Modulators of 14-3-3 Protein-Protein Interactions

Direct interactions between proteins are essential for the regulation of their functions in biological pathways. Targeting the complex network of protein-protein interactions (PPIs) has now been widely recognized as an attractive means to therapeutically intervene in disease states. Even though this is a challenging endeavor and PPIs have long been regarded as 'undruggable' targets, the last two decades have seen an increasing number of successful examples of PPI modulators resulting in a growing interest in this field. PPI modulation requires novel approaches and the integrated efforts of multiple disciplines to be a fruitful strategy. This Perspective focuses on the hub protein 14-3-3, which has several hundred identified protein interaction partners and is therefore involved in a wide range of cellular processes and diseases. Here, we aim to provide an integrated overview of the approaches explored for the modulation of 14-3-3 PPIs and review the examples resulting from these efforts in both inhibiting and stabilizing specific 14-3-3 protein complexes by small molecules, peptide-mimetics and natural products

Chironomids can be reliable proxies for Holocene temperatures. A comment on Velle et al., 2010

Velle et al. (2010) discussed discrepancies between Scandinavian Holocene chironomid-inferred temperature estimates, which they attribute to the response of chironomids to environmental variables other than temperature and to taxonomic shortcomings. They suggest ways in which the reliability of chironomid-based paleotemperature reconstructions could be improved by taking into account ecological complexity. While we agree with many of their recommendations, based on the results of other work, we think their paper is unnecessarily pessimistic regarding the ability of existing chironomid-based temperature inference models to provide reliable estimates of past temperature. We offer a critique of the main points discussed by Velle et al. (2010) and provide evidence that chironomid-based temperature inference models can reliably reconstruct mean July air temperature in the Lateglacial and Holocene over millennial and centennial timescales

Evaluation of an Objective Measurement Tool for Stress Level Reduction by Individually Chosen Music During Colonoscopy—Results From the Study “ColoRelaxTone”

Background and Aims: Colonoscopy as standard procedure in endoscopy is often perceived as uncomfortable for patients. Patient's anxiety is therefore a significant issue, which often lead to avoidance of participation of relevant examinations as CRC-screening. Non-pharmacological anxiety management interventions such as music might contribute to relaxation in the phase prior and during endoscopy. Although music's anxiolytic effects have been reported previously, no objective measurement of stress level reduction has been reported yet. Focus of this study was to evaluate the objective measurement of the state of relaxation in patients undergoing colonoscopy. Methods: Prospective study (n = 196) performed at one endoscopic high-volume center. Standard colonoscopy was performed in control group. Interventional group received additionally self-chosen music over earphones. Facial Electromyography (fEMG) activity was obtained. Clinician Satisfaction with Sedation Instrument (CSSI) and Patients Satisfaction with Sedation Instrument (PSSI) was answered by colonoscopists and patients, respectively. Overall satisfaction with music accompanied colonoscopy was obtained if applicable. Results: Mean difference measured by fEMG via musculus zygomaticus major indicated a significantly lower stress level in the music group [7.700(±5.560) μV vs. 4.820(±3.330) μV; p = 0.001]. Clinician satisfaction was significantly higher with patients listening to music [82.69(±15.04) vs. 87.3(±15.02) pts.; p = 0.001]. Patient's satisfaction was higher but did not differ significantly. Conclusions: We conclude that self-chosen music contributes objectively to a reduced stress level for patients and therefore subjectively perceived satisfaction for endoscopists. Therefore, music should be considered as a non-pharmacological treatment method of distress reduction especially in the beginning of endoscopic procedures

Red pigmentation can be used to reliably distinguish between live Calanus finmarchicus and Calanus glacialis females in the Fram Strait

Copepods from the genus Calanus provide an important lipid-rich food source in the Arctic marine foodweb. Despite extensive research on Calanus finmarchicus and Calanus glacialis, accurately identifying adults to species level remains challenging due to similar morphologies. Although these species co-occur in many regions, the distribution of C. finmarchicus and C. glacialis correspond to Atlantic and Arctic water masses respectively and are frequently used as climate indicators. Correct identification is therefore vital for understanding the phenotypic plasticity of these species and the impacts climate change will have on Calanus-dominated marine ecosystems. In this study, prosome length and percentage of red pigmentation (redness) of genital somites, the antennae, and throughout the whole body were determined for 139 females of C. finmarchicus and C. glacialis from the Fram Strait. Molecular analysis of a 16S rDNA barcode confirmed that the best morphological features for resolving the identity of these two species were the redness of the antennae and the redness of the genital somites. Overall accuracy of using antennae redness and genital somite redness to discriminate between the two species were the same, yet each of these explanatory variables had different specificity; C. finmarchicus were more accurately identified by the absence of redness in the genital somites, whereas C. glacialis were more accurately identified using antennae redness. Given the ecological importance of these congeners, these findings contribute to a better understanding of the reliability of using morphological characteristics to identify Calanus to species level, especially when sorting live specimens for climate-related ecological experiments

Prohibitin plays a critical role in Enterovirus 71 neuropathogenesis

<div><p>A close relative of poliovirus, enterovirus 71 (EV71) is regarded as an important neurotropic virus of serious public health concern. EV71 causes Hand, Foot and Mouth Disease and has been associated with neurological complications in young children. Our limited understanding of the mechanisms involved in its neuropathogenesis has hampered the development of effective therapeutic options. Here, using a two-dimensional proteomics approach combined with mass spectrometry, we have identified a unique panel of host proteins that were differentially and dynamically modulated during EV71 infection of motor-neuron NSC-34 cells, which are found at the neuromuscular junctions where EV71 is believed to enter the central nervous system. Meta-analysis with previously published proteomics studies in neuroblastoma or muscle cell lines revealed minimal overlapping which suggests unique host-pathogen interactions in NSC-34 cells. Among the candidate proteins, we focused our attention on prohibitin (PHB), a protein that is involved in multiple cellular functions and the target of anti-cancer drug Rocaglamide (Roc-A). We demonstrated that cell surface-expressed PHB is involved in EV71 entry into neuronal cells specifically, while membrane-bound mitochondrial PHB associates with the virus replication complex and facilitates viral replication. Furthermore, Roc-A treatment of EV71-infected neuronal cells reduced significantly virus yields. However, the inhibitory effect of Roc-A on PHB in NSC-34 cells was not through blocking the CRAF/MEK/ERK pathway as previously reported. Instead, Roc-A treated NSC-34 cells had lower mitochondria-associated PHB and lower ATP levels that correlated with impaired mitochondria integrity. <i>In vivo</i>, EV71-infected mice treated with Roc-A survived longer than the vehicle-treated animals and had significantly lower virus loads in their spinal cord and brain, whereas virus titers in their limb muscles were comparable to controls. Together, this study uncovers PHB as the first host factor that is specifically involved in EV71 neuropathogenesis and a potential drug target to limit neurological complications.</p></div

Roc-A treatment of EV71-infected NSC-34 cells.

<p>NSC-34 cells were first infected with EV71 at M.O.I. 10, followed by Roc-A treatment for 48 hours. At 48 h.p.i. the culture supernatant was collected for viral titer determination by plaque assay (a). Furthermore, the cell lysate was prepared for Western blot analysis (b). Relative band quantification (below Western blot) was determined by ImageJ, by normalizing to loading control, β-actin. Cell viability was assessed using alamarBlue assay. (c) Immunostaining of Roc-A treated NSC-34 cells. At 48 hours incubation the cells were fixed with ice cold methanol and probed with specific antibodies. Scale bar denotes 20 μm. (d) Mitotoxicity of Roc-A in NSC-34 cells. Cells were incubated with various concentrations of Roc-A for 48 hours before assessment of cytotoxicity (fluorescence) and mitotoxicity (luminescence) using Mitochondrial ToxGlo Assay. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test (*, <i>p</i><0.05; **, <i>p</i><0.005; ***, <i>p</i><0.001; ****, <i>p</i><0.0001). One representative from two independent experiments is shown.</p

Role of PHB in EV71-infected SK-N-SH cells.

<p>(a) SK-N-SH cells were transfected with PHB siRNA at various concentrations for 48 hours. The efficiency of siRNA knockdown was verified by Western blot. PHB-knockdown SK-N-SH cells were infected with EV71 at M.O.I. 1. Viral titers in the culture supernatant were determined by plaque assay at 48 h.p.i. Statistical analysis was performed using two-tailed student’s t-test (** <i>p</i><0.005, *** <i>p</i><0.005). Non-targeting siRNA (NTC) served as control. (b) SK-N-SH cells were pre-treated with anti-PHB antibodies followed by EV71 infection at M.O.I. 1. Culture supernatant was harvested at 48 h.p.i. for viral titer determination by plaque assay. IgG isotype antibodies served as control. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test (***, <i>p</i><0.0005; ****, <i>p</i><0.0001). (c) SK-N-SH cells were reverse-transfected with 50 nM PHB siRNA, siNTC or left untreated prior to transfection with 0.25 μg of lucEV71 RNA. Luminescence signal was read at 48 h.p.t. Statistical analysis was performed using one-way ANOVA with Dunnet’s post test (**, <i>p</i><0.005). (d) SK-N-SH cells were first infected with EV71 at M.O.I. 1 followed by Roc-A treatment for 48 hours. At 48 h.p.i. the culture supernatant was collected for viral titer determination by plaque assay, and the cell lysate was harvested for Western blot analysis. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test (**, <i>p</i><0.005). Relative band quantification (below Western blot) was determined by ImageJ, by normalizing to loading control, β-actin. Error bars represent mean ± standard deviation. Cellular cytotoxicity was assessed using alamarBlue assay. One representative from two independent experiments is shown.</p

siRNA-mediated gene silencing in NSC-34 cells.

<p>NSC-34 cells were transfected with various concentrations of ON-TARGET PLUS siRNA SMARTpools. (a) At 48 h.p.t., the cell viability of transfected cells was assessed using alamarBlue assay. (b) Knocked-down NSC-34 cells were infected with EV71 at M.O.I. 10. The viral titers in the culture supernatant were determined by plaque assay at 48 h.p.i.. Statistical analysis was performed using one-way ANOVA test with Dunnett’s posttest (* <i>p</i><0.1, ** <i>p</i><0.01, *** <i>p</i><0.001, **** <i>p</i><0.0001). (c) NSC-34 cells were transfected with PHB siRNA pool or with non-targeting siRNA (NTC) control at various concentrations. The efficiency of siRNA knockdown was verified at 48 h.p.t. by Western blot. (d) PHB siRNA- or siNTC-transfected cells were infected with EV71 at M.O.I. 10. The viral titers in the culture supernatants were determined by plaque assay at 48 h.p.i.. Cell viability of the transfected cells was assessed using alamarBlue assay. Statistical analysis was performed using two-tailed student’s t-test (** <i>p</i><0.005, *** <i>p</i><0.005). Relative band quantification (below Western blot) was determined by ImageJ, by normalizing to loading control, β-actin. Error bars represent mean ± standard deviation. One representative of two biological repeats is shown.</p

Involvement of intracellular PHB in viral genome replication.

<p>(a) NSC-34 cells were reverse-transfected with 50 nM of PHB siRNA, followed by transfection with EV71 purified RNA genome. Culture supernatant was harvested at the indicated time points post-transfection for viral titer determination by plaque assay. Statistical analysis was performed using two-way ANOVA test with Sidak’s multiple comparisons test (****, <i>p</i>< 0.0001). Viral RNA only transfection served as control. A representative experiment is shown from two independent repeats. (b) Schematic drawing of lucEV71 replicon. (c) NSC-34 cells were reverse-transfected with 50 nM of PHB siRNA, siNTC or left untreated prior to transfection with 1 μg of lucEV71 RNA. Luminescence signal was read at 48 h.p.t. Statistical analysis was performed using one-way ANOVA with Dunnet’s post-test (****, <i>p</i><0.0001). Error bars represent mean ± standard deviation. One representative of two biological repeats is shown.</p