27 M Sucrose, 0.1% β-Mercaptoethanol and complete protease inhibitor tablets (Roche). Protein content was determined using Bradford reagent (Bio-Rad). Extracts were mixed with SDS sample buffer and heated at 90°C for 10 min before being separated by 10% SDS-PAGE.HEK293T cells were recovered by gentle pipetting in PBS1X, centrifuged at 5000 Lysates or immunocomplexes subjected to SDS-PAGE were transferred to nitrocellulose membranes . Nitrocellulose blots were incubated at room temperature for 30 min in blocking buffer [Tris-buffered saline with 0.1% Tween containing 5% bovine serum albumin (BSA) and then incubated overnight at 4°C in 5% BSA with the recommended concentrations of indicated antibodies]. After washing three times for 15 min each with Tris-buffered saline containing 0.1% Tween, the blots were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG , followed by washing. Immunoreactive bands were visualised with enhanced chemiluminescence substrate . Developed films were scanned using a flat-bed scanner and densities of bands were measured using ImageJ software (NIH).Membranes were immunoblotted with the following antibodies at the indicated dilutions: tau T46 , tau phospho S262 , tau phospho T231 , AMPKα , Phospho-AMPKα (Thr172) (40H9) , anti-FLAG M2 , Anti-beta Tubulin , anti-actin , anti-Ref(2)P  (kind gi2O), incubating on a rotator in 1 ml of solution for 15 min at room temperature (RT). Flies were transferred to 100% acetone, for at least 2 days at 4°C, and then attached to stubs using double-sided tape, sputter coated in gold for 60 s and imaged using a Zeiss Supra55 VP SEM at 150× magnification.To analyse external eye structure using SEM, female flies were fixed overnight in 4% PFA in PBS1X at 4°C. Flies were then dehydrated through an acetone series  in order to produce cumulative plots of ommatidial distortion versus fly count . Significance levels were calculated with the Mann–Whitney U-test.Software for assessing the level of ommatidial disruption in adult fly eyes (QED) was developed in collaboration with the Statistics Department at Warwick University, UK . For theRapamycin  was dissolved in DMSO (500 μM stock) and added to fly food at 0.1 μM and 1 μM. For control food DMSO alone  was added.


Organic-free MnO2 aerogels with ultralow density have been achieved by self-assembly of two dimensional MnO2 nanosheets via an ice-templating approach. 2 aerogels with ultralow density have been achieved by the self-assembly of two dimensional (2D) MnO2 nanosheets via an ice-templating approach. To the authors' best knowledge, it is the first reported case of constructing a high-purity inorganic aerogel from preformed NPs without using any functionalization or stabilization agents. Moreover, it has been demonstrated that an ultralight MnO2 aerogel with a density as low as ∼0.53 mg cm–3, which is the lightest metal oxide aerogel to date, can be well obtained by such an approach. The successful formation of the aerogel can be attributed to the enhanced van der Waals force between the 2D building blocks that have been more orderly arranged by the squeezing of ice crystals during the freezing process. Hence, this work shows a pioneering example of assembling inorganic NPs into aerogels relying only on the weak interactions between NPs . It has also been demonstrated that the obtained MnO2 aerogel can function as an effective absorbent for toxic reducing gas, owing to its strong oxidation ability and high porosity. The strategy presented herein holds good potential to be applied to the fabrication of other high-purity inorganic aerogels, especially those with 2D building blocks readily available.Self-assembling inorganic nanoparticles (NPs) into macroscopic three dimensional (3D) architectures often requires the assistance of organic components, leaving residual organics in the resultant. In this work, organic-free MnO Nonetheless, the aerogels formed by destabilization normally contain trace amounts of impurities as a consequence of an incomplete removal of the surface stabilizing agents and suffer from reduced performance  due to the residual organics.A common strategy of organizing NPs into a 3D percolating network is to install organic functionalities onto the surface of inorganic NPs to effect cross-linking. For instance, organics, such as 2-[2-(2-methoxyethoxy)ethoxy]acetic acid, polyethylenimine, sodium dodecyl benzene sulfonate (SDBS), sodium cholate (SC), 2 have been constructed by ice-templating from polyvinylpyrrolidone (PVP)-stabilized Ag/Cu nanowires and dimethylformamide (DMF)-capped MnO2 nanoflowers, respectively.Ice-templating is an inexpensive, convenient and scalable technique that exploits endogenous ice crystals as templates to shape and press building blocks to achieve a desired structure.2, MoS2, BN, and so forth, have emerged as interesting functional materials,etc., and thus, have offered promising starting building blocks to assemble pure inorganic aerogels. However, due to the difficulty and/or limited options of manipulating inorganic interactions,In recent years, two-dimensional (2D) nanosheets, such as MnOvia ice-templating starting with monodispersed organic-free 2D MnO2 nanosheets. To the best of our knowledge, it is the first reported case of achieving a pure inorganic aerogel from preformed NPs without using any functionalization or stabilization agents. The resultant MnO2 aerogels also show extremely low density (as low as ∼0.53 mg cm–3), indicating the lightest metal oxide aerogels to date. The successful formation of the aerogel can be attributed to the enhanced van der Waals force between the 2D building blocks that have been more orderly arranged by the squeezing of ice crystals during the freezing process, demonstrating a novel approach to construct inorganic aerogels by only relying on weak interactions. It has also been shown that the obtained MnO2 aerogel can function as an effective absorbent for toxic reducing gas, owing to its strong oxidation ability and high porosity. The strategy presented herein has good potential to be applied to the fabrication of other high-purity inorganic aerogels, especially for those with 2D building blocks readily available.In this contribution, we have successfully self-assembled a high-purity inorganic aerogel 2 aerogels were self-assembled from monodispersed MnO2 nanosheets by fully manipulating the interaction between inorganic NPs via an ice-templating approach. Building blocks of an MnO2 nanosheet, which displayed a 2D morphology with a lateral dimension mainly in the range of 170 to 240 nm and a typical thickness of 2–4 nm .High-purity MnOf 2–4 nm , were ob see S2.1 was cultinset of . The obt2 building blocks (bottom curve in 2 (JCPDS no. 18-0802) and the broad and low intensity XRD peaks indicate a poor crystalline or polycrystalline feature.001, d002, d100 and d110) at the same positions as those of the starting MnO2 nanosheets. It should be noted that the interlayer peaks (corresponding to d001 and d002) of the aerogel are sharper and stronger, suggesting higher crystallinity of the MnO2 assembly and a more ordered arrangement of the nanosheets as conferred by ice-templating.The X-ray diffraction (XRD) peaks of the MnO–1, which can be assigned to the vibrations of an octahedral [MnO6] framework, and no characteristic bands of organic compounds can be identified. Corroboratively, the elemental analysis of the MnO2 nanosheets (see S2.2The Fourier transform infrared (FT-IR) spectrum of the aerogel  only exh see S2.2. Thus, t2 aerogel was examined in more detail by SEM and transmission electron microscopy (TEM). It has been found that the 3D network of the aerogel consists of two major types of microstructure: one-dimensional (1D) rods and 2D flakes, as revealed in both the top-view and sectional-view SEM images  and (110) planes of δ-MnO2, respectively. Therefore, the obtained 3D aerogel demonstrates a hierarchical structure, that is, the 3D network is constructed of 1D rods and 2D flakes, which have been assembled from MnO2 nanosheets.The micro-morphology of the MnOM images  and S8†.M images  and S9†.M images  and S10†M images  and S10†M images  and S11†nosheets  and S4†.nosheets  reveals 2 colloids freezes below the freezing point, nucleation of ice crystals occurs randomly on the frozen surface of the colloid. Then, the ice nuclei gradually grow in the MnO2 colloids, eventually reaching a cellular morphology regime.2 nanosheets, excluded from the ice crystals at the early stage of freezing, are repelled and concentrated by the growing ice crystals  displays abundant 2D flakes accompanied by a few 1D rods  is crucial to preserve the 3D network assembled in the freezing process.In order to verify that the microstructures of 1D rods and 2D flakes were formed by the concentrating and squeezing effect of ice during the freezing process, the icy chunk of the frozen colloid was thawed at room temperature and characterized by SEM. In contrast to the homogeneous colloid before freezing, the freeze–thawed sample shows colourless liquid with a large amount of brown sediment at the bottom Fig. S14, indicat 1D rods  and S15† 1D rods . This obET) can be expressed as:ER is the repulsive energy  and EA is the attractive energy .Based on the Derjaguin–Landau–Verwey–Overbeek (DLVO) theory,2 nanosheets demonstrate a zeta potential of around –10 mV and can form an aqueous colloid due to the electrostatic repulsion of the negative charge on their surfaces , also excluded and concentrated by the expanding ice crystals, can change the ionic atmosphere of the nanosheets and partially mitigate the rising repulsion between the negatively charged surfaces as they approach each other. As a result, although both the EA and ER increase in absolute value , as the distance (D) between the nanosheets decreases; EA surges up in absolute value more quickly than ER. Therefore, whereas the increment in ER dominates over EA as ice crystals start to reduce D, the net incremental effect on ET becomes zero at a certain critical point (D0), because the increment of ER is neutralized by the increment of EA. As a consequence, once ET reaches a maximum (E0) it begins self-declining, which further reduces D (D < D0) until steric hindrance stops this. At the macroscopic level, a stable assembly of nanosheets can be achieved as ET declines.Our MnOs Fig. S6.53,54 Whue of EA . SimultaIt should be noted that the ice-squeezing effect plays a critical role in forming a stable assembly of 2D nanosheets, especially due to its rearrangement of the squeezed nanosheets. It has been found that the spontaneously formed sediments driven by gravity can be easily sonicated into a homogenous colloid by ultrasound-treatment (100 W) of a few seconds, whereas the freeze–thawed sediments from ice-squeezed assembly can not be redispersed into a colloid under the same ultrasound-treatment even after a longer period of 30 minutes Fig. S14, indicat2 nanospheres with a smaller effective surface for van der Waals interaction were chosen as building blocks to construct aerogels under otherwise identical conditions as used for 2D MnO2 nanosheets  in parallel, and then observed by naked eyes and SEM ,ρ ≥ 0.18 mg cm–3),ρ ≥ 0.16 mg cm–3),ρ ≥ 0.12 mg cm–3),56etc. Our MnO2 aerogel (ρ ≥ 0.51 mg cm–3) adds the first member of metal oxide to the family of ultralight materials below 1 mg cm–3.Encouraged by the successful construction of an ultralight aerogel with a density of 1.0 mg cm and SEM . It has 2 colloid  was chosen for its easy evaporation from N2H4·H2O liquid at low temperature. The colour of MnO2 aerogels gradually changed from dark brown (2 into Mn(OH)2 by the reducing gas.2H4-sensitive colorimetric probe  powders, have also been tested as absorbents under otherwise identical conditions. The colour of the colorimetric probe changed from yellow to light yellow . The resultant morphology and microstructures of the aerogel (e.g. 1D rods and 2D flakes) are in good consistency with the ice forming mechanism as determined in glaciology. The successful formation of the aerogel can be attributed to the enhanced van der Waals force between the 2D building blocks that have been more orderly arranged by the squeezing of ice crystals during the freezing process. It has also been demonstrated that the obtained MnO2 aerogel can function as an effective absorbent for toxic reducing gas, owing to its strong oxidation ability and high porosity. The ice-templating approach presented here provides a general strategy that holds good potential to be applied to the fabrication of other high-purity inorganic aerogels, especially those with 2D building blocks readily available.In conclusion, it has been demonstrated that a high-purity inorganic aerogel can be assembled from 2D nanosheets Supplementary informationClick here for additional data file.


Mitogen-activated protein kinases (MAPKs) are conserved protein kinases that regulate a variety of important cellular signaling pathways. Among them, c-Jun N-terminal kinases (JNK) are known to be activated by various environmental stresses including virus infections. Previously, activation of the JNK pathway has been detected in cells infected with several coronaviruses. However, detailed characterization of the pathway as well as its implication in host–virus interactions has not been fully investigated. Here we report that the JNK pathway was activated in cells infected with the avian coronavirus infectious bronchitis virus (IBV). Of the two known upstream MAPK kinases (MKK), MKK7, but not MKK4, was shown to be responsible for IBV-induced JNK activation. Moreover, knockdown and overexpression experiments demonstrated that JNK served as a pro-apoptotic protein during IBV infection. Interestingly, pro-apoptotic activity of JNK was not mediated via c-Jun, but involved modulation of the anti-apoptotic protein B-cell lymphoma 2 (Bcl2). Taken together, JNK constitutes an important aspect of coronavirus–host interaction, along with other MAPKs. So far, four subgroups of MAPKs are identified, namely extracellular regulated kinase 1/2 (ERK1/2), ERK5, p38, and c-Jun N-terminal kinases (JNK)3. Among them, ERK1/2 is activated by growth factors and mitogens, whereas p38 and JNK respond to cellular stresses and/or environmental stimuli2. MAPKs are activated by kinase cascades. In particular, JNK is activated by MAPK kinases 4(MKK4) or MKK7, which are phosphorylated by upstream MAPK kinase kinases4. JNK activation requires dual phosphorylation of Thr and Tyr within a conserved Thr-Pro-Tyr motif4. Active JNK phosphorylates c-Jun and other substrates to modulate their activities5. For example, phosphorylated c-Jun dimerizes with other transcription factors to form activator protein-1(AP-1) complex, thereby activating transcription of target genes6.Mitogen-activated protein kinases (MAPKs) are conserved kinases regulating critical signaling pathways, such as apoptosis, differentiation, and immune response7. JNK-dependent activation of AP-1 upregulates expression of pro-apoptotic genes such as Bcl2 homologous antagonist killer, Fas ligand, and tumor necrosis factor-alpha8. Some transcription factors, such as p53 and p73, are also activated by JNK and promote cell death10. Also, JNK can translocate into mitochondria and modulate the function of Bcl2 family proteins, such as BH3-interacting domain death agonist11, Bcl2-interacting mediator of cell death12, and Bcl2-associated death promoter14. JNK can also directly phosphorylate Bcl2, inhibiting its anti-apoptotic activity15.JNK pathway modulates apoptosis by two mechanisms: transactivation of pro-apoptotic genes and interactions with B-cell lymphoma 2 (Bcl2) family proteins16. For example, p38 phosphorylation during IBV infection upregulates the expression of pro-inflammatory cytokines interleukin 6 (IL-6) and IL-817, while ERK1/2 is activated and plays a pro-survival role in ER stress-induced apoptosis during IBV infection18. JNK phosphorylation was detected in cells infected with MHV or SARS-CoV20, and in cells overexpressing the N, 3a, 3b, or 7a protein of SARS-CoV24. JNK and Akt are required for establishing persistent SARS-CoV infection25. In cells overexpressing the SARS-CoV spike protein, JNK phosphorylation is mediated by protein kinase C epsilon26, and the expression of IL-8 depends on the activity of AP-127. However, detailed mechanisms of JNK activation during coronavirus infection and its involvement in coronavirus-induced apoptosis are largely unknown.MAPK activation is observed during coronavirus infection, modulating various aspects of virus–host interaction28. Here we determined upstream MKKs of IBV-induced JNK activation and characterized its involvement in regulating IBV-induced apoptosis. We found that IBV infection activated the MKK7/JNK/c-Jun pathway in two mammalian cells (H1299 and Huh-7). IBV-induced JNK activation was mediated by MKK7, and required both its ATP binding and phosphorylation sites. We also showed that JNK-promoted apoptosis during IBV infection, and this activity was not mediated via c-Jun, but involved modulation of Bcl2. Taken together, our data demonstrate an important pro-apoptotic function of JNK during coronavirus infection.Previously, we showed that ER stress sensor inositol-requiring enzyme 1 (IRE1) protects cells from IBV-induced apoptosis partly by modulating JNK phosphorylationActivation of JNK pathway was determined in H1299 cells. Total JNK remained unchanged in both IBV-infected cells and UV-IBV control Fig. . PhosphoIn Huh-7 cells, IBV infection induced similar JNK phosphorylation, though phos-JNK signal was stronger and remained high even at 24 hpi Fig. . SignifiTo determine which MKK activates JNK, H1299 cells were transfected with MKK4 or MKK7 plasmids before infected with IBV. FLAG-tag MKK4 and MKK7 were determined by Western blot Fig. . As expeCompared to vector control, a slightly higher phos-MKK7 was detected in FLAG-MKK7-transfected Huh-7 cells Fig. . SimilarAs JNK activation is associated with IBV-induced apoptosis, we then tested the effect of MKK7 overexpression on apoptosis. Also, to determine the functional domain required, several MKK7 mutants were generated. ATP-binding site (K149) was mutated in the KM mutant. Residues phosphorylated by MKKKs  were mutated to glutamates in the 3E mutant, which served as a “phosphomimetic” of active MKK7. In the 3A mutant, the same three residues were mutated to alanines, rendering it resistant to phosphorylation by upstream kinases. Transfected H1299 cells were irradiated with UVC, a condition known to activate JNK pathway. UVC-induced moderate JNK phosphorylation in vector control Fig. . Wild-tySimilarly transfected H1299 cells were infected with IBV. Expression of wildtype and mutant MKK7 were determined by Western blot Fig. . Phos-MKIn Huh-7 cells, IBV-induced phos-MKK7 was slightly higher in cells transfected with wild-type MKK7, KM, or 3A. Nonetheless, phos-JNK showed a similar pattern as in H1299 cells: only wild-type MKK7 and 3E, but not KM or 3A, significantly enhanced IBV-induced JNK phosphorylation compared with vector control Fig. . PARP cl29. As a negative control, conserved Thr-Pro-Tyr motif in JNK was modified to Ala-Pro-Phe (APF), rendering it unable to be phosphorylated. H1299 cells were transfected with pcDNA-FLAG-MKK7-JNK1 or pcDNA-FLAG-MKK7-JNK1(APF), before infected with IBV or mock infected. Expression of MKK7-JNK1 was slightly higher than APF, possibly due to differences in protein stability . Treatment of 10 μM SP600125 already reduced phos-JNK by ~60%, but higher concentration did not further reduce phos-JNK level  expression was lower than wild-type Bcl2, possibly because phosphorylation at Ser70 stabilized Bcl2 in this cell line Fig. . Similar26. Expression levels of these proteins could be quite different in an actual infection. Moreover, interactions between viral proteins could not be recreated using the over-simplified overexpression approach. In two studies, JNK phosphorylation was detected in cells infected with MHV19 and SARS-CoV20. However, activation of upstream kinases and downstream substrates was not determined, leaving the complete pathway largely unexplored.Previous studies on coronavirus-induced JNK activation focused on overexpression of individual viral proteinsIn this study, we used IBV as a model and characterized MKK/JNK/c-Jun pathway activation in a time course infection. We found that IBV-induced phosphorylation of MKK7, JNK, and c-Jun in the infected cells. Transfection of MKK7, but not MKK4, promoted IBV-induced phosphorylation of JNK and c-Jun. Using various MKK7 mutants, we showed that both ATP-binding activity and phosphorylation sites in MKK7 were required for optimal JNK activation in IBV-infected cells. Finally, gain-of-function and loss-of-function studies demonstrated that JNK phosphorylation plays a pro-apoptotic role during IBV infection. Thus, this study established a crucial involvement of JNK in regulating apoptosis induced by coronavirus infection.31. However, our data showed that SP600125 above 10 µM inhibited IBV replication in a dosage-dependent manner. In fact, SP600125 inhibits replication of poxviruses32 and VSV33, and non-specifically inhibits entry of HCV34. With the antiviral activity of SP600125 for coronavirus remaining uncharacterized, cautions should be taken to interpret these inhibitor studies.Previous studies used SP600125 at 10–40 µM to inhibit JNK phosphorylation in coronavirus-infected cells35. Previously we reported that hyper-phosphorylation of JNK was associated with increased apoptosis observed in IRE1-knockdown cells infected with IBV37. Here we showed that overexpression of MKK7 or constitutively active JNK-promoted IBV-induced apoptosis, while inhibition or knockdown of JNK suppressed apoptosis, further supporting the pro-apoptotic role of JNK during IBV infection. Interestingly, our data suggested that, opposite to JNK, c-Jun actually promotes survival during IBV infection. This is not totally unexpected, because under certain situations the pro-apoptotic activities of JNK was mediated independent of c-Jun38. For example, both vinblastine and taxol induce JNK-dependent apoptosis with similar kinetics and induce c-Jun expression, but c-Jun phosphorylation is only observed in cells treated with vinblastine, but not taxol39. Also, DNA damage-induced apoptosis is mainly mediated by JNK-dependent phosphorylation of p53 or p7310. Thus, JNK-mediated apoptosis during IBV infection might also involve similar c-Jun-independent mechanisms.Studies on the involvement of JNK in coronavirus-induced apoptosis are limited. In one study, JNK inhibition was found to suppress apoptosis induced by overexpression of SARS-CoV protein 67. We showed that Bcl2 was upregulated in JNK-knockdown cells infected with IBV. Overexpression of Bcl2 or other anti-apoptotic Bcl2 family proteins can protects cells from coronavirus-induced apoptosis41. However, our data showed that phosphorylation at Ser70 of Bcl2 was not essential for its anti-apoptotic function during IBV infection. Nonetheless, JNK might still mediate its pro-apoptotic function by indirectly modulating Bcl2 and/or other Bcl2 family proteins. Previous studies showed that JNK signaling is necessary for maintenance of persistent SARS-CoV infection in cells42. Establishment of persistent infection was observed after the apoptotic events, so it is possible that JNK first serves as a pro-apoptotic protein in the acute phase of SARS-CoV infection, but switches to a pro-survival factor in persistently infected cells.JNK can translocate into mitochondria and modify Bcl2 family proteins43, but it can still effectively infect chicken fibroblast cell line DF144. DF1 is not used here because phos-JNK antibody could not detect the expression of avian homolog(s) (data not shown). Also, the cleaved form of PARP could not be strongly detected by PARP antibody. Several studies by our group on stress response have used H1299 and Huh-7 cells28. To be consistent with these previous experiments and to make study on signaling cross-talk between pathways possible, we chose to use the same cell lines in this study.The IBV strain we used has been passaged in Vero cells for 65 times20, has no effect on apoptosis induced by porcine epidemic diarrhea virus45, and promotes IBV-induced apoptosis in this study. Since the outcomes are so dramatically different, we consider it scientifically important to investigate in detail the activation of JNK pathway induced by IBV, a prototype gammacoronavirus. Unlike previous studies that rely on transfection of viral proteins, we examined the activation of JNK pathway in the context of an actual infection. Moreover, to our knowledge, this is also the first study to investigate potential involvement of JNK in coronavirus-induced apoptosis.Cellular response against different virus infections can be substantially different. Within the coronavirus family, JNK is required for persistent SARS-CoV infection in Vero E6 cells46. Also, vaccine strains that induced reduced apoptosis may be desirable, because viral epitopes are displayed longer for immune stimulation, favoring activation of cell-mediated immune response. IBV infection leads to a significant economic loss to poultry industry worldwide, with new variants emerging every year. A deeper understanding of how this important virus interacts with host cells, including the JNK pathway, provides new guidelines for construction of more suitable viral and cell systems for vaccine development.Virus-induced apoptosis is an important event that determines virulence and pathogenicity. At early infection, apoptosis can serve as an antiviral strategy, but apoptosis induced at late stage of infection may facilitate virus spreading and immune evasion. Therapeutical manipulation of JNK may be a viable approach to control viral replication or tissue damage associated with infection, as shown in a recent study for dengue virus43. To prepare the virus stock, monolayers of Vero cells were infected at a multiplicity of infection (MOI) of ~0.1 and cultured in plain Dulbecco Modified Eagle Medium  at 37 °C for 24 h. After three freeze/thaw cycles, cell lysate was clarified by centrifugation at 1500 × g at 4 °C for 30 min. The supernatant was aliquot and stored at −80 °C as virus stock. The titer of the virus stock was determined by plaque assays. Mock lysate was similarly prepared by subjecting uninfected Vero cells to three freeze/thaw cycles and clarified by centrifugation.The egg-adapted Beaudette strain of IBV (ATCC VR-22) was obtained from American Type Culture Collection (ATCC) and adapted to Vero cells as described2 of 254-nm shortwave UV radiation for 15 min with a CL-1000 cross-linker (UVP)47. To demonstrate that IBV had been inactivated, the Vero cells were incubated with UV-IBV and the cell lysates were analyzed by Western blot to confirm that no viral proteins can be detected.Inactivation of IBV was performed by exposing the virus stock to 120,000 mJ/cm2. Huh-7 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco). In all the experiments, cells were washed twice with serum-free medium before infected with IBV at an MOI of ~2 or incubated with equal volume of UV-IBV in serum-free medium. After 2 h of absorption, the cells were washed twice with serum-free medium and incubated at 37 °C before harvested.H1299 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 5% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco). All cells were grown in a 37 °C incubator supplied with 5% CO49.The antibodies against β-actin (#4967), FLAG-tag (#2044), PARP (#9532), total JNK (#9252), phospho-JNK (#9251), total c-Jun (#9165), phospho-c-Jun (#9261), Total MKK4 (#9152), phospho-MKK4 (#9151), Total MKK7 (#4172), phospho-MKK7 (#4171), total Akt (#4691), phospho-Akt (#4060), total Bcl2 (#2876), and phospho-Bcl2 (#2827), were purchased from Cell Signaling Technology. The anti-serum against IBV S protein and N protein were from rabbits immunized with bacterial expressed fusion proteins as previously describedpXJ40-FLAG at the BamHI and KpnI restriction sites. The resulting constructs were named pXJ40-FLAG-MKK4 and pXJ40-FLAG-MKK7, respectively. The cDNA of human Bcl2 (RefSeq NM_000633) was amplified from total RNA of H1299 cells by RT-PCR using the forward primer 5′-CGCAGATCTGCGCACGCTGGGAGAACAGGGTAC-3′ and reverse primer 5′-GCCGCTCGAGTCACTTGTGGCCCAGATAGGCACC-3′. The PCR product was digested and inserted into pXJ40-FLAG at the BamHI and XhoI sites to generate pXJ40-FLAG-Bcl2.The complementary DNA(cDNA) of human MKK4 (RefSeq NM_003010) was amplified from total RNA of H1299 cells by reverse transcription-polymerase chain reaction (RT-PCR) using the forward primer 5′-CCCGGATCCATGGCGGCTCCGAGCCCGAG-3′ and reverse primer 5′-CTTGGTACCTCAATCGACATACATGGGAGAGCTGGGAG-3′. The cDNA of human MKK7 (RefSeq NM_145185) was amplified similarly using the forward primer 5′-CCCGGATCCATGGCGGCGTCCTCCCTGGAAC-3′ and reverse primer 5′-CTTGGTACCCTACCTGAAGAAGGGCAGGTGGGG-3′. The PCR products were digested and inserted into pXJF40-FLAG-MKK7 using the forward primer 5′-CATTGCCGTTATGCAAATGCGGC-3′ and reverse primer 5′-GCCGCATTTGCATAACGGCAATG-3′. The MKK7-3E mutant was generated by replacing amino acids serine 271, threonine 275, and serine 277 with glutamic acid using forward primer 5′-GAGAAAGCCAAGGAGCGGGAAGCCGGCTGTGCCGCC-3′ and reverse primer 5′-TTCCCGCTCCTTGGCTTTCTCGTCCACCAGGCGGCC-3′. The MKK7-3A mutant was generated by replacing the same sites with alanine using forward primer 5′-GCCAAAGCCAAGGCGCGGGCCGCCGGCTGTGCCGCC-3′ and reverse primer 5′-GGCCCGCGCCTTGGCTTTGGCGTCCACCAGGCGGCC. The serine 70 in pXJ40-FLAG-Bcl2 was replaced with alanine using forward primer 5′-GTCGCCAGGACCGCGCCGCTGCAGAC-3′ and reverse primer 5′-GTCTGCAGCGGCGCGGTCCTGGCGAC-3′ to generate the Bcl2-S70A mutant. The constructs pcDNA-FLAG-MKK7-JNK1 and pcDNA-FLAG-MKK7-JNK1(APF) were obtained from Addgene as previously descried29.The ATP-binding mutant of MKK7-KM was generated by site-directed mutagenesis to replace amino acid lysine 149 with methionine in Transfection of plasmids DNA was performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Briefly, the cells were plated to a 12-well plate the day before transfection. For each well, 0.8 µg of plasmid DNA and 2 µl of Lipofectamine 2000 were each diluted with 100 µl of plain medium and incubated for 5 min. Then the diluted plasmid and transfection reagent were mixed by brief vortex and incubated for another 20 min. The H1299 cells were changed with 800 µl of medium containing 5% FBS and the transfection mixture was added to each well. The cells were incubated at 37 °C for 6–8 h before replacing with complete medium. At 24 h post-transfection, the cells were infected with IBV at an MOI of 2 or were mock infected, and were continued to be incubated before being harvested for protein and/or RNA analysis at indicated time points.50 and control EGFP siRNA (+): 5′-GCUGACCCUGAAGUUCAUC dTdT-3′ were purchased from Sigma. Transfection of siRNA was performed using DhamaFECT2 transfection reagent  according to the manufacturer’s instructions. At 48 h post-transfection, the cells were infected with IBV at an MOI of 2 or were mock infected, and were continued to be incubated before being harvested for protein analysis at the indicated time points.JNK1/2 siRNA (+): 5′-AAAGAAUGUCCUACCUUCU dTdT-3′g for 1 min, the supernatant was discarded and the pellets were lysed in 1× RIPA buffer. After clarifying by centrifugation and determination of protein concentration by spectrophotometer, the cell lysates were mixed with Laemmli sample buffer containing 100 mM dithiothreitol51. The protein samples were boiled at 90 °C for 5 min and centrifuged at 16,000 × g for 5 min. Equal amount of protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.2 µm nitrocellulose membranes (Bio-Rad). After the nonspecific antibody binding sites were blocked with 5% skim milk in Tris-buffered saline  containing 0.1% Tween 20, the membranes were incubated with 1 µg/ml primary antibodies at 4 °C overnight. After washing with Tris-buffered saline, the membranes were incubated with 1:2000 diluted anti-mouse or anti-rabbit IgG antibodies conjugated with horseradish peroxidase (DAKO) at room temperature for 2 h. The membranes were washed and the proteins detected with a chemiluminescence detection kit (Amersham Biosciences) and medical X-ray films (Fujifilm) according to the manufacturer’s instructions. The films were scanned as gray scale 8-bit images and the density of bands were determined by the NIH software ImageJ. All experiments were repeated for at least three times with similar result, and one of the representative results is shown.Cells were infected with IBV and harvested at indicated times points using cell scrapers (Corning). After centrifugation at 16,000 × Cell-free supernatants of IBV-infected cells collected at different time points were clarified by centrifugation and 10-fold serially diluted using serum-free DMEM. The viral titers were determined by plaque assay. Briefly, 250 µl of diluted supernatants were applied to confluent monolayers of Vero cells in 6-well plates. The plate was agitated every 10 min to ensure proper coverage of the monolayers. After 2 h of adsorption, unbound viruses were removed and cells were washed twice with DMEM. A total of 2 ml overlay medium (0.4% agarose in DMEM) was added to each well and the plates were incubated at 37 °C for 2 days before plaques formed. Agarose overlay was removed and the cells were fixed with 4% formaldehyde before staining with crystal violet. Finally plaque numbers were counted and the titers of individual samples were expressed in the unit of logarithm of plaque-forming units per ml. Each sample was titrated in triplicate in each experiment.


Pseudomonas syringae is found in a variety of terrestrial and aquatic environments. Some strains of P. syringae express an ice nucleation protein (hereafter referred to as Ice+) allowing them to catalyze the heterogeneous freezing of water. Though P. syringae has been sampled intensively from freshwater sources in France, little is known about the genetic diversity of P. syringae in natural aquatic habitats in North America. We collected samples of freshwater from three different depths in Claytor Lake, Virginia, USA between November 2015 and June 2016. Samples were plated on non-selective medium (TSA) and on medium selective for Pseudomonas (KBC) and closely related species to estimate the total number of culturable bacteria and of Pseudomonas, respectively. A droplet freezing assay was used to screen colonies for the Ice+ phenotype. Ice+ colonies were then molecularly identified based on the cts (citrate synthase) gene and the 16S rDNA gene. Phylogenetic analysis of cts sequences showed a surprising diversity of phylogenetic subgroups of P. syringae. Frequencies of Ice+ isolates on P. syringae selective medium ranged from 0 to 15% per sample with the highest frequency being found in spring. Our work shows that freshwater lakes can be a significant reservoir of Ice+ P. syringae. Future work is needed to determine the contribution of P. syringae from freshwater lakes to the P. syringae populations present in the atmosphere and on plants and, in particular, if freshwater lakes could be an inoculum source of P. syringae-caused plant disease outbreaks.The bacterium  Pseudomonas syringae has been studied as a plant pathogen since the early Twentieth century  allowing the bacterium to catalyze the freezing of water at temperatures much warmer than the temperature at which pure water freezes. This activity is due to expression of a lipoglycoprotein, called the INA protein  examine the frequency of P. syringae within the lake, (2) determine the relative proportion of Ice+ colonies of Pseudomonas in the lake, and (3) examine the phylogenetic relationship within and among strains of P. syringae collected from the lake. This research has applications in understanding how P. syringae is distributed in natural bodies of water, and may work toward greater understanding of the potential for a lake to release aerosolized Ice+ bacteria into the atmosphere.The overall goal of this study was to examine the relative abundance of Ice+ strains of Table 2). The samples were taken from four different locations selected to provide a variety of location types and within a reasonable rowing distance from the adjacent beach where the kayak was launched; (1) deep water in the middle of the lake, (2) shallow water near the mouth of an inlet, (3) shallow water in the inlet, (4) the mouth of a boat launch inlet. Samples were collected at the surface of the water at locations 2–4. At location 1, samples were collected at the water surface, at 4.6 and at 9.1 m. Two Van Dorn samplers were used to collect the surface and 4.6 m samples simultaneously in location 1. Table Samples were collected from Claytor Lake, VA, USA on nine different calendar dates. Fall/winter sampling days were: 5 November 2015, 18 November 2015, 16 December 2015, 21 January 2016, and 4 February 2016. Spring sampling days were: 2 May 2016, 16 May 2016, 25 May 2016, and 7 June 2016. Van Dorn (3.2 L)  water samplers were used to collect samples of lake water off the side of a kayak. For each sample, the time, GPS coordinates, and water temperature were recorded  to obtain counts of culturable bacteria which grow on TSA. We define culturable as any bacteria that grew on a given media under environmental conditions of ~20°C. The plates were incubated for 48–72 h at ambient room temperature (~20°C) and the colonies were counted.The lake water samples were placed on ice in a cooler immediately following collection, and transported back to the laboratory for analysis. In the laboratory, 1 L of each sample was filtered on to 0.2 μm porosity nitrocellulose filters. The filters were placed in 5 mL of the filtrate for 10 min with a stir bar to resuspend the bacteria at a 200X concentration. King's medium B modified with cephalexin (80 mg/L), cycloheximide (200 mg/L), and boric acid  (KBC)  and transferred to 140 μL of water with a sterile toothpick to perform a droplet freezing assay. The samples were stored at 4°C for 24 h. Two droplets of 12 μL of each sample were loaded onto Parafilm® M floating on an Alpha 12 cooling bath . Sterile 0.2 μm filtered water was used as a negative control. The temperature of the bath was set at −5°C during loading of the samples, and was then lowered to −12° in 1°C increments. The temperature at which each of the droplets froze was recorded. Strains for which both droplets froze were selected for further analysis.cts) housekeeping gene to identify colonies of P. syringae and related species. A 1% TBE Ethidium Bromide gel was used to visualize products from the PCR reaction. Samples that did not produce a product specific for P. syringae were subjected to PCR with 16S primers . PCR was performed with GoTaq® Green Master Mix (Promega M712) under the following parameters: 1x cycle denaturation 95°C for 10 min, 5x cycle denaturation 95 for 1 min, annealing 53°C for 30 s, extension 72°C 1 min 40 s, 25x cycle 94°C 30 s, 53°C 20 s, 72°C 1 min, 1x cycle 72°C 10 min, hold 4°C). The samples were purified using ExoI/rSAP prior to sequencing . The sequences from 16S primers were used to search GenBank (http://www.ncbi.nlm.nih.gov/blast) using the Blastn algorithm to identify the sequence with the closest match. The sequences from cts primers along with 38 reference strains of P. syringae  and a BioSprint15 DNA Plant Kit (Qiagen #941517) following manufacturer's protocols. PCR was conducted using a Mastercycler ep Gradient S thermocycler  with the extracted DNA using primers  for the citrate synthase (http://www.r-project.org). Because data were not normally distributed, non-parametric Kruskal-Wallis rank sum tests, were used to test for significant differences (P < 0.05) between percent of Ice+ strains between colony forming units (CFU) of samples at different depths (n = 55) and locations (n = 17). A Wilcoxon rank sum test for non-parametric data was used to compare between percent of Ice+ strains in fall/winter and spring collections (n = 66).Statistical analyses were conducted in R  and Pseudomonas in lake water was estimated from the counts of colonies on TSA and KBC media, respectively  for each sampling location for each day of collection showed considerable variation across all days and across all sampling locations. Figure −1) of Pseudomonas from all locations that grew on KBC media for all days of sampling, indicating that Pseudomonas was consistently found throughout Claytor Lake. However, the mean concentration for each collection day varied dramatically from a low of 110 CFU Liter−1 on 5 November 2015 to 16,240 CFU Liter−1 on 25 May 2016.The concentration of bacteria  was between 15 and 20% on each day of sampling with the exception of 4 February 2016 for which 47% of the Ice+ colonies were not P. syringae. The spring collections were not considered because the sample size of Ice+ strains was too small to obtain an accurate percentage. The 16S sequence results confirmed that these colonies were not P. syringae; they were identified as Xanthomonas, P. fragi, P. fluorescens, P. viridiflava, and P. plecoglossicida.The 2,750 colonies from KBC media were tested for the Ice+ phenotype with an ice nucleation assay. The freezing temperature between −5°C and −12°C was recorded for each droplet, and 58% froze at −5°C, 14% at −6°C, 4% at −7°C, 3% at −8°C, 4% at −9°C, 7% at −10°C, 5% at −11°C, and 6% at −12°C. The percent of colonies for which both tested droplets froze  or locations (P = 0.567). There was a significant difference between the percent of Ice+ strains in the fall collections and spring collections (P < 0.001).There was no significant difference between the CFU on KBC media at different depths , with most collections between 0.1 and 1.5%, indicating Pseudomonas was a small minority of the bacteria present. The concentration of Pseudomonas appears to be variable and subject to change day to day and even in different locations on the same day. Previous studies examining the relative abundances of bacteria in freshwater lakes found between 5 and 10% of the bacteria sampled were γ-Proteobacteria  or locations (P = 0.567). The first 2 days of sampling five different locations in the lake were sampled, but with this finding of non-significance one location was chosen for the remaining 7 days of sampling. In general, more variation was seen in the concentrations of CFU on KBC media in the spring collections since both, the highest and lowest concentrations, were obtained in the spring collections. The total bacteria count on non-selective TSA medium did not show as much variation between the two seasons of collection, suggesting that Pseudomonas is likely more subject to variation than the bacteria cultured on TSA.There was no significant difference in the concentration of Pseudomonas colonies were Ice+ on each sampling day, indicating Ice+ strains are consistently present throughout Claytor Lake. The Ice+ colonies were not evenly distributed across samples on a given day. Some collections did not have any Ice+ colonies and others had up to 50%. Joly et al. . The five fall/winter collections had a higher frequency of Ice+ bacteria ranging from 6 to 15% while the spring collections had a frequency of 0–2%. This significant difference between these collections suggests a potential ecological role for the ice nucleation phenotype; Ice+ strains of P. syringae may be better adapted to colder climates.Between 0 and 15% of the y et al.  sampled P. syringae were identified from diverse phylogroups. Berge et al.  under Grant Numbers DEB-1241068 [Dimensions: Collaborative Research: Research on Airborne Ice-Nucleating Species (RAINS)] and DGE-0966125 [IGERT: MultiScale Transport in Environmental and Physiological Systems (MultiSTEPS)]. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mesenchytraeus solifugus was analyzed by amplicon sequencing of 16S rRNA genes and their transcripts. Ice worms were collected from two distinct glaciers in Alaska, Harding Icefield and Byron Glacier, and glacier surfaces were also sampled for comparison. Marked differences were observed in bacterial community structures between the ice worm and glacier surface samples. Several bacterial phylotypes were detected almost exclusively in the ice worms, and these bacteria were phylogenetically affiliated with either animal-associated lineages or, interestingly, clades mostly consisting of glacier-indigenous species. The former included bacteria that belong to Mollicutes, Chlamydiae, Rickettsiales, and Lachnospiraceae, while the latter included Arcicella and Herminiimonas phylotypes. Among these bacteria enriched in ice worm samples, Mollicutes, Arcicella, and Herminiimonas phylotypes were abundantly and consistently detected in the ice worm samples; these phylotypes constituted the core microbiota associated with the ice worm. A fluorescence in situ hybridization analysis showed that Arcicella cells specifically colonized the epidermis of the ice worms. Other bacterial phylotypes detected in the ice worm samples were also abundantly recovered from the respective habitat glaciers; these bacteria may be food for ice worms to digest or temporary residents. Nevertheless, some were overrepresented in the ice worm RNA samples; they may also function as facultative gut bacteria. Our results indicate that the community structure of bacteria associated with ice worms is distinct from that in the associated glacier and includes worm-specific and facultative, glacier-indigenous lineages.The community structure of bacteria associated with the glacier ice worm  Glaciers, which are harsh environments that are permanently covered with ice and snow, have unique ecosystems composed of psychrophilic/psychrotolerant organisms , 33. RecMesenchytraeus solifugus , is the largest metazoan and regarded as the predominant consumer in North American maritime glaciers . Some collected ice worms were kept alive in melted glacier surface ice. All fixed samples were kept frozen with ice during transportation to a laboratory in Japan. Specimens preserved in RNAlater® were stored at −80°C, and live ice worms were reared at 4°C.Sampling expeditions were carried out in the upper area of Exit Glacier located in Harding Icefield (August 2014) and in Byron Glacier (August 2015). Both glaciers are located in the Kenai Peninsula, Alaska, and are approximately 90 km apart. In Harding Icefield, sampling was conducted at two sites; an ice surface site  and snowpack site  . In Byrolater® solution were slowly thawed on ice. Five ice worm individuals were arbitrarily selected from the specimens and placed into one tube. DNA and RNA were both simultaneously extracted using the PowerViral® Environmental RNA/DNA Isolation Kit , according to the manufacturer’s protocol with modifications. Briefly, ice worms were suspended in 600 μL of solution PV1 (lysis buffer), in which 24 μL of 1M dithiothreitol (DTT) was added instead of 6 μL β-mercaptoethanol. The suspension was homogenized with a sterile plastic pestle and subjected to a bead-beating procedure. Two-milliliter aliquots from glacier ice samples were centrifuged at 10,000×g at 4°C for 20 min. The pellets were suspended in solution PV1 with DTT, as described above, and processed according to the manufacturer’s protocol. Residual RNA or DNA was digested using RNaseA  or the TURBO DNA-free™ Kit (Ambion), respectively. DNA extracts were further purified with the DNeasy® Blood and Tissue Kit  to remove RNaseA. The amplification of 16S rRNA genes was not observed from purified RNA samples in 30-cycle PCR with a Bacteria-specific primer set, performed as described previously  with random hexamers. PCR was conducted using Phusion® High-Fidelity DNA Polymerase  with the Bacteria-specific 341F and 785R primer set, which targets the V3–V4 region (approx. 400 bp) of 16S rRNA. Primer sequences contained the Illumina overhang adapter sequence (http://support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.html). The PCR program was as follows: initial denaturation at 98°C for 30 s, 25 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, and a final extension at 72°C for 7 min. Nextera® XT indices  were attached to the amplicons by eight additional PCR cycles. After purification with the Agencourt® AMPure® XP , DNA was quantified with the Qubit® dsDNA High Sensitivity Assay Kit (Invitrogen). Sequencing was conducted on the Illumina MiSeq™ platform with MiSeq reagent kit v3.RNA samples were reverse-transcribed into cDNA using the SuperScriptfastq filter command with the following parameters: minimum length=350 bp, no ambiguous bases allowed, and maximum expected errors for all bases in a read were less than 1. The quality of reads was evaluated using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).Paired-end sequence reads were merged, primer trimmed, and quality filtered using the program package USEARCH v8.0 . Qualityhttps://www.arb-silva.de/aligner/) using the SILVA 126 database with default parameter settings, except for the minimum identity with the query sequence, set to 0.80. OTUs assigned to Eukarya, Archaea, mitochondria, chloroplasts, and “unclassified” were removed. We also removed OTUs affiliated with the genera Halomonas and Shewanella; we concluded that these two bacterial groups were reagent contaminants because they commonly appeared in distinct samples, including those not related to this study (data not shown). We further eliminated OTUs that could not be aligned with other 16S rRNA sequences.16S rRNA sequences were sorted into OTUs with a criterion of 97% sequence identity using the program UCLUST implemented in the QIIME v1.8.0 package with the furthest-neighbor clustering algorithm . Chimerimothur v1.37.0  based on the Bray–Curtis similarity index. Differences among clusters were statistically tested by an analysis of similarity (ANOSIM) with 999 permutations. The separation of clusters in the NMDS plot was correlated to OTU abundance by calculating Spearman’s correlation coefficient with  v1.37.0 .R v3.2.2 using the DESeq2 package with the negative binomial Wald test  and 1,218 (for Herminiimonas) unambiguously aligned nucleotide sites, which corresponded to positions 69–1440 and 94–1390 in Escherichia coli (J01695), respectively, were used for a phylogenetic analysis. Maximum likelihood (ML) and neighbor-joining (NJ) trees were constructed in MEGA7 , which corresponds to positions 1251–1272 in E. coli (J01695), was designed using ARB and labeled with Texas Red at the 5′ end. A non-labeled helper probe (5′-GGTTTTGTAGATT GGCACT-3′) was used to improve the efficiency of hybridization  . Specimens were observed under an Olympus BX51 epifluorescence microscope . Procedures were described in detail previously , respectively, were subjected to 16S rRNA amplicon sequencing. In addition, two and three sets of the glacier surface samples from Harding and Byron (designated as Harding surface/Byron surface), respectively, were similarly analyzed. In total, 4,208 OTUs were identified from 1,965,105 paired-end reads. The mean number (with SD) of the observed OTUs in each sample category was as follows; 79±16 (Harding worm DNA), 546±350 (Harding worm RNA), 101±44 (Byron worm DNA), 1,105±583 (Byron worm RNA), 230±160 (Harding surface DNA), 757±297 (Harding surface RNA), 116±30 (Byron surface DNA), and 810±274 (Byron surface RNA). Thus, the observed number of OTUs as well as the Chao1 species richness estimate and reciprocal Simpson’s diversity index were higher in RNA samples than in DNA samples in any of the worm or glacier surface samples . The ranEntomoplasmatales and Mycoplasmataceae sequences were abundantly detected in ice worm samples, whereas only a few were recovered from the glacier surface samples. Most of the Mycoplasmataceae OTUs in the ice worm samples were identical or closely related to the “Ca. Vermiplasma” sequences obtained in our previous study (Mycoplasmataceae bacteria in the NCBI nr/nt database: the hemotrophic cattle pathogen “Candidatus Mycoplasma haemobos” (KJ883514)  with 93.3% sequence similarity. Cytophagaceae, predominated by the genus Arcicella , were frequently detected in both worm and glacier surface samples, but were more abundant in the former, particularly in Byron worms   and uncuJ883514) . Entomopus study . Lachnoson worms .R=0.96, P<0.001). The contribution of the 10 most abundant OTUs to the separation of the plots is also shown in Ca. Vermiplasma” OTU11203 greatly contributed to separation between the ice worm and glacier surface samples. Entomoplasmatales OTU23265 and Arcicella OTU19485 contributed to the grouping of Byron worm samples, and the Harding worm-specific Lachnospiraceae OTU742 correlated with the grouping of Harding worm samples. Ferruginibacter OTU2436, Nakamurella OTU1618, and Methylophilaceae OTU15614, which were detected in all sample categories, but were more abundant in glacier surfaces, drove the grouping of the glacier surface plots  OTUs dominant in both Harding and Byron worm samples (designated as “core OTUs”); (ii) OTUs dominant in Harding worm samples only; (iii) OTUs dominant in Byron worm samples only; (iv) other dominant OTUs, which did not fit into groups (i) to (iii), but were included in the 10 most abundant OTUs , OTU11203 (“Ca. Vermiplasma”), OTU19485 (Arcicella), and OTU17386 (Herminiimonas) were only rarely found in the glacier surface samples , OTU9924 (Parachlamydiaceae), OTU3927 , and OTU18119 , were specifically detected in either the Harding or Byron worm samples, but were almost never found in any of the glacier surface samples. Parachlamydiaceae OTU9924 showed the highest sequence similarity (92.5%) to “Candidatus Metachlamydia lacustris” (GQ221847), which is a parasite of the aquatic amoeba, Saccamoeba lacustris  isolated from Ny-Alesund, Svalbard. OTU3927 was closest to the Entomoplasmatales core OTU, OTU23265.The majority of OTUs that were only dominant in either the Harding or Byron worm samples were also abundantly recovered from their corresponding habitat glacier surface samples, but were less abundantly detected in the other glacier surface samples . HoweverRickettsiales OTUs , were almost exclusively detected in ice worm samples  (Diophrys oligothrix strain DS12/4 (FR823003)  . OTU1360Flavobacterium) was also specifically detected in the ice worm samples, particularly in Byron worms , which was exclusively detected in the Byron worm samples, shared 91.1% sequence identity with clone MspOz-28-Sw-1-012 (AB991225) obtained from Mesenchytraeus nivalis, which was collected from snow in Japan (Brevinema andersonii strain CT11616 (NR_104855); however, sequence identity was only 84.2%.OTU23058 , 42FlavoMollicutes OTUs  were significantly more abundant in DNA samples than in RNA samples. Conversely, several glacier-indigenous OTUs of Alphaproteobacteria, Betaproteobacteria, and Planctomycetacia, including OTU11712 and OTU20203 (Acetobacteraceae), OTU8825 (Comamonadaceae), OTU15614 (Methylophilaceae), and OTU25211 (Planctomycetaceae), were more abundantly detected in RNA samples. The dominant Lachnospiraceae OTU742 was also significantly more abundant in RNA samples  also indicated that Lachnospiraceae and Ruminococcaceae bacteria in the mammalian gut are more specialized for the degradation of plant-derived recalcitrant substrates, such as cellulose, than Clostridiaceae bacteria  more abundant in RNA samples than in DNA samples , al, alAlphaMollicutes, Rickettsiales, Parachlamydiaceae, and Lachnospiraceae, but also glacier-indigenous bacteria, including Arcicella and Herminiimonas, benefit from the presence of the ice worm. The body surface and intestinal tract of the ice worm appear to provide a unique habitat, which is potentially rich with nutrients, to microbes in glacier ecosystems with limited resources, thereby driving the formation of a worm-associated bacterial microbiota that is distinct from that in glaciers. Since ice worms are the predominant metazoans in Alaskan maritime glaciers, the relationships between ice worms and bacteria potentially have an impact on the geochemical cycles in glaciers, as reported previously for terrestrial earthworms (The results of the present study strongly suggest that not only animal-associated bacterial lineages, such as rthworms . Future


Sargassum serratifolium C. Agardh  is a marine brown alga that belongs to the family Sargassaceae. It is widely distributed throughout coastal areas of Korea and Japan. S. serratifolium has been found to contain high concentrations of plastoquinones, which have strong anti-cancer, anti-inflammatory, antioxidant, and neuroprotective activity. This study aims to investigate the anti-diabetic activity of S. serratifolium and its major constituents through inhibition of protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, and ONOO−-mediated albumin nitration. S. serratifolium ethanolic extract and fractions exhibited broad PTP1B and α-glucosidase inhibitory activity . In an attempt to identify bioactive compounds, three plastoquinones  were isolated from the active n-hexane fraction of S. serratifolium. All three plastoquinones exhibited dose-dependent inhibitory activity against PTP1B in the IC50 range of 5.14–14.15 µM, while sargachromenol and sargaquinoic acid showed dose-dependent inhibitory activity against α-glucosidase . In the kinetic study of PTP1B enzyme inhibition, sargahydroquinoic acid and sargaquinoic acid led to mixed-type inhibition, whereas sargachromenol displayed noncompetitive-type inhibition. Moreover, plastoquinones dose-dependently inhibited ONOO−-mediated albumin nitration. Docking simulations of these plastoquinones demonstrated negative binding energies and close proximity to residues in the binding pocket of PTP1B and α-glucosidase, indicating that these plastoquinones have high affinity and tight binding capacity towards the active site of the enzymes. These results demonstrate that S. serratifolium and its major plastoquinones may have the potential as functional food ingredients for the prevention and treatment of type 2 diabetes. Diabetes mellitus (DM) has become a major growing public health problem worldwide. DM is a chronic disease that arises when the pancreas does not produce enough insulin or when the body cannot effectively use it, resulting in hyperglycemia. Type II DM (non-insulin-dependent diabetes) results from the ineffective use of insulin due to excess body weight, physical inactivity, and genetic susceptibility . Protein− action and the production of ONOO− can be indirectly inferred by the presence of nitrotyrosine residues  of determining the type of enzyme inhibition and dissociation constant (iK) of an enzyme-inhibitor complex. iK values of 2.21, 5.20, and 5.85 μM, respectively  resulted in markedly dose-dependent inhibition of ONOO−-mediated nitration of albumin (2-carboxyphenyl)amino]-1-naphthyl}-l-alanyl)amino] pentyl}oxy)-2-naphthoic acid (compound 23) and -2-ethyl-benzofuran-6-sulfonic acid 4-sulfamoyl-phenyl)-amide (compound 2) were considered the standard ligand for validating the AutoDock 4.2 results , indicating that additional hydrogen bonding might stabilize the open form of the enzyme and potentiate tighter binding to the PTP1B active site, resulting in more effective PTP1B inhibition.Molecular docking simulation is a good option for investigating protein-ligand interaction geometries at the molecular level. In these studies, molecular docking simulation of sargahydroquinoic acid, sargachromenol, and sargaquinoic acid with PTP1B was performed, where 3-({5--1-naphthyl}-l-alanyl)amino]pentyl}oxy)-2-naphthoic acid (compound 23) were obtained from the RCSB Protein Data Bank website [d-glucose (PDB ID: 3A4A) and the structure of acarbose and (Z)-3-butylidenephthalide (BIP) were obtained from the RCSB Protein Data Bank website [23, compound 2, acarbose, and BIP of the protein were considered to be the most convenient regions for ligand binding in the docking simulation. The 3D structures of sargahydroquinoic acid, sargachromenol and sargaquinoic acid were obtained from PubChem Compound (NCBI) and protonated using the MarvinSketch . A Lamarckian genetic algorithm (GA) method was used for docking. Gasteiger charges were added by default, the rotatable bonds were set with ADT, and all torsions were allowed to rotate. Grid box size was set to maximum with a default spacing. The X, Y, Z center was 37.303, 30.97, 33.501 for PTP1B and 21.272, −0.751, 18.633 for α-glucosidase. The docking simulation was conducted 10 independent GA with the default parameters. The results were analyzed using UCSF Chimera (http://www.cgl.ucsf.edu/chimera/), while the hydrogen bond interacting residues and van der waals interacting residues were visualized using LigPlot+.The structure of PTP1B, with its selective allosteric inhibitor 3--2-ethyl-benzofuran-6-sulfonic acid (4-sulfamoyl-phenyl)-amide , and was noted at S. serratifolium and its constituents may have the potential for therapeutic treatment of DM through PTP1B and α-glucosidase inhibition. Of the three isolated plastoquinones, sargahydroquinoic acid showed potent PTP1B inhibitory activity, while sargachromenol and sargaquinoic acid showed potent α-glucosidase inhibitory activity. These plastoquinones also effectively suppressed ONOO−-mediated albumin nitration in a dose-dependent manner. In addition, enzyme kinetics analysis of the type of enzyme inhibition and molecular docking simulation between enzymes and active compounds supported the above results. Therefore, S. serratifolium and its constituents should be further explored for the development of novel therapeutic or preventive agents for the treatment of diabetes.In conclusion, our results demonstrated that


The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity. Control of enzyme activity by phosphorylation of protein hydroxyls is universal in biology. The collective kinome in eukaryotes regulates enzyme activity by phosphorylating protein substrates at tyrosine, serine or threonine residues. This phosphorylation alters the electrostatic properties of an enzyme to modify its conformation to activate or inhibit the enzyme, induce interactions with other proteins, and to stabilize or, alternatively, promote degradation. All cellular processes, including cell cycle, growth, metabolism, gene expression, cell differentiation and death, as well as responses to the environment, are orchestrated by protein kinases. This utility means that there is intense interest in developing pharmaceuticals to regulate the activity of kinases. This has impelled efforts to determine the structure and function of kinases and their distinct attributes that might enable specific therapy.Structural analysis of many different protein kinase domains has identified that they adopt a broadly similar conformation when active. In contrast to this equivalence, different kinase domains appear to adopt diverse conformations in their inactive state13PKR is a member of a small family of kinases that induce a universal translational checkpoint, which enables eukaryotic cells to respond to environmental stress. Each member of this kinase family responds to separate stress stimuli to phosphorylate the eukaryotic translation initiation factor 2α (eIF2α), thereby inhibiting recharging of the initiation complex and suppressing general protein translation. PKR responds to duplex RNA molecules that are characteristic of viral transcripts. Accordingly, PKR constitutes an important arm of the antiviral response, which is underscored by its induction by the type I and III interferons. PKR is otherwise constitutively expressedPKR encodes an amino-terminal RNA-binding domain, which consists of tandem RNA-binding motifs (RBM1 and RBM2), and a carboxyl-terminal kinase domain that are linked by unstructured regions. It was initially asserted that the activity of PKR was regulated by an auto-inhibitory interaction between RBM2 and the kinase domain67101112Here we identify novel processes in the regulation of kinase activity. Our findings corroborate that PKR is controlled by auto-inhibition. However, in contrast to the initial report of the mechanism, we demonstrate that both motifs in the RNA-binding domain are involved. Unexpectedly, we identify that auto-phosphorylation of RBM1 is required to instigate the intra-molecular interactions with the kinase domain. This interaction alters the ternary state and activity of the protein. Our findings identify another innovative mechanism that has evolved to control the activity of kinases and provide insight for a strategy to develop inhibitors against kinase activity.Escherichia coli and purified as described by us previously1516Activation of PKR is accompanied by and contingent upon auto-phosphorylation. To gain insight into this process, we conducted mass spectroscopic analysis to identify phosphor-residues in recombinant human PKR produced in To determine the consequence of the phosphorylation of S33 and T42, these residues were mutated to alanines and the degree of auto-phosphorylation assessed as a measure of kinase activity. Auto-phosphorylation was assessed as the charge of the recombinant protein by isoelectric focusing. An unphosphorylated kinase-dead PKR molecule, in which the lysine residue number 296 that catalyzes transfer of the phosphate from ATP to a substrate is mutated to an arginine (K296R)7As kinase activity was advanced by mutation of the S33 and T42 to a non-phosphorylatable residue, it suggests that, paradoxically, auto-phosphorylation at these sites represses activity. As auto-phosphorylation is crucial in establishing the conformation of the active enzyme, we conducted a series of biochemical experiments to assess whether modifying S33 and T42 within RBM1 affects the ternary structure of PKR.per se. This was confirmed by RNA-binding assays with truncated peptides that encompass just the RNA-binding domain  through a conserved glutamate from the helix-α1; (2) residues in the motif GPxH from the β1-β2 loop; and (3) positively charged residues in the motif KKxAK at the N-terminus of the helix-α2 . As the g domain . As the A putative intra-molecular association would be expected to alter the state of other regions of PKR outside of the RNA-binding domain. The linker region between the RNA-binding and kinase domains contains a caspase-3 cleavage site at asparagine residue number 251The experiment was repeated to confirm the specificity of the cleavage site by engineering a unique protease site within the linker. A tobacco etch virus (TEV) protease cleavage site was introduced at residue number 225 by mutating the amino acid sequence DSLNSSS to ESLYFQS. The influence of mutating S33 and T42 to glutamic acids for proteolysis was reduced in this TEV-PKR molecule . AlthougAccordingly, the phosphor-S33 and -T42 residues change the state of the linker region that links the RNA-binding and kinase domains.To detect whether the effects of S33 and T42 residues extend to the kinase domain, we measured its association with the nucleoside analogue 2-aminopurine25We conducted a second ligand binding experiment that measured the extent of binding of the non-ATP competitive kinase inhibitor sunitinib, which has previously been reported to bind to PKRNext, we tested an association between PKR and a peptide inhibitor, coded P2, which was reported to bind to the kinase domain outside of the catalytic pocketTogether these data demonstrate that the phosphor-S33 and -T42 residues in RBM1 have allosteric effects that change the state of the following linker region and kinase domain. Together with the preceding findings, this is consistent with a putative intra-molecular interaction.nd following RPScore , so that an association between the separately tagged peptides restores the full-length fluorophore to produce a fluorescent signal. These assays were conducted in HEK293 cells in which the endogenous PKR was reduced by RNA interferences as described by us previouslyVenus fluorescence was produced in cells expressing the RNA-binding domain separately tagged with the split Venus fluorophore. Accordingly, we initially assessed the association between the RNA-binding domain and a truncated kinase domain to circumvent this signal from either homo-oligomerization of RBMs or, co-localization of the domain on RNA. A fluorescent signal was generated by co-expression of the RNA-binding domain and the kinase domain tagged with the split Venus tags . This siOur preceding data predict that the RNA-binding domain primarily associates with the kinase domain as a substrate, then after phosphorylation, in an alternative inhibitory conformation. To try to distinguish these alternatives, we examined fluorescence generated by dimerization of the full-length PKR with the truncated kinase domain. As before, we examined the consequence of kinase activity using the inhibitor 2-aminopurine. Additionally, we sought to reinforce the putative intra-molecular interaction by reducing RNA-binding to RBM1. Directed by structural and mutagenic analysis of RBMs, we mutated the histidine residue number 37 (H37K) within the GPxH motif from the β1-β2 loop of RBM1 343536. V3435Dimerization is an essential step in forming the active enzyme and so the role of the phosphor-residues in RBM1 in regulating this state would be anticipated to affect kinase activity. To demonstrate the consequence of this, we measured the effect on protein translation using a fluorescent reporter that we have described previouslyCollectively the data identify that the S33 and T42 are phosphorylation sites that affect the state of the enzyme, as demonstrated by altered binding of ATP-homologue and peptide ligands at the kinase domain, changed proteolysis of the linker region, and by masking of RNA-binding at the amino-terminus. As the masking of RNA-binding in the monomeric molecule was relieved by protomer dimerization, it suggested an intra-molecular interaction between RBM1 and the established dimer interface at the N-lobe of the kinase domain. Mutagenic analysis confirms modelling that predicted the association between specific residues within RBM1 and the N-lobe of the kinase domain. Correspondingly, we demonstrate that the phosphor-residues inhibit dimerization and repress PKR-dependent control of protein translation.We are not aware of another instance whereby auto-phosphorylation has been shown to repress kinase activity, although inhibition by a second kinase has been identified. For instance, the proto-oncogene tyrosine-protein (Src) kinases are phosphorylated at their carboxyl-terminus by the c-Src tyrosine protein kinase. These phosphorylated residues then associate with the protein’s first Src homology (SH) domain, which stabilizes an association between the second SH domain and residues that link these to the kinase domain. This then induces an association between tryptophan residue number 260 in the linker and αC-helix in the N-lobe of the kinase domain, which represses kinase activity2224In parallel with the SH domains of Src kinases, we propose that PKR is auto-inhibited through an intra-molecular interaction that engages both of its N-terminal RBMs. For the RBMs to co-operatively repress kinase activity, these homologous peptides must interact with separate surfaces on the kinase domain. Residues that vary between RBM1 and RBM2, between positions 119 and 129 of RBM2, likely determine this specificity. Consistent with these residues mediating discriminatory interactions, this variance is conserved in RBM2 but not RBM1 in PKR from separate species, or RBMs from other proteins . As some34et al.Analysis by others had demonstrated that the RBM2 associates with the C-lobe of the kinase domain in a charged region between the αD, αE and αG-helices6It had initially been contended that only RBM2 instigated auto-inhibition and that RBM1 must remain free in order to initiate RNA binding that then recruits RBM2 to relieve auto-inhibition. However, we propose that the association between RBM1 and the kinase domain does not involve the same face of the molecule that binds RNA, as is the case for RBM2. Instead we contend that the intra-molecular interaction is regulated by phosphor-residues within the domain’s β1–β2 loop. A structure of the RBM from the mitochondrial ribosomal protein L44, in association with the 39S ribosomal subunit, supports a role for residues in this loop in interactionsOur demonstration that phosphor-residues affect intra-molecular interaction offers an explanation for why earlier studies identified only a role for RBM2 in auto-inhibition and, also, how more recent findings controverted regulation of PKR by auto-inhibition. The initial studies used peptides that were separately expressed and purified and/or used the kinase-dead construct (K296R), and so did not account for phosphorylation. More recent studies that refute auto-inhibitory control of PKR used a wild-type construct that is co-expressed with a lambda protein phosphatase (PKR-PPase)et al.4748et al.There is precedence for inhibition of PKR activity through protein association at the N-lobe, as we propose for RBM1. The chaperone protein DNAJC3  associates with a similar region of the kinase domain (within the residues 244–296), as predicted for RBM14345Alternatively, it has been proposed that RBM2 obstructs an undetermined allosteric effect in the activation of PKR. The proposed positioning of RBM2 and the rigidity of much of the C-lobe in this region would seem to prescribe that such a mechanism involves residues within the flexible catalytic loop of the kinase domain. Accordingly, RBM2 might prevent essential auto-phosphorylation of the catalytic site (T446), thereby controlling allosteric effects. Perturbing an interaction between the catalytic loop and residues in the αC-helix could also prevent reposition of the αC-helix to establish the active kinase confirmation. Notably, replacing the activation loop of PKR with that from the constitutively active phosphorylase kinase 1, which contains a charged glutamic acid (E182) at the catalytic site, rescues the activity of dimerization mutantsIn summary, we propose a model for the regulation of PKR in which auto-phosphorylation of the S33 and/or T42 residues in RBM1 instigates an association with the N-lobe of the kinase domain that stabilizes a second association between the RBM2 and the C-lobe of the kinase domain. This co-operative, intra-molecular association restrains conformational changes required to constitute the active kinase molecule. The mechanism implies that PKR is inherently active and so must be constrained. We believe this is in keeping with the sensor and effector function of PKR within the innate immune response. It is also in keeping with the identification of a number of cellular suppressors that appear to target the monomeric PKR protein. The proposed mechanism would appear to be common to the other eIF2α kinases, with the Heme-regulated eIF2α kinase restrained by heme-binding, the PKR-like endoplasmic reticulum kinase by binding of the Heat Shock 70 kDa protein 5  and GCN2 by an intra-molecular auto-inhibition, although the precise mechanisms of suppression for these proteins is still unclear. As the cytosolic inhibitor of PKR, DNAJC3, has also been reported to suppress the activity of two other eIF2α kinases52Escherichia coli BL21 was from Sigma-Aldrich (USA). Caspase-3 was used as recommended by the manufacturer (Immunochemistry Technologies). Cells were transfected with Lipofectamine®2000 (ThermoFisher Scientific). Specific residues within PKR were altered by polymerase chain reaction (PCR), using mutagenic oligomers and the PfuTurbo DNA Polymerase (Agilent Technologies). PKR constructs were generated by point mutations introduced by PCR.2-Aminopurine was purchased from GE Healthcare Life Sciences. Anti-PKR (sc-6282) and -eIF2α (sc-81261) antibodies were from Santa Cruz Biotechnology, Inc. (USA). The anti-pSWestern blot analysis was conducted by transferring whole-cell lysates to Immobilon-FL, probing with primary antibodies followed by Alexa 680 nm or IRdye800 nm secondary antibodies, then visualizing by Licor-Odyssey (Biosciences). Primary antibodies consisted of the rabbit polyclonal eIF2α[pS52]PAb, eIF2αPAb (Invitrogen), rabbit monoclonal antibody YE350 (Abcam), rabbit polyclonal EIF2B2 (Abcam) and mouse monoclonal GAPDH (Abcam). Western blots were quantified with NIH Image J using sum intensity of the OD profile plots, with background correction calculated per lane.Purified recombinant PKR proteins were trypsin digested, and analysed using matrix-assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry and analytical high performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ESI-MS). Potential phosphor-peptides were identified by tandem mass spectrometry by searching for a characteristic mass loss equating to the loss of the phosphate group. To detect low intensity phosphor-peptides, the digest was then applied to a commercial phosphor-peptide affinity enrichment kit, and enriched phosphor-peptides were subsequently eluted and analysed by the above mass spectrometry methods. To characterize the site of phosphorylation, the phosphor-peptides were analysed on an ESI mass spectrometer with electron transfer dissociation (ETD) capability, giving sequence-specific information with the phosphor-peptides remaining intact. The phosphor-analysis was conducted on wild type and, as a control for auto-phosphorylation activity, kinase-dead (K296R) molecules.2 humidified incubator5/well) were plated into a 24-well plate , then transfected with separately tagged PKR constructs the next day and Venus fluorescence detected after 48 h. Total fluorescence was quantified using ImageJBimolecular complementation assays were performed as previously described in HEK293:shPKR cultured in DMEM supplemented with 10% fetal bovine serum under puromycin (10 μg/ml) selection, at 37 °C in a 5% COA fluorescent reporter was generated that expressed monomeric red fluorescent protein (RFP) under control of an RNA polymerase III U6 promoter enhanced with the RNA polymerase II cytomegalovirus promoter in a pBluescript (Stratagene) backbone plasmid. Control of translation by PKR was assessed by co-transfecting HEK293 cells with this reporter and plasmid that expressed different PKR constructs, then RFP production was measured after 60 h using a Typhoon Trio .Escherichia coli cells as described previouslyHis-tagged PKR proteins and the tobacco etch virus TEV protease were prepared in BL21-basic Purified single (K296R) or triple (K296R-S33E-T42E) mutant PKR proteins were incubated at molar ratios of 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20, 50, 100 and 200 with 100 μM of 2-aminopurine nitrate salt (SIGMA) or, alternatively, FITC-P2-peptide in 100 μl of buffer ), at room temperature for 20 min. Fluorescence was measured in an opaque, black 96-well plate (Millipore), at a 5 nm spectral band pass with excitation and emissions set to 310 and 340–550 nm, respectively, for 2-aminopurine, or 413 and 480–580 nm for the FITC-peptide, using a SpectraMAxi3 (Molecular Devices) microplate reader.g for 1 min, then electrophoretically separated through a native 10% polyacrylamide gel at 200 V, conducted at 4 °C for 20 min in TBE buffer. RNA was stained with a 10,000× dilution of SYBR Gold (ThermoFisher Scientific) in 1 × TBE for 10 min, then visualized with a Typhoon image scanner  using excitation with the 488 nM blue laser and emission with the LPB filter (≥510 nM). Images were analysed using ImageQuant 1.2 software (Molecular Dynamics).Purified preparations of single or triple mutant PKR proteins were incubated at molar ratios of 0.5, 1.0, 2.0, 5.0 and 10 with 50 ng of a duplex 57 nucleotide RNA species derived from the human immunodeficiency virus type I (HIV-1) trans-activation response element (TAR). Protein-RNA complexes were formed in 20 μl of a binding buffer  for 1 h at 4 °C, centrifuged at 10,000 Human recombinant caspase-3 (Abcam) was diluted to 1 U/μl aliquots in 1× phosphate-buffered saline (PBS) with 15% glycerol and stored at −70 °C before use. His-tagged PKR peptides were purified and 15 μg of protein was digested with caspase-3 in digestion buffer  or, alternatively, TEV in digestion buffer  at 37 °C with caspase-3 (1 U/μl), or TEV (1 U/μl), respectively, for 80 min. His-tagged peptides were recovered by incubation with 1 μl Ni-Sepharose beads  at 4 °C overnight with a gentle rotation and centrifugation at 500 rpm for 1 min in a microcentrifuge. The pellet was resuspended in 100 μl loading buffer and 30 μl was loaded onto a polyacrylamide gel for electrophoretic separation and peptides detected by immunoblotting.Two-dimensional gel electrophoresis used the ZOOM Benchtop Proteomic System (ThermoFisher Scientific).Circular dichroism spectra were measured at 20 °C using a Jasco J815 CD spectrophotometer. All scans were run on proteins concentrated to 400 μg/ml in PBS. Scans were run between 190 and 260 nm and the average ellipticity of triplicate scans recorded. Data were converted to mean residue ellipticity (MRE) by the equation of Correa and Ramoshttp://R-project.org) were used to analyse the data. All statistical analysis was performed with GraphPad Prism or Microsoft Excel software. Differences were calculated using the unpaired, two-tailed t-test for pairwise comparisons, or by linear regression calculated for each genotype, for the temporal data.The statistical applications within Microsoft Excel or the program R-2.14.0 .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.