Chronic tinnitus seems to be caused by reduced inhibition among frequency selective neurons in the auditory cortex. One possibility to reduce tinnitus perception is to induce inhibition onto over-activated neurons representing the tinnitus frequency via tailor-made notched music (TMNM). Since lateral inhibition is modifiable by spectral energy contrasts, the question arises if the effects of inhibition-induced plasticity can be enhanced by introducing increased spectral energy contrasts (ISEC) in TMNM. Eighteen participants suffering from chronic tonal tinnitus, pseudo randomly assigned to either a classical TMNM or an ISEC-TMNM group, listened to notched music for three hours on three consecutive days. The music was filtered for both groups by introducing a notch filter centered at the individual tinnitus frequency. For the ISEC-TMNM group a frequency bandwidth of 3/8 octaves on each side of the notch was amplified, additionally, by about 20 dB. Before and after each music exposure, participants rated their subjectively perceived tinnitus loudness on a visual analog scale. During the magnetoencephalographic recordings, participants were stimulated with either a reference tone of 500 Hz or a test tone with a carrier frequency representing the individual tinnitus pitch. Perceived tinnitus loudness was significantly reduced after TMNM exposure, though TMNM type did not influence the loudness ratings. Tinnitus related neural activity in the N1m time window and in the so called tinnitus network comprising temporal, parietal and frontal regions was reduced after TMNM exposure. The ISEC-TMNM group revealed even enhanced inhibition-induced plasticity in a temporal and a frontal cortical area. Overall, inhibition of tinnitus related neural activity could be strengthened in people affected with tinnitus by increasing spectral energy contrast in TMNM, confirming the concepts of inhibition-induced plasticity via TMNM and spectral energy contrasts. The human nervous system changes dynamically with experience. This phenomenon, named plasticity, was first described by James in 1890 ,3. One pTinnitus, a syndrome assumed to derive from imbalanced excitation and inhibition in the auditory cortex –11, is aOne major cause for chronic tinnitus is reduced inhibition in the auditory cortex ,20,21, tSince tinnitus seems to be caused by reduced inhibition, a possible treatment strategy is the induction of inhibition onto the over-activated neurons representing the tinnitus frequency (TF) . The stiThe direct effect of lateral inhibition decreases as a function of reduced masker duration  and withThe mechanisms of inhibition-induced plasticity were applied as a treatment strategy against tinnitus by inducing inhibition onto neurons coding the TF. Okamoto et al. presented music with a notch filter centered at the individual TF to participants affected by tonal tinnitus for two hours each day over a period of one year  . They deThe question arises, if the above mentioned treatment effects of inhibition-induced plasticity could be further enhanced. Lateral inhibition was found to be modifiable by spectral energy contrasts variation in notched noise . More prRecently, we demonstrated inhibition-induced plasticity in participants affected with tinnitus . We adapThe current study compared inhibition-induced plasticity by classical TMNM versus ISEC-TMNM. Here we hypothesized:Overall, neural activity evoked by the TF should be reduced after TMNM exposure in a tinnitus network comprising temporal, parietal and frontal regions in the N1m time window.Reduction of neural activity after music exposure should be greater in the ISEC-TMNM group as compared to the classical TMNM group within the N1m time window.Overall, subjectively perceived tinnitus loudness should be reduced after TMNM exposure.Tinnitus loudness reduction after TMNM exposure should be greater in the ISEC-TMNM group as compared to the classical TMNM group.The aim of this study was therefore twofold. On the one hand we wanted to test our concept of inhibition-induced plasticity by manipulating the degree of lateral inhibition, hereby testing the transferability of effects of lateral inhibition on inhibition-induced plasticity. Additionally, we wanted to improve the tailor-made notched music training (TMNMT), by transferring basic research into clinical practice.Eighteen subjects  were recruited from a database of the Institute for Biomagnetism and Biosignalanalysis of the University of Muenster. All subjects in the data base previously participated in a diagnostic screening including an anamnestic interview, an ear examination conducted by an otolaryngologist, a hearing test and a TF determination. The hearing test and the TF determination were done using an appropriate audiometer  and calibrated headphones (Sennheiser HDA 200). TF determination consisted of three steps including a presentation of seven predefined sine tones (between 1 and 12.5 kHz), a two-forced choice task until only one frequency remained and an octave confusion test . 2. TF between 1.5 kHz and 8.5 kHz. 3. Maximal hearing loss of 65 dB in a range of ½ octaves below or above the individual TF. 4. No acute or chronic otological, neurological or psychiatric disorders. 5. No drug abuse. 6. Subjects were right handed. These criteria were defined to ensure an optimal effect of TMNM. A hearing level of equal or less than 65 dB should guarantee an adequate perception of TMNM. The tinnitus pitch limitation was defined due to limitations in our magnetoencephalographic system (MEG). However, according to the data and literature available, we cannot state which exact levels of hearing loss or tinnitus pitch might be limitations of the general effectiveness of TMNM.The study was conducted according to the Declaration of Helsinki and the study protocol was approved by the ethics committee of the Medical Faculty of the University of Muenster. Subjects were fully informed about the study’s content and gave written consent before participating.t-tests for the factors (i) age, (ii) TF, (iii) hearing loss at TF and (iv) maximum hearing loss ½ octaves above or below TF. The values for the TF are naturally logarithmic and were transformed into octaves (with 1000 Hz as reference frequency) to achieve a linear distance between values, making a test of group homogeneity possible. The transformation formula was: Log2*(Hz/1000). Before and after each training session, auditory evoked fields were recorded by means MEG recordings.The study design is depicted in Training session: Subjects were seated in a silent room and were supplied with an MP3 player. Before participation, subjects provided CDs and digital music files with approximately three hours of their favorite music. This music was filtered online during the training session. The filtering procedure differed between the two groups in the following manner: in the classical TMNM group, music was modified on-line in two steps. First, music energy spectrum was equalized by redistributing energy of lower frequencies to higher frequencies, which is also called “flattening” . This prMEG stimulation: In the MEG, subjects were stimulated with a reference tone of 500 Hz (RT) and a tinnitus tone (TT), representing their individual TF. Both stimuli consisted of a combined sound stimulus of 1000 ms duration. The first 300 ms consisted of a sine tone; the last 700 ms were 40 Hz amplitude modulated. We demonstrated previously that only the N1m response is modulated by spectral energy contrasts, not the ASSR evoked by the 40 Hz amplitude modulated tone  could noTinnitus loudness assessment: Tinnitus loudness was judged before and after each MEG measurement via visual analog scales (VAS). The scale had a length of 10 cm and ranged from “tinnitus is absent” to “highest perceived tinnitus loudness ever” which was rated by marking a cross onto the scale line. In order to be sure that no Zwicker tone (a transient phantom sound) has appeared after notched music exposure , subjectContinuous MEG data was recorded in a magnetically shielded room with a 275 channel whole head MEG system  and digitally sampled at a rate of 600 Hz. The MEG pickup coils had an axial gradiometer configuration . Subjects were sitting in an upright position while their head was stabilized by cotton patches inside the MEG dewar. The stability of the head position during the MEG sessions was monitored by repeatedly measuring the magnetic fields of three head localization coils, set up inside the ear canals and upon the nasion .The current study investigates the impact of increased spectral energy contrasts onto inhibition-induced plasticity and therefore the concept of inhibition-induced plasticity. It additionally investigates parametrically a possible improvement of the effectiveness of the tailor-made notched music training. These research questions can be answered by analyzing the neural activity evoked by the TT. The RT is completely irrelevant for these research questions. However, we needed to present the RT to guarantee an adequate loudness matching and to keep paradigms between classical TMNM group  and ISECData was preprocessed using Brain Electrical Source Analysis Software . First, an artefact correction was applied by calculating an independent component analysis (ICA) implemented in BESA  on the rwww.emegs.org), a MATLAB based Software . A spherical shell with 8 cm radius and 350 evenly distributed dipole locations, tangentially oriented to the spherical model, was used as a source model. The positions of the MEG sensors relative to the head were used to calculate individual lead-field matrices for each subject. The Tikhonov regularization parameter was set to λ = .1 for each subject and condition. To roughly represent the grey matter depth, a source shell radius of 87% of the individually fitted spherical conductivity model was chosen. A distributed source reconstruction of MEG signals does not reveal the precise location of neural generators; nevertheless, it results in a reasonably good approximation of cortical sources and corresponding assignment to larger cortical structures. The averaged magnetic field distributions and individual sensor positions for each subject were used to calculate topographies of source direction independent neural activities (i.e. the vector length of the estimated source activities at each position) for each time point, condition and individual subject.Neural sources underlying the auditory evoked fields were estimated using the least square minimum-norm estimation  implemeDay  and Session  and the between-subject factor Group  was calculated for the L2-MNE values. A correction for multiple comparisons was applied on the results of interest of the ANOVA (main effect Session and interaction effect Session x Group) using a cluster-based permutation test for F-values [P-value of. 05. Significant spatiotemporal clusters in the N1m time interval (70–130 ms), which survived the cluster-based permutation test, for the main effect of Session and the interaction effect Session x Group were further analyzed with post-hoc t-test to disentangle the significant interaction effects.A 3 x 2 x 2 mixed model Analysis of Variance (ANOVA) over all dipoles and time points with the within-subject factors F-values . The corHemisphere  was performed. Depending on the effect of interest, the ANOVA had either the factors Session x Hemisphere, or the factors Session x Group x Hemisphere, to calculate possible hemispheric differences.For the case that significant effects were found in one hemisphere only, a lateralization analysis was conducted. This included the calculation of a lateral-symmetric spatiotemporal cluster by mirroring the significant dipole locations sagittally. An ANOVA for the mean neural activation of both clusters including the factor Group and the within subject factor Session for the mean VAS ratings before and after MEG measurements averaged across all three days.Statistical analysis of behavioral data included a 2 x 2 mixed model ANOVA with the between subjects factor p-values of independent t-tests.On average, groups did not differ significantly in age, TF, hearing loss at TF, maximum hearing loss ½ octaves above or below TF and VAS ratings on the first training day before music exposure. F-values of the mixed model ANOVA Session  x Group  confirms the above mentioned observation by revealing a significant main effect for the factor Session  in five different regions  = 13.14, p = .002, η2 = .45. This effect could also be shown in a left parietal region  = 5.62, p = .031, η2 = .26. Additionally, a right orbitofrontal cluster  = 15.91, p = .001, η2 = .50), as well as a right cerebral region with extents towards occipital and temporal areas  = 13.39, p = .002, η2 = .46). Furthermore, a right frontal region showed a significant main effect between 90 and 113 ms, F  = 10.21, p = .006, η2 = .39. However, here TF evoked neural activity increased after music exposure.There was a significant reduction of neural activity evoked by the TF in a left temporal cluster  for the l region  between  cluster  in the tal areas  for the Session x Hemisphere for the temporal, parietal and occipito-temporal clusters within the respective time intervals  and thus lateralization effects for these spatiotemporal clusters are not further regarded. However, there was a significant interaction effect of Session x Hemisphere for the frontal  = 6.78, p = 0.019) and the orbitofrontal cluster  = 7.69, p = 0.014). Hence, both frontal clusters showed lateralization effects and only the right hemisphere should be further interpreted.To test for lateralization effects, the above mentioned data was reanalyzed taking the sagittally mirrored data into account. There was no significant interaction effect of F-values of the interaction effect (Session x Group) revealed two significant spatiotemporal clusters in the predefined N1m time interval shown in Considering the type of TMNM, the cluster-based permutation test for the F  = 6.77, p = .019, η2 = .30. There was a reduction of neural activity after music exposure in the ISEC-TMNM group, which was absent in the classical TMNM group  = 2.62, p = .031), but not in the classical TMNM group (t (8) = -0.95, p = .370).The first cluster was located in the left inferior temporal lobe  = 10.11, p = .006, η2 = .39. Paired t-tests again resulted in a significant reduction of neural activity only for the ISEC-TMNM (t (8) = 3.521, p = .008) but not for the classical TMNM group (t (8) = -0.384, p = .711; right panel of A second significant spatiotemporal cluster for the interaction effect Session x Group x Hemisphere  resulted in non-significant differences between hemispheres for both clusters . Hence a lateralization effect can be excluded for both regions.Testing for lateralization effects by calculating the interaction effect for the factors Session x Group were found in the P2 time interval (150–250 ms). The first cluster was located in a central-parietal region for the time interval 241–250 ms, F  = 8.27, p = .01, η2 = .34. Paired t-tests resulted in a significant increment of neural activity in the classical TMNM group (t (8) = -3.15, p = .014), while the ISEC-TMNM group showed no significant differences. Additionally, a broad frontal regions revealed a significant interaction effect in the time interval between 190 and 248 ms, F  = 9.97, p = .01, η2 = .38. Again we calculated paired t-tests to disentangle the interaction effect. While the ISEC-TMNM group showed a significant reduction of tinnitus-related neural activity after TMNM exposure (t (8) = 1.94, p = .045 (one-sided)), the classical TMNM group showed a significant increment of neural activity (t (8) = -2.76, p = .025). Results in the P2 time-window were reported for the sake of completeness and will not be discussed further, since this would extend the scope of this study.Inspecting the global field power depicted in Session x Group for the VAS ratings revealed a significant main effect for the factor Session, F  = 49.69, p <. 001, η2 = .76. As depicted in Session and Group, F  = 1.52, p = .235. Hence, the amount of tinnitus loudness reduction did not differ between both groups. No participant reported to perceive a Zwicker-tone after TMNM exposure.The mixed model ANOVA with the factors Plasticity is defined as the dynamical adaptation of our nervous system due to experience –3. We coclassical TMNM group in a previous study by Stein and Engell et al. (2014) [Another spatiotemporal cluster revealing a significant reduction of tinnitus-related neural activity was located in a cerebral region with extents into occipital and temporal areas. We assume this cluster as reflecting activity in the cerebellum, which extends to neighboring areas as consequence of the L2MNE bias towards distributed and superficial sources . Int. (2014) , though The main aim of the current study was to investigate whether inhibition-induced plasticity is modifiable by varying spectral energy contrasts in music. More precisely we asked the question if amplifying the edge frequency bands around the notch results in increased inhibition of neurons coding the TF as compared to classical TMNM. We found two regions, both part of the tinnitus network, which showed a reduction of neural activity evoked by TF after TMNM exposure for the ISEC-TMNM group only. This outcome argues for a superior effect of amplified edge frequency bands around the notch on inhibition-induced plasticity in addition to the general inhibition-induced plasticity effect in the tinnitus network.One significant spatiotemporal cluster was located in the inferior temporal lobe, reflecting inhibition in the auditory cortex due to increased spectral energy contrasts in TMNM. These results confirm previous findings that lateral inhibition is modifiable in the auditory cortex ,33, espeInterestingly, the second significant spatiotemporal cluster revealing a greater inhibition of tinnitus related neural activity for the ISEC-TMNM group was found in a right prefrontal cortical region. Most research investigating modulation of inhibition via spectral energy contrasts in sounds so far focused on the auditory cortex ,33,37,40Our study aimed at testing the concept of inhibition-induced plasticity. We argued, following the theory of lateral inhibition in the auditory cortex, that neurons coding the edge frequency bands around the notch should have the greatest influence on lateral inhibition of neurons coding the center frequency of the notch. Therefore, the ISEC-TMNM group should have a superior inhibition-induced plasticity as compared to the classical TMNM group. In addition to the reduction of TF related neural activity in the tinnitus network after TMNM exposure, we also found an improvement of inhibition-induced plasticity within the N1 time window in two regions of this network due to spectral energy contrast amplification. Thus, the current results confirm our hypothesis and, accordingly, the concept of inhibition-induced plasticity.The second aim of this study was to find a parameter which possibly improves TMNMT . BehavioOne reason for the missing modulation of inhibition-induced plasticity due to spectral energy contrast variation on subjective tinnitus loudness might be due to the low sensitivity of the VAS. Even if the VAS seems to be a valid and reliable instrument for tinnitus loudness change measurements , it mighAnother limitation of this study is that we could not analyze the exact loudness level which our participants chose to listen to TMNM. However, we assume that the loudness level should be balanced between subjects. Our participants had to listen to TMNM in a comfortable loudness so that they were able to enjoy the music. Since hearing loss values and possible recruitment mechanisms varied between subjects, it was not possible to present the music with equal loudness for all subjects. If the loudness level would have differed between groups, it should have been lower for the ISEC-TMNM group, since here, the edge frequency bands were amplified at about 20 dB. On the other hand, we found greater inhibition-induced plasticity in this group. So if the comfortable loudness in the ISEC-TMNM group was lower as compared to classical TMNM, than increased spectral energy contrasts would even have greater influence, since lateral inhibition would have been induced, even though there was less overall energy in the music. This would be in line with our hypotheses. Overall, the current results confirmed the involvement of a distributed tinnitus neural network in inhibition-induced plasticity by TMNM exposure. We were able to demonstrate an improvement of inhibition-induced plasticity in people suffering from chronic tinnitus as a consequence of spectral energy contrasts. This improvement was found in temporal as well as frontal cortical regions, thereby confirming the concept of inhibition-induced plasticity.S1 DatasetSession (pre TMNM exposure vs. post TMNM exposure) which survived the applied cluster-based permutation test for all subjects . Significant clusters were located in a left temporal (left_temp), a left parietal (left_par), a right orbitofrontal (right_orb_front), a right cerebral region with extents towards occipital and temporal areas  and a right frontal region .Mean activity of significant clusters for the main effect (XLSX)Click here for additional data file.S2 DatasetSession (pre TMNM exposure vs. post TMNM exposure) x Group  vs. increased spectral energy contrast TMNM (coded with ‘1’)) which survived the applied cluster-based permutation test for all subjects. Significant clusters were located in a left inferior temporal (left_temp) and a right frontal region .Mean activity of significant clusters for the interaction effect (XLSX)Click here for additional data file.S3 DatasetMean ratings of the subjectively perceived tinnitus loudness averaged for each session and across all three days before (pre) and after (post) TMNM exposure. Scale ranged from ‘tinnitus is absent’ (0.00 cm) to ‘loudest tinnitus ever perceived’ (10.00 cm). ‘Group’ is coded as follows: 0 = classical TMNM; 1 = increased spectral energy contrast TMNM.(XLSX)Click here for additional data file.S1 FigSession x Group and survived the applied cluster-based permutation test.Continuous lines show the source power evoked by the tinnitus tone before tailor-made notched music (TMNM) exposure; dashed lines indicate the tinnitus related source power after TMNM exposure. Grey lines reflect the source power of the classical TMNM group while the black lines indicate the source power of the ISEC-TMNM group. The averaged source waveforms depict only the activity of temporal sources, which showed a significant interaction effect (TIF)Click here for additional data file.S2 FigSession x Group and survived the applied cluster-based permutation test.Continuous lines show the source power evoked by the tinnitus tone before tailor-made notched music (TMNM) exposure; dashed lines indicate the tinnitus related source power after TMNM exposure. Grey lines reflect the source power of the classical TMNM group while the black lines indicate the source power of the ISEC-TMNM group. The averaged source waveforms depict only the activity of frontal sources, which showed a significant interaction effect (TIF)Click here for additional data file.S3 FigContinuous lines show the global field power of measurements before exposure to tailor-made notched music ; dashed lines indicate the global field power after TMNM exposure (post). Black lines reflect neural activity evoked by the reference tone, while grey lines indicate tinnitus related neural activity. Since the reference tone had a far lower carrier frequency (500 Hz) as compared to the tinnitus frequency (ranging from 1500–8500 Hz), the global field power for the lower tone is overall greater than for the tinnitus tone. Comparisons of pre and post measures indicate, that tinnitus related neural activity decreased after TMNM exposure, while neural activity evoked by the reference tone remained the same.(TIF)Click here for additional data file.


Interaction of integrin β3 with c-Src plays critical roles in cellular signaling which is heavily implicated in platelet adhesion and aggregation, as well as in tumor cell proliferation and metastasis or in osteoclastic bone resorption. Selectively blocking integrin αIIbβ3 outside-in signaling in platelets has been a focus of attention because of its effective antithrombotic potential together with a sufficient hemostatic capacity. The myristoylated RGT peptide has been shown to achieve this blockade by targeting the association of c-Src with the integrin β3 tail, but the lack of key information regarding the mechanisms of action prevents this strategy from being further developed into practical antithrombotics. Therefore, in-depth knowledge of the precise mechanisms for RGT peptide in regulating platelet function is needed to establish the basis for a potential antithrombotic therapy by targeting c-Src.The reduction-sensitive peptides were applied to rule out the membrane anchorage after cytoplasmic delivery. The c-Src activity was assayed at living cell or at protein levels to assess the direct effect of RGT targeting on c-Src. Thrombus formation under flow in the presence of cytoplasmic RGT peptide was observed by perfusing whole blood through the collagen-coated micro-chamber.416 and Y527, consistent results were also from in vitro kinase assays. Under flow conditions, the myr-AC ~ CRGT peptide caused an inhibition of platelet thrombus formation predominantly at high shear rates.The RGT peptide did not depend on the membrane anchorage to inhibit outside-in signaling in platelets. The myr-AC ~ CRGT peptide readily blocked agonist-induced c-Src activation by disrupting the Src/β3 association and inhibited the RhoA activation and collagen-induced platelet aggregation in addition to the typical outside-in signaling events. The myr-AC ~ CRGT had no direct effect on the kinase activity of c-Src in living cells as evidenced by its inability to dissociate Csk from c-Src or to alter the phosphorylation level of c-Src YThese findings provide novel insights into the molecular mechanisms by which the RGT peptide regulates integrin signaling and platelet function and reinforce the potential of the RGT peptide-induced disruption of Src/β3 association as a druggable target that would finally enable in vivo and clinical studies using the structure-based small molecular mimetics.The online version of this article (doi:10.1186/s13045-015-0159-8) contains supplementary material, which is available to authorized users. Arterial thrombosis developing on the atherosclerotic lesions causes heart attack and stroke that are currently the most common cause of death. Platelets play central roles in hemostasis and in arterial thrombosis by forming thrombi . Conside1 or P2Y12/adenosine diphosphate (ADP) and protease-activated receptor (PAR)/thrombin, etc. The transduction of inside-out signals transforms the integrin αIIbβ3 receptor from low to high affinity that facilitates ligand (fibrinogen) binding and thus platelet aggregation  adenosine triphosphate (ATP) incorporation assays , which respectively represented flow conditions in stenotic arteries and, or in arterioles or slightly stenotic arteries, thrombus formation in the presence of myr-AC ~ CRGT was between that of DMSO or αIIbβ3 antagonists where thrombus formation scarcely occurred , mimicking medium-sized arteries, myr-AC ~ CRGT showed no inhibitory effect in contrary to a substantial inhibition by 7E3 , which modeled the flow in venules, neither myr-AC ~ CRGT nor 7E3 inhibited platelet adhesion  in contrast to an unequivocal inhibition induced by 7E3  myr-AC ~ CRGT partially inhibited thrombus formation, as evidenced by the smaller and thinner thrombi and less thrombus coverage as well , myr-AC ~ CRGT-treated platelets may adhere normally, while in arterioles and capillaries (1500 s−1 or greater), the unaffected inside-out signaling may still enable these platelets to form thrombi to a size sufficient to seal the vessel damages in contrast to a much more profound inhibition of 7E3  incorporation assay [32P]ATP and 10 μM of ATP at 30 °C overnight. Reaction mixtures were transferred to P81 phosphocellulose membranes and washed with 0.75 % phosphoric acid. These membranes were rinsed briefly in acetone, air dried, and transferred into scintillation vials. The [γ-32P]ATP incorporation of the peptides were counted using a Beckman LS6500 liquid scintillation counter . We also measured β3 Y747 and Y759 phosphorylation by active c-Src in CHO cell models. Tac-β3 or Tac-β3-ΔRGT was transfected or co-transfected together with c-Src Y527F at a ratio of 1:1 into CHO cells using the Lipofectamine 2000 kit (Invitrogen). Twenty-four hours later, the cells were lysed at 4 °C in ice-cold RIPA buffer  and clarified by centrifugation at 12,000 rpm for 15 min and then were analyzed by Western blot.First, the Yon assay . Brieflyw/v) trisodium citrate. Platelet-rich plasma (PRP) was obtained by a centrifugation at 200 × g for 20 min. For preparation of washed platelets, the PRP was acidified by adding 1/7 volume of acid citrate dextrose  and platelets were collected by a centrifugation at 1500 × g for 20 min. The platelet sediments were washed twice with CGS , and were finally resuspended at required concentrations in HEPES-Tyrode’s buffer . The platelet suspensions were left at room temperature for 60 min prior to being used in experiments. Platelet spreading on immobilized fibrinogen was assayed as previously described ATP and 10 μM of ATP and incubated at 30 °C overnight. [γ-32P]ATP incorporation was counted using a Beckman LS6500 liquid scintillation counter.Reactions were carried out using a Src kinase assay kit (Upstate Biotechnology). Briefly, 150 μM of substrate peptide KVEKIGEGTYGVVYK was mixed with purified active c-Src in the presence or absence of CRGT, CGRT, or PP2 in Src kinase reaction buffer containing 5 μCi of [γ-527F, or Tac-β3-ΔRGT and c-Src Y527F, or peptide-pretreated CHO cells co-expressing Tac-β3 and c-Src Y527F were lysed for 30 min at 4 °C in ice-cold RIPA buffer containing the protease inhibitor mixture. Protein concentrations were determined using the bicinchoninic acid protein assay kit . The lysates were centrifuged for 5 min at 4 °C at 12,000 rpm, and then, 100 μL aliquots of the supernatants containing equal amounts of protein (ranging from 400 to 500 μg between experiments) were immunoprecipitated by the indicated antibodies for 2 h at 4 °C with gentle agitation and were further incubated with protein G plus-agarose beads overnight at 4 °C. After washing three times with RIPA buffer, the immunoprecipitates were eluted by boiling the beads in Laemmli sample buffer and were electrophoresed with 8 % or 12 % SDS-PAGE, transferred onto PVDF membrane. Proteins were detected using the indicated primary antibodies and HRP-conjugated secondary antibodies. Immunoreactive bands were detected by ECL with the reaction time ranging from 30 s to 5 min. The signal intensity of the respective bands was measured by the NIH Image J software.Preparation of platelet lysates or cell lysates, immunoprecipitation, SDS-PAGE, and immunoblotting were performed according to the procedures published elsewhere . In brie−1 for 4 min. Adherent platelet aggregates were monitored using an inverted fluorescent microscope with a × 20 long-working-distance objective and CCD camera (Nikon eclipse Ti-s).The experiments were performed as described . Biofluxt test. When three or more groups were compared, we used ANOVA . Data are presented as mean and SD, unless otherwise specified.Differences between groups were assessed using the Student’s


Transformation of the glaciated isthmus between Sørkapp Land and the rest of Spitsbergen since 1900 is described. The landscape–seascape dynamics depends on the glacial recession determined by climate warming after the Little Ice Age . The isthmus has been narrowed from 28 km in 1899–1900 to 6.2 km in 2013, and lowered by 60–200 m from 1936 to 2005. Two isthmus’ glaciers will have melted, given the current thermic conditions, by 2030–2035. It cannot be ruled out that Sørkapp Land will become an island after that period, because the altitude of the glaciers’ bedrock is close to the sea level. The disappearance of this huge ice mass, even without origin of a sound and island, will lead to a great transformation of the landscape and the ecosystem. It consists of a thick glacial cover settled on bedrock, and it undergoes a quick transformation of its landscape. The isthmus, as the whole Spitsbergen, the biggest island of the Svalbard archipelago, divides the Barents Sea in the east from the Greenland Sea in the west  ended in Spitsbergen with a cold period during the 1890s Brázdil . A globaThe isthmus makes a depression between the mountains in the north and south and is also an elevation between the two fjords in the east and west. The isthmus was a part of the extensive ice cover  during the LIA, and consisted of two long glaciers being fed by glaciers from the mountains to the south and north of it , its head has belonged geologically to Spitsbergen east, being situated 32–33 km east of the open Greenland Sea and only 14–15 km from the open Barents Sea coast. Nowadays, the whole isthmus’ bedrock consists of relatively soft Cretaceous and Tertiary clastic sedimentary rocks  to 15 m (TerraASTER). These images are useful for landscape investigations at a scale of 1:100 000  and even at a 1:50 000–1:25 000 scale under conditions of scant cloudiness, lack of fog, scant ice cover on the sea, and a lack of snow on the land at the moment of the satellite scanning. A simultaneous occurrence of these conditions is very rare near the isthmus. Hence, only 14 satellite images were selected (from 1976 to 2013), and the glaciers’ extents on them were manually vectorized.Satellite images LANDSAT and TerraASTER (from 1976 to 2013), and the terrain model Aster Global Digital Elevation Model Global Version 2  were taken from the collections opened by U.S. Geological Survey (USGS) in www pages The terrain model was elaborated on for NASA and Japan’s Ministry of Economy, Trade and Industry, which thereafter was released by them on October 17, 2011. This Aster GDEM model was created by stereo-correlating all scenes in the ASTER archive, covering the Earth’s land surface between 83°N and 83°S latitudes. The GDEM is produced with 30-m postings, and is formatted in 1 × 1 degree tiles as GeoTIFF files. The second version of this model was improved after including additional scenes of ASTER. In the ASTER GDEM ver. 2 model, the medium horizontal error is estimated to be less than 10 m, and the medium vertical error may reach up to ca. 15 m .The map of changes in the glaciers’ elevations and extents Fig.  were elaOur field survey was made using a GPS 12 Garmin and altimeter Paulin. Geographic coordinates and altitudes of many points along profiles and research routes, including extent of glaciers, were measured. The use of these both traditional and modern methods of localization enabled us to minimize mistakes which are specific to each of the methods.The isthmus with its surroundings has undergone a dramatic transformation since the end of the LIA Figs. , 2013, dIt should be emphasized that the frontal retreat of the isthmus’ ice coasts has been a final result of the negative net mass balance, i.e., a loss of ice mass, of its former and present tributary glaciers Ziaja . The uplDuring 1899–1900, the isthmus was 28 km wide and fed by 21 glaciers (Hornbreen by 15 glaciers and Hambergbreen by 6 glaciers); in 1936, 22.4 km wide and fed by 15 glaciers (Hornbreen by 12 glaciers and Hambergbreen by 3 glaciers); and in 2005, 7.9 km wide and fed by 5 glaciers (Hornbreen by 4 of them and Hambergbreen by only one). Feeding from the south was very insignificant in 2005 because two tributary glaciers’ tongues from this area  had narrowed and thinned considerably since the 1980s .Nowadays 2013), the isthmus is only 6.2 km wide and only three glaciers are weakly supplying the isthmus with ice, all of them from the north . Moreover, the delivery of ice from these glaciers is becoming smaller and smaller due to decreases in their thickness and surface elevation , in spite of surge episodes mentioned by Pälli et al. . A more Over a period of ca. 70 years (since 1936), the isthmus’ ice thickness decreased by ca. 60 m in the ice shed between both glaciers and by more below it, down to their fronts. Decrease in the ice thickness reached ca. 125 m near the present (2013) Hambergbreen’s front and 150–175 m near the present (2013) Hornbreen’s front  and Sørkapp Land will be transformed into an island. If not, the isthmus will change its character (from icy to rocky) but Sørkapp Land will remain as the peninsula.nunataks (isolated rocky peaks which protrude above the glaciers) near the ice-shed.After the discoverer Poole who mistakenly named the fjord “Hornsund”, thinking that it is a sound, in 1610  at 50 MHz were made by the Finnish–British–Polish team , led to the conclusion that “under current environmental conditions” the Hornbreen–Hambergbreen “ice isthmus will disappear by 2020” Sharov  and “Sør2 and are at least ca. 70 times smaller than Sørkapp Land which would become the biggest new Arctic island in the event of its splitting out from the rest of Spitsbergen.Transformation of the Arctic peninsulas to new islands due to the glacier shrinkage is usually described in preliminary or summarizing notes on the Internet or in popular press cited below in this paragraph, being omitted, as it is considered to be an obvious phenomenon, in the main stream of scientific literature on glacial recession. The appearances of new islands in both the eastern Greenland and the western Greenland were noted on the Internet  isthmus’ edge at the foot of the Ostrogradskijfjella mountainous group, from August 21 to 24, 2005 when the lower parts of the glaciers were devoid of snow Figs. , 2009. OThe altitude of the ice pass between Hornsund and Hambergbukta decreased (lowered) from 241 m during 1899–1900  of the Hornbreen and Hambergbreen glaciers’ surface were very clear . Contrary to the maps elaborated by Sharov , c, d, tOur synthesis of the research results has determined that the future loss of the Hornbreen–Hambergbreen glacial ice isthmus is certain to occur in the current thermic conditions. The low location of the isthmus’ glaciers excludes the possibility of their being rebuilt due to an increase in precipitation. According to Pälli et al. , “HornbrIt can be asked, “What will be the timing of these glaciers’ definite decline?” The time as predicted by Pälli et al.  that “HoIn our opinion, the ice isthmus will disappear in ca. 20 years, i.e., by the period of 2030–2035 or several years earlier. Our calculation is very simple: the smallest width of the isthmus decreased from 12 328 m in 1990 to 6167 m on June 24, 2013 Table , 2010 thThe question of whether Sørkapp Land becomes an island after the disappearance of the ice from the isthmus is impossible to answer given the current state of knowledge (as outlined above), as the research results which refer to altitude of the isthmus’ bedrock are contradictory. Nevertheless, a possibility of the appearance of at least a shallow and narrow sound between the Barents Sea and the Greenland Sea (which would be very interesting not only from a scientific point of view) is not excluded yet. It is worth noting that the Koryakin’s  thesis o−1 during the last 2000–3000 years  and “partially inundated” . In contrast, when the climate becomes colder (and the ELA becomes lower) such removed ice masses melt slowly, which increases the extent of glaciers.


The porcine model is frequently used during development and validation of new spinal devices, because of its likeness to the human spine. These spinal devices are frequently composed of pedicle screws with a reputation for stable fixation but which can suffer pullouts during preclinical implantation on young animals, leading to high morbidity. With a view to identifying the best choices to optimize pedicle screw fixation in the porcine model, this study evaluates ex vivo the impact of weight (age) of the animal, the level of the vertebrae (lumbar or thoracic) and the type of screw anchorage  on pedicle screw pullouts. Among the 80 pig vertebrae (90- and 140-day-old) tested in this study, the average screw pullout forces ranged between 419.9N and 1341.2N. In addition, statistical differences were found between test groups, pointing out the influence of the three parameters stated above. We found that the the more caudally the screws are positioned (lumbar level), the greater their pullout resistance is, moreover, screw stability increases with the age, and finally, the screws implanted with a mono-cortical anchorage sustained lower pullout forces than those implanted with a bi-cortical anchorage. We conclude that the best anchorage can be obtained with older animals, using a lumbar fixation and long screws traversing the vertebra and inducing bi-cortical anchorage. In very young animals, pedicle screw fixations need to be bi-cortical and more numerous to prevent pullout. New medical devices require not only biocompatibility but also “biofunctionality”, with a proof of concept. Thus, the pre-clinical phase of development frequently involves the use of animal experimentation. However, this raises the familiar issue of the pain and distress caused to the animals used for research. The “Three Rs” ethical concept, standing for Replacement, Reduction and Refinement, was first described by Russel and Burch in 1959 ,3 becausPedicle screws are widely used in treating spinal diseases such as scoliosis –9 and otAlthough the pedicle fixation technique is not new, the literature contains little information on the pullout resistance of pedicle screws inserted in young pigs. Existing studies evaluate the impacts either of screw design  or of inThe objective of this study is to evaluate pedicle screw fixation and thereby identify the most relevant parameters to be optimized in future experimentation on this porcine model. To do so, we assessed the influence of 1) the level of the vertebrae (lumbar and thoracic), 2) the weight of the animals  and 3) the cortical anchorage  on the ultimate pedicle screw pullout forces.We chose as animal model the landrace pig, known to most closely approximate the size and shape of human vertebrae . Eight iThe pedicle dimensions of the animal model at these ages are given in All pedicle screw insertions were performed with a free-hand technique, with a convergent direction (Magerl insertion technique ) by the Each vertebra was then casted in polyurethane resin  in a PVC circular container 100mm in diameter and between 25mm and 35mm high, depending on the size of the vertebra. The heating associated with the hardening of this particular resin was previously proved not to degrade bone [vs displacements during the tests, and noted the maximum pullout force for each tested vertebra.The screws were tested on a mechanical testing machine (MTS INSTRON 5566A), using a load cell with maximum capacity 10 kN and precision ±0.5%. A special assembly was developed to firmly maintain the embedded vertebra with kind of metallic shelf brackets see . The heaMean values and standard deviations (SD) were calculated for each set of 10 repeats. A total of 8 different configurations of type of anchorage, vertebra level and weight of animal were tested. A non-parametric statistical study was realized on the results in pairs, using the Mann Whitney test.During the tests, two vertebrae were eliminated because of poor resin casting (the vertebra slipped), in the 50kg, bi-cortical, lumbar configuration. At the end of the tests, 78 vertebrae (thus 78 screws) were available for analysis. Pullout forces were averaged from the ten repeats for each level , weight and type of anchorage; results are shown in Results of the statistical tests on the pullout forces are presented in The following three graphs highlight the respective influence of vertebra level , weight Current medical device development processes frequently require an experimental phase using animals, to assess efficacy before implantation in humans. During previous studies on the pig model to validate spinal devices, some veterinarians and researchers noted screw pullouts ,17 leadiYazici et al  studied When we investigated how the level of the vertebra affects screw pullout forces, the results for a given weight and anchorage were statistically different between the thoracic and lumbar level (p<.01) . We founLooking at how animal weight (age) of the animals affects pedicle screw pullout resistance, results for a given level and anchorage were statistically different between 30kg and 50kg (p<.01) . Screw sExamining the impact of type of cortical anchorage, we obtained results that, for a given age and vertebra level, were statistically different between mono- and bi-cortical anchorage (p<.01) . The scrThis study sought the most relevant parameters for pedicle fixation in a porcine model being used to develop new human implant systems. We found statistical evidence of the impact of age of pigs, level of the implanted vertebra and type of screw anchorage. It would be interesting to extend these tests to osteointegrated screws, to assess the influence of bone healing on their stability. Previous work on the creation of scoliotic deformities suggests that the conservation of an osteointegration period before applying stresses to screws limits the risk of pullouts .One limitation of our study is that pure axial loading does not faithfully represent the in vivo loading sustained by screws used in spinal systems. However, axial loading is commonly used to evaluate screw stability . MoreoveTo limit screw pullouts, we conclude that the best choices are implantation in older animals, using a lumbar fixation and long screws traversing the vertebra and inducing bi-cortical anchorage. However, bi-cortical anchorage implies that the screw goes beyond the anterior aspect of the vertebral body, and has to be performed with care to avoid any vascular issue. If very young animals are required, pedicle screw fixations need to be bi-cortical to enhance stability, and as many screws as possible should be implanted to prevent avulsion.


A/γA-fibrin or glycoprotein Ibα peptide via exosite 1 and 2, respectively, and thrombin binding to γA/γ′-fibrin or factor Va, which is mediated by both exosites. Whereas dabigatran attenuated binding, argatroban increased thrombin binding to γA/γA- and γA/γ′-fibrin and to factor Va. The results with immobilized fibrin were confirmed by examining the binding of radiolabeled thrombin to fibrin clots. Thus, dabigatran modestly accelerated the dissociation of thrombin from γA/γA-fibrin clots, whereas argatroban attenuated dissociation. Dabigatran had no effect on thrombin binding to glycoprotein Ibα peptide, whereas argatroban promoted binding. These findings not only highlight functional effects of thrombin allostery, but also suggest that individual active site-directed thrombin inhibitors uniquely modulate exosite function, thereby identifying potential novel mechanisms of action.Thrombin is a highly plastic molecule whose activity and specificity are regulated by exosites 1 and 2, positively-charged domains that flank the active site. Exosite binding by substrates and cofactors regulates thrombin activity by localizing thrombin, guiding substrates, and by inducing allosteric changes at the active site. Although inter-exosite and exosite-to-active-site allostery have been demonstrated, the impact of active site ligation on exosite function has not been examined. To address this gap, we used surface plasmon resonance to determine the effects of dabigatran and argatroban, active site-directed inhibitors, on thrombin binding to immobilized γ A/γ′-fibrinogen [A-chain lacks a thrombin binding site, thrombin binds the bulk γA/γA-fibrinogen with lower affinity solely via exosite 1 [The substrate specificity of thrombin is dependent on exosites 1 and 2, positively-charged domains that flank the active site ,4 and, bbrinogen . In contxosite 1 ,7. Both xosite 1 –10.+ to a conserved site on thrombin increases the catalytic activity of thrombin by altering the conformation of the active site [A/γA-fibrin, γA/γ′-fibrin, factor Va, and GpIbα peptide. In addition, the effects of these inhibitors on the binding of radiolabeled thrombin to fibrin clots and its subsequent dissociation were examined.Numerous studies have shown that thrombin is subject to allosteric modulation. For example, binding of Naive site ,12. Likeive site –15, plative site ,16, or tive site ,18 to exive site  or prothive site  to exosiive site –24. Previve site ,22. Howeive site , 19 nM [ive site , and 45 ive site , respectA/γA- and γA/γ′-fibrinogen were isolated and characterized as previously described [Bathrops atrox moojeni was from Pentapharm . D-Phe-Pro-Arg (FPR) chloromethyl ketone was from Calbiochem . Chromozym-Thrombin (Chz-Th) was from Hyphen BioMed . Active dabigatran was generously provided by Dr. J. van Ryn , whereas argatroban was a gift from Dr. D. Stump . Recombinant hirudin was from Dade-Behring . Chloramine T and sodium metabisulfite were from Sigma-Aldrich and Bolton-Hunter reagent was from Pierce Biotechnology . The synthetic peptide corresponding to the thrombin exosite 2-specific binding site at residues 269–286 on GpIbα, (Asp-Glu-Gly-Asp-Thr-Asp-Leu-Tyr(PO3)-Asp-Tyr(PO3)-Tyr(PO3)-Pro-Glu-Glu-Asp-Thr-Glu-Gly; GpIbα269-286ppp) was synthesized with three phosphorylated Tyr residues by Mimotopes  [Human thrombin and plasminogen-free fibrinogen were from Enzyme Research Laboratories . Fibrinogen was immunodepleted of factor XIII as described . Human fescribed ,28,29. Blis, MN) ,31.125I  was added 5 μl of 1.5 mM Bolton-Hunter reagent in DMSO. The reaction was initiated by adding 10 μl of 5 mg/ml chloramine T in 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 (PBS). After 1 min at 23°C, the reaction was stopped by addition of 10 μl of 12 mg/ml sodium metabisulfite in PBS. To the reaction mixture was added 150 μg of thrombin and after incubation for 1 hr at 23°C, the reaction was terminated by addition of 100 μl of 0.2 M glycine in 0.2 M sodium borate, pH 8.0. Labeled thrombin was isolated on a PD-10 column , and eluted with 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 (TBS) containing 0.01% Tween-80 (TBS-Tw). The specific activity of the labeled thrombin was 3.8 x 108 cpm/mg.To 10 μl of 0.2 M sodium borate, pH 8.0, and 10 μl (1 mCi) of NaThrombin (3–3.5 mg/ml) was incubated with 2-fold molar excess of FPR chloromethyl ketone at 23°C. After 30 min, absence of residual activity of 2 μM FPR-thrombin was determined at 405 nm with 200 μM Chz-Th in a Spectramax plate reader . The sample was washed with TBS and concentrated using an Amicon Ultra 4 ml– 3000 MW centrifugal cartridge (Millipore) .A/γA-fibrinogen, γA/γ′-fibrinogen, and factor Va were immobilized to separate flow cells of a CM5 sensor chip using the amine coupling kit from GE Healthcare [A/γA- or γA/γ′-fibrinogen or 28 μg/ml factor Va in 10 mM acetate buffer, pH 4.5, was injected at a rate of 5 μl/min until about 8000 response units (RU) were immobilized. High levels of protein were adsorbed because previous studies indicate that only 30% of bound fibrinogen is in an accessible orientation, and because of the large difference in molecular weight between the bound ligands and the analyte thrombin [2. To convert the immobilized fibrinogen to fibrin, flow cells were subjected to successive 60-min injections of up to 1 μM thrombin [Studies were performed using a Biacore T200 . γalthcare . Brieflythrombin ,28,32. Qthrombin .A/γA-fibrin, γA/γ′-fibrin or factor Va, increasing concentrations of up to 5 μM thrombin or FPR-thrombin were injected into the flow cells. To measure the effects of dabigatran, argatroban, and DAPA on thrombin binding to fibrin or factor Va, 250–500 nM thrombin or FPR-thrombin was incubated with 0–5 μM inhibitor and injected into the flow cells. Because of the fast on- and off-rates, all SPR experiments were performed at a flow rate of 25 μl/min [2.To measure binding to immobilized γ5 μl/min . Associad values for one-site binding of thrombin to γA/γA-fibrin were determined from plots of RU values at equilibrium (Req) versus thrombin or FPR-thrombin concentration using Biacore T200 Evaluation software v 1.0. The rapid approach to equilibrium at the low flow rate nullified any potential mass transport concerns. Nonspecific binding was accounted for by subtracting the control RU values obtained from the unmodified flow cell. To calculate two-site binding of thrombin to γA/γ′-fibrin or factor Va, plots were subjected to kinetic analysis using a two-site binding model. EC50 values for the effect of dabigatran, argatroban, or DAPA on thrombin binding were determined by plotting Req values against the concentration of active site inhibitor and using non-linear regression to fit these to a rectangular hyperbola.The K2-terminus by incubation with a 10-fold molar excess of Hook-Sulfo-NHS-LC biotin  for 1 h at 23°C. After separation from unincorporated reagent by chromatography on Sephadex G15, fractions containing biotinylated peptide were identified by monitoring absorbance at 204 nm. Streptavidin (Sigma) was attached to flow cells of a CM5 chip to about 12000 RU and biotin (b)-GpIbα269-286ppp was then adsorbed to about 150 RU above the streptavidin background. Aliquots of thrombin or RA-thrombin up to 4 μM in HBS-Tw were injected into the flow cell at 25 μl/min for 80 s, followed by HBS-Tw for 200 s. Between runs, flow cells were regenerated with 1 M NaCl for 30 s. To measure the effect of dabigatran, argatroban, or DAPA on this interaction, 250 nM thrombin was injected in the presence of 0–5000 nM dabigatran, argatroban, or DAPA. EC50 values were determined as above. To quantify the effects of inhibitors on binding affinity, 0–2 μM thrombin was injected in the presence of 2.5 μM active site inhibitor at 25 μl/min for 60 s. Binding affinities were calculated as described above.To examine the effect of the active site inhibitors on exosite 2-mediated binding, 1 mg of GpIbα269-286ppp in HBS was biotinylated at its NH125I-thrombin, 3 μM γA/γA-fibrinogen or 1 μM γA/γ′-fibrinogen, 2 mM CaCl2 and 1 unit/mL batroxobin in TBS-Tw in a total volume of 100 μl [2 2470, Perkin-Elmer) to determine the concentration of unbound 125I-thrombin. EC50 values for the effect of the inhibitors were calculated by fitting plots of bound 125I-thrombin versus the inhibitor concentration by non-linear regression analysis to an equation for a rectangular hyperbola. Ki values were then determined using the Cheng-Prusoff equation and the dissociation constant of 125I-thrombin for fibrinogen as determined in a separate experiment.To a series of 1.5-ml micro-centrifuge tubes containing 0–1 μM dabigatran or argatroban, 0–4 μM DAPA, or 0–0.1 μM hirudin were added 10 nM f 100 μl ,28,32. B125I-thrombin from preformed fibrin clots was performed as described [A/γA-fibrinogen and 30 nM factor XIII in TBS-Tw containing 2 mM CaCl2 with 10 nM 125I-thrombin. After incubation for 1 h, clots were immersed in 50 ml plastic tubes containing 10 ml of 100 nM dabigatran, argatroban or DAPA, or 2 M NaCl in TBS-Tw at 23°C. At defined intervals, aliquots were removed, counted for radioactivity, and returned to the tubes. Plots of residual radioactivity versus time were analyzed using a 2-phase exponential decay equation [Dissociation of escribed . Brieflyequation .Statistical analysis–All experiments were performed at least three times. Results are presented as the mean ± standard deviation (SD). Paired data were compared using Student t-tests; whereas group means were compared using one-way ANOVA followed by Tukey's test for multiple comparisons. For all analyses, p values less than 0.05 were considered statistically significant.A/γA-fibrin, γA/γ′-fibrin, or factor Va [d  as in its absence, the affinity of thrombin for GpIbα269-286ppp significantly (p < 0.001 by ANOVA) increased by 25–35% in the presence of argatroban or DAPA . These findings demonstrate that occupation of the active site by some, but not all, small molecules increases the affinity of ligands for exosite 2, similar to the observation for exosite 1-dependent interactions.Thrombin bound to the immobilized GpIbα269-286ppp peptide with a K ± 11 nM ; Table 1 ± 11 nM ; Table 2A/γA- and γA/γ′-fibrin determined by SPR are comparable with those obtained with fibrin clots [125I-thrombin bound γA/γA- and γA/γ′-fibrin clots with Kd values of 3.6 ± 0.3 μM and 1.2 ± 0.2 μM (not shown), respectively; values comparable with those obtained using SPR and with those published previously [125I-thrombin bound to fibrin, demonstrating that labeling had little effect on the capacity of thrombin to bind to fibrin.Although the affinities of thrombin and FPR-thrombin for γin clots , it was eviously . Up to 7125I-thrombin binding to γA/γA- and γA/γ′-fibrin clots in a concentration-dependent manner with Ki values of 3.8 ± 1.5 nM and 26.0 ± 4.0 nM, respectively ; a difference that likely reflects the higher affinity of thrombin for γA/γ′-fibrin. To verify the role of exosite 1 in binding, the effect of hirudin also was examined. As expected, hirudin fully inhibited 125I-thrombin binding to γA/γA- and γA/γ′-fibrin. In agreement with the SPR observations, argatroban and DAPA enhanced the binding of 125I-thrombin to γA/γA- and γA/γ′-fibrin clots to a similar extent (~15–25%) (Dabigatran reduced ectively . The Ki ic assay . The Ki exosites . As obse~15–25%) , Table 3A/γA-fibrinogen in the presence of 125I-thrombin were incubated in buffer in the absence or presence of active site inhibitors and the amount of 125I-thrombin released from the clot over time was quantified. Biphasic analysis was used to evaluate the fast non-adsorbent and slow bound phases of dissociation of thrombin from fibrin. In the control, the bound fraction dissociated with a half-life of 20.5 ± 3.1 h, whereas in the presence of 2 M NaCl where ionic interactions are abrogated, the rate of dissociation was significantly faster (p = 0.02) at 14.2 ± 1.2 h (To determine whether the alteration in the affinity of thrombin for fibrin in the presence of active site inhibitors influences the amount of thrombin associated with fibrin, dissociation from fibrin clots was examined . Clots f ± 1.2 h . The halA/γA-fibrin and GpIbα269-286ppp is solely dependent on exosite 1 and exosite 2, respectively, whereas thrombin binding to γA/γ′-fibrin and factor Va requires both exosites. Third, the use of native ligands such as fibrin and factor Va would complement previous results obtained with peptide ligands, and the use of fibrin would enable confirmation in functional assays. Dabigatran and argatroban were chosen because they are highly specific for thrombin and their interactions with the active site of thrombin have been extensively investigated and because both agents are in clinical use. DAPA was chosen to complement the results with argatroban because both incorporate an Arg group that is essential for specificity [The purpose of this study was to determine whether occupation of the active site of thrombin by small molecules induces functional changes at its exosites. We chose fibrin, factor Va and GpIbα269-286ppp as targets for three reasons. First, their use enabled studies with unmodified, active thrombin. Second, the mode of interaction of thrombin with these ligands has been well characterized; thus, binding of thrombin to γcificity ,37.A/γA-fibrin and factor Va by 28–48%, but has no effect on the interaction of thrombin with Gp1bα269-286ppp. Surprisingly, argatroban and DAPA increase thrombin binding to γA/γA-fibrin, γA/γ′-fibrin, factor Va and GpIbα269-286ppp by 13–47%. The observations with fibrin were confirmed using three-dimensional fibrin clots and were not dependent on the affinity of the inhibitors for thrombin because dabigatran, argatroban and DAPA inhibit thrombin with similar nanomolar Ki values. DAPA and argatroban also slow the dissociation of thrombin from fibrin clots in a manner consistent with their enhancement of the affinity of thrombin for fibrin. Thus, these studies demonstrate allosteric effects of active site binding on thrombin, and provide evidence that responses are ligand specific.Dabigatran saturably reduces thrombin binding to γ1-4 subsites and the 60- and autolysis-loops contribute to this allosteric network [Investigation into the allosteric regulation of thrombin has shown that binding of a variety of effectors to the exosites modulates the active site. This phenomenon has been attributed to a complex allosteric network within thrombin that is subject to modulation through surface residues ,16. With network ,38,39. T network –37,40. T network ,39,41,42 network –46. Cons network . These oThe observation that binding of dabigatran, argatroban or DAPA to the active site of thrombin induces changes at the exosites is consistent with the hypothesis that changes transmitted over the allosteric network are bidirectional. Most studies have focused on the effect of exosite ligands on active site function. However, calorimetry and fluorescence studies using peptide ligands demonstrate that structural changes that result from ligand binding at the active site or exosite 1 can run in both directions ,47,48. TThe capacity of dabigatran to attenuate thrombin binding to fibrin and factor Va reveals a unique mechanism by which this inhibitor might function as an anticoagulant. Thus, in addition to inhibiting the catalytic activity of thrombin, dabigatran may displace thrombin from fibrin or platelets. This would reduce the protection of thrombin from inhibition afforded by such binding, and could further attenuate the thrombogenic potential of the thrombus ,57. SuchIn summary, our results suggest that active site inhibitors modulate thrombin function mediated by the exosites. This reverse direction of allosteric signaling provides greater insight into the dynamic nature of thrombin. Furthermore, different active site-directed agents evoke unique responses, demonstrating that their inhibitory function can be refined. Thus, exploitation of its allosteric network endows thrombin with additional mechanism of regulation without compromising its repertoire of substrates. This demonstrates that the versatility of thrombin is a consequence of the intricate connections between the active site and the exosites.


Mycobacterium tuberculosis, is an obligate pathogen that evolved to exclusively persist in human populations. For M. tuberculosis to transmit from person to person, it has to cause pulmonary disease. Therefore, M. tuberculosis virulence has likely been a significant determinant of the association between M. tuberculosis and humans. Indeed, the evolutionary success of some M. tuberculosis genotypes seems at least partially attributable to their increased virulence. The latter possibly evolved as a consequence of human demographic expansions. If co-evolution occurred, humans would have counteracted to minimize the deleterious effects of M. tuberculosis virulence. The fact that human resistance to infection has a strong genetic basis is a likely consequence of such a counter-response. The genetic architecture underlying human resistance to M. tuberculosis remains largely elusive. However, interactions between human genetic polymorphisms and M. tuberculosis genotypes have been reported. Such interactions are consistent with local adaptation and allow for a better understanding of protective immunity in TB. Future ‘genome-to-genome’ studies, in which locally associated human and M. tuberculosis genotypes are interrogated in conjunction, will help identify new protective antigens for the development of better TB vaccines.The causative agent of human tuberculosis (TB),  Co-evolution between a host and a pathogen occurs when evolutionary changes in the pathogen that increase infectivity are counteracted by evolutionary changes in the host that increase resistance to infection. Such reciprocal changes have been demonstrated in experimental systems such as bacteria and their bacteriophages ,2. HowevMycobacterium tuberculosis complex (MTBC) and Homo sapiens in light of these aspects.For co-evolution to occur between a host and a pathogen: (i) pathogen infectivity and host resistance have to have reciprocal antagonistic effects on the fitness of each; (ii) the variation in pathogen infectivity and in host resistance have to be at least partially genetically determined; and (iii) the outcome of the interactions between the partners has to be dependent on the respective genotypes –8. Here,Tuberculosis (TB) remains a leading cause of morbidity and mortality in the world . DespiteThe purpose of this review was to discuss new ideas and concepts that together could guide future research. We focus on the bacterial side of the association, but always in the context of its relation to the host. We start by introducing MTBC, the different phylogenetic lineages that make up this group of organisms, as well as their biogeographical distribution and primary host range. Next, we discuss how MTBC virulence might have evolved in response to different evolutionary forces, in particular human demography. Local adaptation is a phenomenon expected from a co-evolutionary association, and the evidence for it in human TB is presented. Co-evolution is expected to lead to interactions between host and pathogen loci; hence, we summarize the existing evidence with respect to such interactions in TB. Recent research on the age of MTBC is summarized in light of both genetic and archeological data. In the final section of this review, we discuss how genetic drift and natural selection might influence the ability of MTBC to adapt to its host and end by highlighting how understanding more about the co-evolutionary history of MTBC and humans can guide us in our quest for a better TB vaccine.Mycobacterium canettii. For simplicity, throughout this review, we use the term MTBC to refer to all MTBC lineages except M. canettii.MTBC comprises various bacterial species and sub-species sharing 99.9% DNA sequence identity but differing in their primary host range. We describe below the different members of MTBC, dividing them according to their primary host: human-adapted MTBC lineages, the lineages adapted to wild and domestic mammalian hosts, and Mycobacterium tuberculosis sensu stricto and M. africanum. Humans are the only known host where infection and transmission by M. tuberculosis and M. africanum occur efficiently and where infection cycles are known to be sustainably maintained  that is not present in M. africanum strains  not present in the modern strains or any other strains belonging to M. tuberculosis sensu stricto , and Lineage 6 (M. africanum West African 2), which are geographically restricted to Western African countries  groups of the MTBC phylogeny, respectively, and which have no PhoPR mutations, regulate the secretion of ESAT-6 through PhoPR M. africagh PhoPR . InteresMycobacterium, the most likely bacteria to share the most recent common ancestor with MTBC are the so-called ‘smooth tubercle bacilli’ (STB), which include M. canettii , and the presence of CRISPR spacers matching prophages present in other mycobacteria (e.g. M. marinum)   are consM. canettii-like ancestor, a strain with a loose ability to survive both in the environment and inside a host. The genetic changes associated with the transition to becoming a professional pathogen involved gene loses and gene acquisitions , because at least some degree of pathology is essential for MTBC transmission to occur. This might have marked the beginning of the co-evolutionary association between M. tuberculosis and Homo sapiens. Notably, the trajectory toward host specialization has allowed this MTBC ancestor to spread all over the world and become one of the most successful human pathogens.This finding is in sharp contrast to the other members of MTBC, which are obligate pathogens. The MTBC possibly evolved from a isitions –59,63, afamilies ,65. Currustrates . BetweenIn the process of becoming obligatory pathogens, the ancestors of MTBC must have evolved strategies to overcome the immune response of the host, to transmit between hosts, and to avoid becoming extinct in small human populations. The affected humans must have evolved counter strategies, because for transmission of MTBC to occur, human disease is necessary. Specifically, lung cavitation and increased sputum-smear positivity are associated with more efficient transmission of MTBC –71. Furt+ T-cell counts  and quantitatively (e.g. abundance), temporal and spatial dynamics of host–pathogen associations are expected under a co-evolutionary process such that a snapshot in time or space will reveal that certain host genotypes are more susceptible to certain parasite genotypes ,76. One l counts .Given that TB disease is necessary for MTBC transmission, what are the implications of local adaptation; are local MTBC strains more or less virulent in their local host population? One study in Texas has shown that different levels of pulmonary impairment could not be explained by differences in the causative MTBC lineages alone, but by the pathogen–host association; patients infected with sympatric MTBC lineages suffered less lung impairment than patients infected with allopatric lineages . TransmiM. africanum lineages show lower a prevalence compared to the modern lineages, even within their specific range of distribution in West African  was associated with infection by Lineage 2 (Mannose-binding Lectin (Protein C) 2 gene (MBL2) was associated with TB caused by M. africanum as opposed to M. tuberculosis sensu stricto (Immunity-related GTPase M gene (IRGM) was protective against TB caused by Lineage 4, but not against disease caused by other MTBC lineages  of MTBC, but only recently has the subject been addressed in a quantitative way. Four studies have used DNA diversity to infer the age of MTBC ,29,107 Table ,108. Howe et al. , if for Table et al. . One alternative way to calibrate phylogenetic splitting events, for which bacterial divergence is strictly a consequence of co-divergence with its host, is to use the host fossil record to date these events. This approach has been particularly powerful in the case of vertical transmitted endosymbiotic bacteria and their insect hosts . In Fig. 4A, we added to the internal nodes of the MTBC phylogeny by Pepperell et al. . We highlight here what we consider to be congruent among Figs More reliable phylogenetic calibration points can be obtained by dating nodes or tips of a phylogenetic tree using ancient bacterial DNA (aDNA), but this was until very recently (see below) not possible for MTBC, because only small fragments of aDNA with little or no phylogenetic information were available. In the absence of calibration points, another possibility is to estimate mutation rates experimentally  or from ct hosts . A similsmission . Recentl in vivo gal. Fig. , it is ual. Fig. , we indiet al. , and close to the mutation rate (0.2–0.5 SNPs per year) inferred from molecular epidemiology studies  to support co-divergence. We agree this is an important analysis, but it should be repeated using more isolates spanning more of the global diversity of MTBC.Important differences between the results of Comas et al.  and thos  to calibrate a global MTBC phylogeny. The substitution rate obtained was 4.6 × 10−8 substitutions/site/year, again much higher than the estimate by Comas et al.  than the one obtained with the Peruvian aDNA Fig. et al. Fig. et a. et al.  BC today . Howeverthe past . Similarthe past .et al. et al. et al. ears ago . Interesears ago ,143, whiago Fig. .de novo (adaptive immune system) and will thus not be encoded in their DNA. Alternatively, pathogens may evolve strategies other than avoiding being recognized by the host. In MTBC, this seems to be the case as suggested by one study which studied variation in 491 experimentally confirmed T-cell epitopes in representatives of several lineages of the complex (+ T cells (M. canettii (in vitro passage (In a co-evolutionary association, molecular interactions between the host and the pathogen are expected , such as complex . Surpris complex ,107. One complex . Support complex ; this is T cells . Interescanettii  and in Bvar genes of Plasmodium falciparum and pil genes of Neisseria meningitis (pe_pgrs genes underwent extensive duplication (pe_pgrs genes that encode T-cell epitopes are mostly confined to the PE domains, which exhibit high sequence conservation when comparing different clinical strains of MTBC (pe_pgrs genes cannot be explained by evasion of T-cell recognition. Perhaps, the importance of the genetic diversity of pe_pgrs genes lies not so much on the diversity generated at the population level but rather on the diversity generated within each bacterial cell, for instance by differential expression of these genes under different conditions. Another possibility is that the diversity of pe_pgrs genes might be driven by host antibody recognition (In addition to mutations causing polymorphisms in populations, duplication, and expansion of gene families has evolved in almost all organisms as a way to generate genetic diversity . In partningitis . In MTBClication . Moreovelication ,150. Howlication , the reg of MTBC . Thus, tognition . In summTable Is there any other evidence of adaptation in MTBC genomes? The emergence of drug resistance clearly illustrates that MTBC has the potential to adapt. Yet, except in the context of drug resistance, the current evidence of ongoing adaptive changes in MTBC is scarce. One important limitation to adaptive evolution in MTBC might be the lack of recombination and horizontal gene transfer . Recombiet al. (et al. (vice versa. If so, most mutations responsible for host adaptation would already have reached fixation. New mutations will mostly be neutral or slightly deleterious and hence assume the form of transient polymorphisms, which end up disappearing from the population over time. Such a scenario has been suggested for other genetically monomorphic bacterial pathogens (et al. (Nevertheless, evidence for adaptation is emerging as more full genomes of MTBC clinical strains become available for comparative analyses. A recent analysis by Osorio et al. , reporte (et al.  describeathogens . Thus, mathogens . Contras (et al. , suggestAlternatively, it has been hypothesized that infection by MTBC might have been beneficial to early human populations during times of nutrient shortage  and/or bTable Table Table It has been proposed that the evolutionary trajectory of new mutations in MTBC might be mostly governed by genetic drift, negative selection, and background selection ,60,107 Table . GeneralThe observations we reviewed here favor the hypothesis of co-evolution between humans and certain MTBC lineages. Humans differ in their genetic susceptibility to TB, and the genetic diversity in MTBC is also increasingly recognized as a factor influencing the outcome of TB infection and disease. Moreover, recent studies have reported interactions between human genetic diversity and bacterial diversity, reinforcing the view that in addition to environmental variables, TB susceptibility varies as a function of both the human and MTBC genotype. The age of the association between humans and MTBC is subject to intense debate, but irrespective of the exact duration of this association, different phylogenetic lineages of MTBC are associated with different geographic regions, and possibly adapted to specific human populations. In addition, some lineages seem to behave in a generalist way, while others appear to be specialists restricted to a smaller group of human host populations.H. sapiens could be exploited in vaccine development. For example, as illustrated in the recent genome-to-genome study of HIV mentioned above (Co-evolution is defined as reciprocal adaptive changes in interacting host and pathogen genetic loci. Conceptually, these interacting loci can be seen as in ‘conflict’ with each other . Identifed above , a simil


We will present new modifications for spinal fixation after pedicle subtraction osteotomy (PSO) in aged patients with kyphosis.The pedicle screws in the proximal site of osteotomy are not connected to the rod to permit spinal shortening in the postoperative course. When a connector is used between a pedicle screw and a rod, the connector is not fixed to the rod to allow it to slide on the rod. When a connector is not used, the rod is only placed in the screw head with reinforcement by some sets of cranial sublaminar taping. U-rod is used to not injure the skin when postoperative junctional kyphosis (PJK) occurs.We investigated 6 cases of posttraumatic kyphosis and a case of degenerative kyphosis. We measured a local kyphotic angle and a distance between the center points of the adjacent endplates at the site of osteotomy.Average local kyphotic angle improved from 39 degrees to 6 degrees, and finally increased to 18 degrees. Spinal shortening at the site of osteotomy occurred 8 mm on average . In two cases without using connectors, kyphosis recurred because sublaminar taping couldn't keep the spine in the correct position.Patients with osteoporosis have risks of height loss and new vertebral fractures. A good result demands a stable anterior column support. Unexpected excessive spinal shortening by various reasons can lead to instrumentation failures.Previously we had used rod & wire system as a fixation device after osteotomy for kyphosis with osteoporosis. It has a telescoping function to keep giving the axial load to the anterior column till it attains stability without significant change of sagittal alignment. When we extended the indication of this procedure to the kyphosis or scoliosis with segmental instability, we had to do some re-operations.We thought up some new modifications of pedicle screw system to compensate the weak points of the rod & wire system. Many cases showed gradual spinal shortening at osteotomy site, keeping a good sagittal alignment. However in some of cases a connector was not used, and the proximal part of instrumented spine gradually separated from the rod. Sublaminar taping seems to be insufficient to keep a good sagittal alignment.This new modification of pedicle screw system can be one surgical option for an osteoporotic spine with some morphologically unstable factors.


Escherichia coli, we observed that steady-state levels of betT mRNA, which encodes a transporter mediating the influx of choline, are dependent on cellular concentrations of RNase III. In the present study, we also observed that steady-state levels of betT mRNA are dependent on RNase III activity upon exposure to osmotic stress, indicating the presence of cis-acting elements controlled by RNase III in betT mRNA. Primer extension analyses of betT mRNA revealed two tandem RNase III cleavage sites in its stem-loop region, which were biochemically confirmed via in vitro cleavage assays. Analyses of cleavage sites suggested the stochastic selection of cleavage sites by RNase III, and mutational analyses indicated that RNase III cleavage at either site individually is insufficient for efficient betT mRNA degradation. In addition, both the half-life and abundance of betT mRNA were significantly increased in association with decreased RNase III activity under hyper-osmotic stress conditions. Our findings demonstrate that betT mRNA stability is controlled by RNase III at the post-transcriptional level under conditions of osmotic stress.While identifying genes regulated by ribonuclease III (RNase III) in  Escherichia coli, RNase III, encoded by the rnc gene, consists of a ribonuclease domain (amino acid residues 21–149) and a dsRNA-binding domain (residues 155–209). E. coli RNase III functions as a homodimer in which two ribonuclease domains form a single processing center, and each domain contributes to the cleavage of one RNA strand of the duplex substrate RNase III plays an important role in regulating gene expression by degrading and processing mRNA in both prokaryotes and eukaryotes E. coli mRNA transcripts have been identified whose abundance is dependent on the cellular concentration and activity of RNase III, including rncpnpbdmcorAproUmltDrngE. coli that are downregulated by RNase III. Among them, the mechanisms of RNase III-mediated regulation of bdmproUbdm and proU mRNA is significantly altered in E. coli subjected to hyper- and hypo-osmotic stress. These results indicate that RNase III activity is regulated by external signals in E. coli. However, betT, another important osmoregulator, has not been studied with respect to RNase III-mediated regulation upon osmotic stress although betT mRNA abundance was found to be dependent on cellular RNase III concentrations.Several betT gene forms part of the osmoregulatory system Bet regulon, which participates in the synthesis of glycine betaine from externally supplied choline. The Bet system is composed of the following: BetA, a choline dehydrogenase; BetB, a betaine aldehyde dehydrogenase; BetI, a regulatory protein; and BetT, a choline transporter Km = 8 µM) when the external concentration of choline is low Km = 1.5 mM), choline uptake is mediated by the ProU transport system The betT gene expression and the factors involved have not been fully characterized. In the present study, we investigated the effects of RNase III activity on betT gene expression in E. coli. Our results provide direct evidence that RNase III controls the degradation of betT mRNA by cleaving at two sites in its open reading frame region, thereby suggesting a physiological relationship between the regulation of RNase III activity and osmotic stress resistance.The physiological role of BetT in osmotic stress resistance has been previously reported E. coli strain MG1655rnc has been previously described betT, a DNA fragment encoding betT was amplified using the primers betT-5′  and betT-3′ , and cloned into the NotI site in pCAT924 betT under the control of a constitutive ctrp promoter. To express betT mRNA containing a single nucleotide substitution at the RNase III cleavage sites (C33U and C39U), pBetRS1-C33U and pBetRS1-C39U were constructed as follows: DNA fragments were amplified using overlap extension polymerase chain reaction (PCR) method and cloned into the NotI site in pCAT924. The primers used were betT-5′, betT-48F (5′-GGTGTTTTACACCTCCGCCG), betT-1610R (5′-TAATTCATCAGGCGCGAGAG), and betT-11D-67R  for pBetRS1-C33U, and betT-5′, betT-48F, betT-1610R, and betT-13I-67R  for pBetRS1-C39U. The MG1655betT, MG1655proV, and MG1655betTproV strains were constructed via one-step inactivation of the chromosomal gene by using the method described by Datsenko and Wanner betT-deleted strains were betT-H1  is underlined) and betT-H2  with pKD3 as a template. The primers proV-5′-UTR-Km  and proV-3′-UTR-Km  were used to amplify the kanamycin antibiotic resistance marker in pKD13 for construction of proV-deleted strains.Construction of the betT were betT-F (5′-ATGACAGACCTTTCACACAG) and betT-652R (5′-TAAGCTGCACCACACCGATA). The primers used for M1 were rnpB-F (5′-GAAGCTGACCAGACAGTCGC) and rnpB-R (5′-AGGTGAAACTGACCGATAAG).RT-PCR analysis was performed as previously described betT mRNA and the model hairpin RNA, which spans nucleotides 21–95 in the betT coding region (the first nucleotide of the start codon is at position 1) and encompasses the RNase III cleavage sites, were obtained by amplifying the corresponding sequence in pBetRS1 using the following primers: T7-betT-F  and betT-R  for a full-length betT transcript, as well as T7-betT-21F  and betT-95R  for the model hairpin RNA. Labeled RNA (0.2 pmol) was incubated with 1 ng of purified RNase III in the presence of 0.25 mg ml−1 of yeast tRNA (Ambion) and 20 U of RNase inhibitor (Takara) in 20 µl of cleavage buffer , pH 8.0). Cleavage reactions were initiated by adding 10 mM MgCl2 at 37°C. Samples were removed at various time intervals  and mixed with an equal volume of gel-loading buffer , 0.025% sodium dodecyl sulfate, 0.025% xylene cyanol, and 0.025% bromophenol blue). The samples were denatured at 65°C for 10 min and separated on an 8% or 10% polyacrylamide gel containing 8 M urea and 0.5×Tris-borate EDTA.His-tagged RNase III purification and cleavage assays were performed as previously described 32P-labeled primers were used: betT-120R (5′-GGCCGAGAAGTCGCGAAACA) and betT-273R (5′-TGAATTCCGGTTTGGATTGT). RNA with labeled primers were annealed at 65°C for 15 min, slowly cooled down to room temperature for 2.5 h, and extended at 42°C for 1 h using AMV reverse transcriptase (New England Biolabs). The extended fragments were denatured at 95°C for 10 min and separated on 10% polyacrylamide gels containing 8 M urea.Primer extension analysis was performed using total RNA purified via heated phenol extraction and ethanol precipitation. The following 5′-betT mRNA detection were synthesized using a random-primed DNA labeling kit , in which the PCR products containing the coding region of betT were used as a template. The primers used were betT-F and betT-R. The oligonucleotide probe used for M1 was rnpB-137R (5′-GCTCTCTGTTGCACTGGTCG).Forty micrograms of the total RNA sample prepared as describe above was denatured at 65°C for 10 min in twice the volume of formamide loading buffer and separated by electrophoresis on a 1.2% GTG agarose gel containing 2.3% formaldehyde. The gels were transferred onto nylon membranes  in 20× standard saline citrate. The random hexamer probes used for β-Galactosidase activity in whole cells was determined as previously described betT mRNA, we measured the steady-state levels of betT mRNA in wild-type and rnc-mutant E. coli by using semi-quantitative RT-PCR. Two sets of primers were used to amplify cDNAs (+1 to +652 and +1612 to +2034)  . Consistpe cells . To furtanalysis . The halanalysis . For thebetT mRNA contains cis-acting elements that are responsive to RNase III, we performed an in vitro cleavage assay using purified RNase III and synthetic full-length betT mRNA transcripts. RNase III cleavage of 5′-end labeled betT mRNA generated a major cleavage product that was approximately 80 nt in length and several minor cleavage products whose lengths were approximately 70–130 nt  that was designed to bind to a downstream region of the RNase III cleavage sites that were deduced from the in vitro cleavage assay. Because betT expression is induced at high salt concentrations, total RNA was purified from wild-type and rnc-mutant cells grown in plain (0.17 M NaCl) or 0.50 M NaCl-supplemented Luria-Bertani media. However, we were not able to observe cDNA bands extended from betT mRNA in all reaction mixtures  for primer extension analysis. We observed four distinct cDNA bands that were dependent on RNase III  in the betT overexpression plasmid (pBetRS1) , and primer extension analysis was performed. Whereas the cDNA bands corresponding to RNase III cleavage sites I–IV were clearly visible in the reaction mixture containing wild-type betT mRNA, the abundance and patterns of cDNA products from mutant betT mRNAs differed from those of wild-type betT mRNA  were greatly decreased when these mutations were introduced , resulting in the significant accumulation of uncleaved mutant betT mRNAs. RT-PCR analysis of betT mRNA further confirmed increased steady-state levels of betT mRNA resulting from the mutations (in vivo stability of betT mRNA.To test whether RNase III activity on cleavage sites regulated the stability of pBetRS1) . These metT mRNA . Nucleotutations . These rE. coli cells betT mRNA. First, we confirmed the functional role of betT in E. coli growth by culturing cells under hypo-osmotic (0.01 M NaCl), normal (0.17 M NaCl), and hyper-osmotic (0.50 M NaCl) conditions in the presence of low choline concentration (10 µM)   and S3. betT mRNA under different osmotic stress conditions. The results from semi-quantitative RT-PCR analysis showed that the decay rate of betT mRNA in wild-type E. coli under the hyper-osmotic condition was approximately two times lower than those under normal or hypo-osmotic conditions   or an rnc′-′lacZ (KSC006) reporter gene et al. betT mRNA.Next, we measured the half-lives of nditions . Steady-ondition , indicatis study . We alsoondition . Althoug7.6 min) . Steady-E. coli have been well studied, the regulation mechanisms underlying rapid control of osmosensing factors are not fully understood. Recent studies have demonstrated that RNase III plays roles in the regulation of several stress response factors, such as the biofilm-dependent modulation protein (Bdm) E. coli. The present study showed that under conditions of hyper-osmotic stress, betT expression is also regulated by RNase III at the post-transcriptional level.Although the factors involved in osmoregulation mechanisms in betT mRNA. The results of the in vitro cleavage assay support this notion as the cleavage products of the 3′-32P-end-labeled model hairpin RNA that were generated by RNase III cleavage at sites proximal to the 3′-end of the transcript (III and IV) appear to accumulate at similar rates, indicating that the RNase III cleavage at the two tandem sites  is independent . These results demonstrate that the RNA fragments generated by RNase III cleavage at one dimeric RNase III site in betT mRNA are not likely to be further cleaved by RNase III, indicating the independent cleavage of two tandem RNase III sites in betT mRNA. This result raises the question as to why betT mRNA contains two consecutive RNase III cleavage sites, neither of which is sufficient on its own for efficient betT degradation. This appears to be a unique property, as no other known RNase III substrates show two tandem RNase III cleavage sites. One possibility is that both sites are cryptic and do not provide the structural determinants that are required for full RNase III activity. This possiblity is supported by our mutational analyses at the betT mRNA cleavage sites, which demonstrated that inactivation of RNase III cleavage at either site (I and IV or II and III) was sufficient to inhibit betT mRNA degradation . Semi-quantitative RT-PCR analysis of the cDNA +1-+652 was performed to measure the relative amounts of betT mRNA by setting the amount of betT mRNA in cultures 0 min after the addition of rifampicin to 1. The experiments were repeated three times and averaged. The error bars (standard errors of the mean) were used to indicate the range of assay results. The half-lives of betT mRNA were estimated by fitting and extrapolating the plots in the graphs.Effects of RNase III on the half-life of (EPS)Click here for additional data file.Figure S2betT mRNA. Total RNA was prepared from MG1655, MG1655rnc, MG1655rnc harboring pBetRS1, and either pKAN6B or pRNC1. Total RNA was hybridized with a 5′-32P-end-labeled primer (betT+273R) and extended using AMV reverse transcriptase. Sequencing ladders were produced using the same primer used in the PCR DNA, encompassing the betT gene as a template. −, no expression; +, endogenous expression; ++, overexpression.Primer extension analysis of (EPS)Click here for additional data file.Figure S3betT deletion on E. coli growth upon osmotic stress. The cultures of wild-type and betT and/or proV-deleted MG1655 were grown in M63 supplemented with 22 mM glucose until an OD600 of 0.3 was reached, and either treated with 0.01 M (hypo-osmotic), 0.17 M , or 0.5 M (hyper-osmotic) NaCl with 10 µM choline. Growth was measured by analyzing the cell density (OD600) of cultures.Effects of (EPS)Click here for additional data file.Figure S4E. coli strains KSC004 and KSC006 were grown in M63 supplemented with 22 mM glucose until an OD600 = 0.3 was reached, and then cultures were incubated for a further 3 h in the same medium containing either 0.01 M, 0.17 M, or 0.5 M NaCl with 10 µM choline. Cells were then subjected to β-galactosidase assays (A), and a western blot analysis of RNase III (B). Experiments were performed in triplicate and repeated at least three times.Osmotic regulation of RNase III activity and expression ratio. (TIF)Click here for additional data file.