Balaena mysticetus), northwest of Camden Bay in the Beaufort Sea. The drilling operation involved a number of vessels performing various activities, such as towing the drill rig, anchor handling, and drilling. Acoustic data were collected with six arrays of directional recorders (DASARs) deployed on the seafloor over ~7 weeks in Aug–Oct. Whale calls produced within 2 km of each DASAR were identified and localized using triangulation. A “tone index” was defined to quantify the presence and amplitude of tonal sounds from industrial machinery. The presence of airgun pulses originating from distant seismic operations was also quantified. For each 10-min period at each of the 40 recorders, the number of whale calls localized was matched with the “dose” of industrial sound received, and the relationship between calling rates and industrial sound was modeled using negative binomial regression. The analysis showed that with increasing tone levels, bowhead whale calling rates initially increased, peaked, and then decreased. This dual behavioral response is similar to that described for bowhead whales and airgun pulses in earlier work. Increasing call repetition rates can be a viable strategy for combating decreased detectability of signals arising from moderate increases in background noise. Meanwhile, as noise increases, the benefits of calling may decrease because information transfer becomes increasingly error-prone, and at some point calling may no longer be worth the effort.During summer 2012 Shell performed exploratory drilling at Sivulliq, a lease holding located in the autumn migration corridor of bowhead whales ( Balaena mysticetus) begins its autumn westward migration in late August. Unlike the spring migration, the autumn migration takes place relatively close to the northern shores of Alaska [After summering in the eastern Beaufort Sea, the Bering-Chukchi-Beaufort (BCB) population of bowhead whales  as part of their exploration activities in the Beaufort Sea. In summer 2012 Shell performed exploratory drilling at et al. [Sivulliq in 2012, as these four studies pertain to bowhead whales migrating in the autumn past active drillships and support vessels in the Beaufort Sea. In 1986 and 1993, bowhead whales appeared to avoid an area of radius ~10 km around the drillship, deflecting either seaward or shoreward around the operation, with some whales apparently beginning to divert when 20 km or more from the drill site [Early studies of the reactions of baleen whales to drilling operations, many of which were published in the gray literature, are summarized in detail in Richardson et al. . Four ofet al.  and 1991et al. –8 are paill site , 8. Greeill site .et al. [versus an actual decrease in calling rate.To our knowledge, there is only one other study assessing effects of drilling activities on calling behavior in bowhead whales. Richardson et al.  performeet al. [Two previous studies , 12 haveet al. [2 / Hz @ 10 Hz, 48 dB @ 50 Hz, 44 dB @ 100 Hz, and 37 dB @ 400 Hz. This sensor was used for sound pressure measurements of any sounds of interest, including whale calls, distant airgun pulses, or various types of background noise. The directional channels included two particle motion sensors mounted orthogonally in the horizontal plane for sensing direction to sounds for which a bearing was required, such as whale calls and airgun pulses. DASARs include a signal digitizer with 16-bit quantization. The 1 kHz sampling rate allowed for 116 days of continuous recording in the frequency range 10–450 Hz.The equipment and field methods employed for this study are nearly identical to those presented in Blackwell et al. . Recordiet al. ) sampline.g., 11, 12), DASAR locations in 2012 were slightly changed, to better serve the purpose of industrial sound monitoring during Shell’s drilling operation. For example, the four northernmost DASARs at site 4 and the three site 0 DASARs were new locations for the 2012 field season. The approximate distance from the Sivulliq drilling location to the center of each array was 178 km for site 1, 114 km for site 2, 29 km for site 3, 7 km for site 0, 19 km for site 4 , and 103 km for site 5.During the open-water season of 2012, 40 DASARs were deployed in the Beaufort Sea, in six arrays of 3–13 recorders each that spanned an east-west distance of ~280 km . In eachDASARs were deployed between 16 and 21 August and retrieved between 3 and 7 October 2012. Each DASAR was placed on the seafloor with a 110-m ground line connecting it to a small Danforth anchor, and GPS positions of the recorder and the anchor were obtained on each deployment. DASARs were then retrieved by grappling for the ground line. The Immediately after deployment and preceding retrievals, each DASAR’s clock and orientation on the seafloor were calibrated by transmitting specific signals  at known GPS-determined times and locations, three around each recorder. For more information on the calibration methodology, see , 14.Passive acoustic recording of endangered bowhead whale calls does not typically require a federal permit as it does not have the potential to “take” the animals as defined by the U.S. Marine Mammal Protection Act or the U.S. Endangered Species Act. The research presented here was, however, part of an approved monitoring program around activities conducted under incidental harassment authorizations (IHAs) issued by the U.S. National Marine Fisheries Service. The research was therefore subject to regulatory review and approval under those authorizations and under the terms of lease agreements under the Outer Continental Shelf Lands Act.et al. [i.e., airgun pulses and tones from machinery (the “dose” in a dose-response study), and the number of whale calls detected (the “response” in a dose-response study). Sound from airgun pulses was quantified because many pulses were present in 2012, and previous work had demonstrated that these signals have measurable effects on bowhead calling rates [The analysis strategy used here is similar to that used in Blackwell et al. . The entng rates .The analysis required defining the length of the time interval over which the dose  and response  would be matched. The interval needed to be long enough that many intervals included at least one call, and it needed to be relevant for whale response to received levels of sound. Based on these considerations as well as previous analyses , we chosAfter retrieval, data were transferred to file servers and analyzed using custom MATLAB-based software. An automated call detection algorithm  was applet al. [i.e., were unaffected by masking  above 113 dB re 1 μPa were removed from the analyses (see below), and (2) for the remaining 10-min periods, i.e., with mean background levels below the 113 dB cut-off, only calls localized within two km of a DASAR were included in the analyses. Hereafter, a particular analysis cell (2-km circle) at a particular 10-min time interval will be referred to as a “cell-time interval” (CTI). To address this issue, we took a two-step approach: (1) 10-min periods with a mean broadband (background) level  were determined for each minute of collected data by analyzing a series of 119 one-second-long data segments, overlapped by 50%, and then averaging them. Broadband levels were derived from the narrowband spectral data by integrating the mean square pressures of all frequencies within the 10–450 Hz frequency range. A mean broadband level BB, when (A) using all calls localized versus (B) using only calls localized within 2 km of a DASAR. The four lines show the 97.5th percentile of number of calls based on quantile regressions, for BB greater than 100 dB (the lower cut-off) and four alternative upper cut-offs: 107 dB, 110 dB, 113 dB, and 116 dB re 1 μPa. Several alternative cut-offs were tested since our goal was to use as much of our dataset as possible while still satisfying the constraint of detected calling rates being independent of background levels. Since interest focused on masking, which we had no reason to suspect at the lowest broadband levels, two lower cut-offs were tested, at 95 dB and 100 dB  subset. Between each pair of cut-offs  subset), there were no calls in 95.4–96.7% of all CTIs. Therefore, using a lower percentile, say, the 95th percentile  would have been uninformative.i.e., indicating no relationship between background levels and call detection rates. Section B.1 , listed as follows by site (from west to east):The entire dataset contained 273,522 cell-time intervals (CTIs). At site 0, the DASARs were only 2 km apart instead of 7 km, so to avoid duplicate whale counts at overlapping CTIs, only the data for DASAR 0Y (chosen randomly) were included in the analyses. This decreased the number of CTIs to 258,617. The 113 dB 0.5% for site 1 1.9% for site 2 23% for site 3 27% for site 0 22% for site 4 , and1.9% for site 5 .Sivulliq drilling location . A previous study [Tones index for a 10-min cell-time interval was obtained by summing the two-min values over 10 min. Details of how the Tones index was calculated, with an example, are presented in the Tones index over a period of three days with variable sound levels at DASAR 2D.This index quantifies the intensity of tones that are present in the sound spectrum, relative to the complete background noise intensity. We define a tone as a sound of bandwidth 1 Hz with an amplitude at least 4 dB above that of neighboring frequencies and that occurs continuously over several minutes see the . Tones aet al. [2-s. In 2012, two different seismic exploration operations were identified, to the east and to the north of the DASAR arrays [et al. [2-s. Since this restricted range of RLs from airgun pulses would not have allowed us to replicate the results of Blackwell et al. [Airguns, to describe the presence (1) or absence (0) of detected airgun pulses in each 10-min CTI. Airgun pulses were detected, analyzed, and the detections were quality-controlled as described in Blackwell et al. [Airgun pulses were present on all 40 DASAR records during the 2012 field season. Since airgun pulses were known to affect bowhead whale calling rates [R arrays . Both opTones and Airguns) and Site (represented by five indicator variables for the six sites). We checked for potential collinearity among the candidate covariates using condition indices and variance decomposition proportions [Covariates considered in the negative binomial regression modeling included the two sound metrics  necessarily included the corresponding lower order effects, and models with interactions included the corresponding main effects. Thus, models included the main effects of Site, Tones, and Airguns, with corresponding higher order effects and interactions, for a total of 29 candidate models. We evaluated models based on relative fit .To address potential spatio-temporal correlation in whale call counts and its effects on parameter variances, we conducted block bootstrapping . These pi.e., after removal of CTIs with BB > 113 dB re 1 μPa, included 223,487 cell-time intervals and 14,751 bowhead calls –(D) of Similarly, Section (C) shows that when airgun pulses were present, all sites showed excess calls (range 2–33%), even though the excess was modest for sites 1 and 5. Section (D) compares CTIs with both tones and airguns with CTIs lacking detections of either sound source. Excess calls were present in the range 21–32% at all sites but site 1, where there was no difference.Tones > 0), (2) airguns were present (Airguns = 1), (3) both tones and airguns were present, and (4) neither tones nor airguns were detected in the sound records (Tones = 0 and Airguns = 0). It shows that the percentage of CTIs with detected airguns increased from west (site 1) to east . In addition, the highest ranked model included the interaction between Site and Airguns, which resulted in it having 50% more parameters (15 vs. 10) than the second ranked model, with a modest advantage in BIC (ΔBIC = 16.1). Parameter estimates and associated 95% confidence intervals are listed in The 28 regression models assessing the relationship between the sound metrics and call localization rate had 2–30 parameters and BIC values ranging from 93,515 to 95,979. The Site, Airguns, and Tones). It is important to keep in mind that when presenting modeled data in marginal effects plots, we assess the effect of any single covariate by holding other covariates at fixed values, as this helps in interpretation of the coefficient estimates.Figures illustrating the modeling results are presented below. Figures that summarize the actual data are shown in a gray color scheme, with bar height indicating mean calling rate and error bars (± one standard error) showing variability in the data. In contrast, multi-colored plots are marginal effects plots based on the best regression model. These plots show the predicted calling rates as functions of model parameters (Tones consistently displays a non-linear relationship (cubic term) to call localization rate in the top four models, and that the inclusion of this cubic term generates better fits  than comparable models in which Tones appears as only a linear or quadratic effect  is directly associated with these bar charts, including whether mean calling rate is significantly greater when airgun pulses are present than when absent see . Rather,vs. present, respectively . The predicted number of calls at each site as a function of airgun presence is depicted in Tones metric set to zero (Tones absent). These figures show that predicted calling rates increased in the presence of airgun pulses, but the size of the increase was site-dependent, with evidence of proportionately larger increases at the sites closer to Sivulliq.Observed calling rates in the presence and absence of airgun pulses, for the combined sites, is shown in Tones index initially lead to increased calling by the whales, but as the value of Tones continued increasing, calling rates decreased. In the absence of tones, calling rates were on average 0.056 calls / CTI  had increased to 0.098 calls / CTI. Furthermore, at the peak shown in Tones bins in the range 25–40, the average calling rate was 0.156 calls / CTI.Observed calling rates as a function of the presence and relative “dose” of tones is shown in ls / CTI . In compTones up to a Tones value of ~38 dB, beyond which they dropped precipitously. Peak predicted calling rates were highest at site 5 and lowest at site 4, with intermediate values for the other sites. When airguns were present (Tones (>55 dB), where calling rates were near 0.These trends are confirmed in the modeling results, shown for the top model in When airguns were present , this is not indicative of an interaction between Airguns and Tones. Rather, it is a consequence of the interaction between Airguns and Site . In this case, mean calling rates were 0.040 calls / CTI when neither industrial sound source was present, and 0.142 calls / CTI when both were detected—a 3.5-fold increase.Calling rates were also calculated for two contrasting subsets of the observed data: CTIs that included both airguns and tones BB < 113 dB re 1 μPa) that were enforced in this analysis to avoid masking effects. This result parallels that from a previous study examining the effects of airgun pulses on bowhead calling rates [The primary result of this analysis is that whenever tones from industrial activity were present in the sound environment of bowhead whales during their fall migration, the whales’ calling rates were affected. With increasing dosages of “tone sound”, calling rates increased, peaked, and then decreased, all within the restricted broadband background levels  and 2.05 (0.044 to 0.090) when tones or airguns, respectively, were present in the background. In comparison, Blackwell et al.  reportedet al. ) and a sSite x Airguns interaction  and the drilling-related vessel traffic that was almost entirely west of Sivulliq [The largest increase in calling rates occurred when tones and airguns happened concurrently, compared to times when neither were detected in the sound records: mean calling rates  were 0.142 versus 0.040, respectively, a 3.55 factor difference. In addition the increase was site-dependent, due to the top model’s eraction , and rouSivulliq . We suspSivulliq  and the R) acoustic transmission loss (where R is range in meters between source and receiver), which is an appropriate acoustic propagation model for the study area [i.e., an increase in calling with an increase in noise levels, is the opposite trend from the decrease in calling one would expect if masking were a factor.The confounding of masking effects with behavioral changes is a common problem whenever calls are not localized and thus variations in call detectability cannot be taken into account . We atteudy area , 21, thee.g., increasing wind speeds in penguin colonies, [e.g., plane overflights near amphibian colonies, [Whenever animal communication is hampered by noise, whether it be natural  or antholonies, ), there Increasing the amplitude of the signal—the so-called Lombard effect—has been observed in many vertebrates, see Brumm and Zollinger  for a reThe third strategy, to increase the repetition rate (redundancy) of the signal, is the strategy supported by the results presented here. Again, several examples exist from the bird literature , 37. AmoBoth signal detection theory  and infoe.g., continuous or intermittent, frequency range, etc.).Both signal detection theory and information theory predict that communication ceases whenever noise levels become so large that signals are almost completely masked—at that point no amount of repetition can succeed in increasing the probability of detection of a repeated sequence. Nevertheless, bowhead whales start decreasing their calling rates in response to higher doses of sound from airgun pulses  and toneThe automated call detection algorithm used in this analysis  did not In conclusion, little can be said at this point on the effects of changes in calling behavior on the long-term fitness of bowhead whales. The Bering-Chukchi-Beaufort population of bowhead whales has been increasing steadily over the last 30 years , despiteS1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


An avian infectious bronchitis virus (IBV) was detected from trachea swabs of chickens in Hubei province, China, in 2016. The complete genome of the IBV strain, CK/CH/HB/2016, was characterized and analyzed to better understand IBV epidemiology in China. Gammacoronavirus in the family Coronaviridae of the order Nidovirales (Avian infectious bronchitis virus (IBV) belongs to the genus ovirales . IBV is ovirales . Besidesovirales .To investigate the genetic diversity of IBV, we monitored the intensive farms in Hubei province, China. During 2016, seven batches of chickens from three farms showed typical clinical signs of infectious bronchitis. Out of 127 trachea swab samples, 122 (96.1%) were detected as IBV positive by one-step reverse transcriptase-PCR testing with primers IBVNF (5′ GTTTGARGGTAGYGGYGTYCCTGA 3′) and IBVNR (5′ CAGMACCHTTDRCAGCAACCCACACTA 3′). Subsequently, a complete genome sequence of the IBV strain, CK/CH/HB/2016, was characterized by a previously described method .169T, in the N gene of CK/CH/HB/2016 was detected, which is rare for IBVs , excluding the 3′ poly(A) tail. Genomic alignment showed that CK/CH/HB/2016 shares nt identity percentages of 97.4% and 96.5% with CK/CH/SD09/005 (GenBank accession number KF668605) and GX-NN09032 (GenBank accession number JX897900), respectively (The frequency of recombination was one of the most important reasons for the emergence of novel IBV strains –11. By r–The coronavirus spike, which is also a highly variable gene, is responsible for virus entry through receptor binding and membrane fusion . A sequeMF882923.The genome sequence of IBV strain CK/CH/HB/2016 was deposited in GenBank under the accession number


The use of peripheral locked screws has reduced glenoid baseplate failure rates in reverse shoulder arthroplasty. However, situations may arise when one or more non-locked screws may be preferred. We aimed to determine if different combinations of locked and non-locked screws significantly alter acute glenoid baseplate fixation in a laboratory setting.Twenty-eight polyurethane trabecular bone surrogates were instrumented with a center screw-type glenoid baseplate and fixated with various combinations of peripheral locked and non-locked screws . Each construct was tested through a 55° arc of abduction motion generating compressive and shear forces across the glenosphere. Baseplate micromotion (μm) was recorded throughout 10,000 cycles for each model.p > 0.662) and following 10,000 cycles (p > 0.665), no differences were observed in baseplate micromotion for screw combinations that included one, two, three and four peripheral locked screws. The maximum difference in measured micromotion between the extremes of groups (1-locked and 4-locked) was 29 µm.All constructs survived 10,000 cycles of loading without catastrophic failure. One test construct in the 1-locked fixation group exhibited a measured micromotion >150 μm (177.6 μm). At baseline (Hybrid peripheral screw fixation using combinations of locked and non-locked screws provides secure glenoid baseplate fixation using a polyurethane bone substitute model. Using a glenosphere with a 10-mm lateralized center of rotation, hybrid baseplate fixation maintains micromotion below the necessary threshold for bony ingrowth.N/A/, basic science investigation. Improvements in glenoid baseplate fixation have been critical to the success of the modern reverse shoulder replacement. Recognition of the importance of baseplate tilt –3, optimOne key modification which helped improve baseplate fixation was the introduction of peripheral locked screws. First tested by Harman et al. , locked There are, however, clinical scenarios when a surgeon may prefer to use non-locked peripheral screws. The concept of hybrid fixation, where combinations of locked and non-locked screws are used together, has been a longstanding feature used in locked plate osteosynthesis and has been shown to be biomechanically similar while also providing the potential benefits of compression or aiding with reduction –28. Vari® Model #1522-12, Rigid Cellular Foam; Pacific Research Laboratories, Vashon, WA, USA) with a nominal density of 0.32 g/cm3 (ASTM F1839-08) were utilized in the current investigation. This nominal was chosen as an intermediary between poor quality (0.24 g/cm3) and good quality (0.48 g/cm3) cancellous bone used by others in studies of similar scope [Twenty-eight synthetic trabecular bone surrogate cylinders . The baseplate consists of a single central 6.5-mm lag screw in combination with four peripheral screw holes, which can be used in locked or non-locked fashion. The underside of the baseplate is coated with hydroxyapatite to facilitate bony ingrowth in vivo. All baseplates were fixed perpendicular to the bone block substitutes. Briefly, a 2.5-mm drill was used to define the trajectory for the baseplate’s central screw. A 6.5-mm tap was used to create the threads for the central screw. With the tap still in place, a reamer was used to create the circumferential concavity required to accommodate the underside of the baseplate, which was then inserted and tightened to a maximum torque of 3.5 N-m . For this study, we evaluated baseplate fixation using four combinations of locked (5-mm diameter) and (3.5-mm diameter) non-locked peripheral screws in seven (n = 7) cylinders per fixation group—(1) 1 locked screw + 3 non-locked screws (1-locked), (2) 2 locked screws + 2 non-locked screws (2-locked), (3) 3 locked screws + 1 non-locked screw (3-locked), and (4) 4 locked screws (4-locked). All locked and non-locked screws were the same length as determined by a retrospective analysis of a consecutive series of 100 reverse shoulder arthroplasty surgeries using the same glenoid baseplate. This identified an average peripheral screw length of 21 mm. Thus, 22-mm screws were selected for all locked and non-locked screws.This study used the glenoid components of a single reverse shoulder arthroplasty system   with 3 μm accuracy, was mounted to the instrumented surface of the Sawbones® in the inferior/superior direction with a custom-designed attachment rig such that the sensor was in contact with the periphery of the baseplate immediately adjacent to the superior screw hole  in a constant perpendicular orientation to the baseplate. For non-locked and locked peripheral screws, respectively, 2.5 and 4-mm drills were used to define the screw trajectory in the cylinder. On the circular surface of the baseplate-instrumented Sawbones® cylinder was mounted in a swing arm attached to the torque motor of a servoelectric test frame . Test specimens were cycled through a 55° arc of motion at 1 Hz, while the test frame’s actuator applied a constant 750 N compressive force via a glenosphere size-matched humeral component polyethylene neutral liner, in similar fashion to the methods adopted by previous authors  and inter-group (fixation) comparisons and post hoc comparisons were performed with Bonferroni correction at a significance level of 0.05. The number of test constructs in which micromotion was measured >150 μm threshold commonly accepted for osseous integration was reported.All 28 instrumented cylinders survived 10,000 cycles of loading without catastrophic failure. No gross evidence of baseplate subsidence into the test blocks was noted in any specimen. One test construct in the 1-locked fixation group exhibited a measured micromotion >150 μm (177.6 μm) (Table p > 0.662)  from the 1-locked , 2-locked , or 3-locked fixation groups   Fig. a. After 2 Fig. a.Critical to glenoid component survivability of the reverse shoulder replacement is obtaining sufficient initial fixation on the glenoid bone to facilitate osseous integration , 6, 11. Use of a glenosphere implant with a more lateral center of rotation was met with controversy based on historical implant failures seen with earlier generation implants –21. The p > 0.662) and following 10,000 cycles (p > 0.665) using a bone substrate model that simulates poor glenoid bone quality often seen in reverse shoulder arthroplasty patients [Our study results demonstrate that a variety of hybrid peripheral screw combinations can provide sufficient glenoid component fixation using a glenosphere with a more lateral center of rotation. No statistical differences were observed in baseplate micromotion for peripheral screw combinations that included one, two, three and four peripheral locked screws. This was true at initial fixation  hybrid fixation is a reasonable option for surgeons to consider when placing peripheral screws around a central screw baseplate and that (2) maximizing the number of peripheral locked screws, when clinically feasible, may more consistently maintain baseplate fixation through the postoperative rehabilitation course. Furthermore, use of a glenosphere with a center of rotation 10 mm lateral to the glenoid can be utilized with a glenoid baseplate secured with a combination of locked and non-locked screws without compromising the fixation necessary for ingrowth.Several steps were taken to help maintain uniformity of the tested constructs and strengthen the validity of the data. Non-locked screws have the potential to lose fixation during implantation by stripping out. A pilot study was thus performed to evaluate the torque at which peripheral non-locked screws stripped (documented as a sharp drop in measured torque) within the bone model and lost fixation. All of the peripheral non-locked screws were then implanted within the torque limits observed, ensuring that each screw placed provided similar fixation. Additionally, the orientation of the baseplate was controlled such that the screw orientation remained constant, preventing variability in rigidity based on the orientation of the locked and non-locked screws. Finally, the central screw baseplates were implanted with an identical maximum torque to ensure that uniform compression was achieved with each tested model.The cyclic loading protocol in this study mimics the stresses that might be observed in vivo and was similar to the technique utilized in our previous work  and by RLimitations of this study relate to the use of polyurethane foam. While efforts were made to simulate the clinical environment, including placement of screws within torque limits, variations in patient bone quality may have different effects on hybrid peripheral screw baseplate fixation strength. Additionally, the screws placed were all within the foam model and did not obtain bi-cortical purchase that may occur in vivo. However, even without bi-cortical purchase, all but one non-locked baseplate configurations were able to limit micromotion <150 µm through 10,000 cycles. Finally, this study represents the initial fixation obtained by the glenoid baseplate. With the limitations of postoperative rehabilitation protocols, osseous integration of the glenoid baseplate may occur well before the 10,000 cycles tested in this study. Further investigation of the clinical outcomes using hybrid peripheral screw fixation of a central screw baseplate are warranted to validate the findings of this study.In summary, hybrid peripheral screw fixation using combinations of locked and non-locked screws provides secure glenoid baseplate fixation using a polyurethane bone substitute model. Glenospheres with a more lateral center of rotation can be utilized together with a hybrid combination of peripheral glenoid baseplate screws without compromising fixation necessary for ingrowth of bone into the implant.


Contrasting regional changes in Southern Ocean sea ice have occurred over the last 30 years with distinct regional effects on ecosystem structure and function. Quantifying how Antarctic predators respond to such changes provides the context for predicting how climate variability/change will affect these assemblages into the future. Over an 11-year time-series, we examine how inter-annual variability in sea ice concentration and advance affect the foraging behaviour of a top Antarctic predator, the southern elephant seal. Females foraged longer in pack ice in years with greatest sea ice concentration and earliest sea ice advance, while males foraged longer in polynyas in years of lowest sea ice concentration. There was a positive relationship between near-surface meridional wind anomalies and female foraging effort, but not for males. This study reveals the complexities of foraging responses to climate forcing by a poleward migratory predator through varying sea ice property and dynamic anomalies. Mirounga leonina; SESs) according to regional variability in sea ice and wind patterns across East Antarctica.Over the last 30 years, Earth’s polar regions have experienced significant changes in their sea ice coverage, with predictions of accelerated future change in the coming century2SESs are deep-diving, wide-ranging predators1719202122232426272830313233Around much of Antarctica, variability in sea ice concentration and the timing of annual sea ice advance (and retreat) is linked with variability in wind patterns as they affect both sea ice dynamic and thermodynamic processes. While cold southerly winds tend to drive enhanced equatorward ice advance and (depending on the season) increase the ice concentration, warmer northerly winds can compact the sea ice353637383935404138In this study, we examine an 11-year time-series 2004–2014) of SES movements and diving behaviour to quantify how wind variability and the associated sea ice variability, both forced by large-scale climate variability, affect top predator foraging activity through abiotic and biotic mechanisms. Previous work on this dataset has shown that adult females prefer to forage in high sea ice concentration regions, close to the sea ice edge in the pack ice, while juvenile males remain deep within the sea ice to forage mainly over the Antarctic shelf or within the Antarctic Slope Front and in low sea ice concentration regions (presumably polynya areas)04–2014 o2123Winter post-moult foraging trips of 43 SESs  from Kerguelen Islands to the seasonal Antarctic sea ice zone were monitored using satellite-relayed position and diving data from 2004 to 2014 . Previou19The tracked seals spanned a large region longitudinally ranging from 0 to 150°E , which c437The The mean sea ice concentration and day of sea ice advance exhibit large inter-annual variability across East Antarcticap-value ≈ 0), confirming the significance of negative sea ice concentration anomalies in influencing male hunting times. Hunting times were 1.9 min/dive longer  but the impact of earlier sea ice advance for males was relatively low.For males, hunting times were on average 4.6 min/dive longer  when sea ice concentration was lower (negative) than when positive sea ice concentration anomalies were observed ; Fig. 3ai.e. ~3 times lower than the test condition. While the difference between sea ice concentration anomalies was significant , the impact of sea ice concentration anomalies on female hunting times was less important than for males. However, the effect of earlier sea ice advance on female hunting times was more marked, with their foraging time increasing by ~5.3 min/dive, i.e. 41% of the median hunting time .In contrast to males, positive sea ice concentration anomalies were associated with longer hunting times for females  controlled by local near-surface winds35363839t-value = 3.5, p-value = 0.001) between the near-surface meridional wind anomalies and female hunting times, but not for male hunting times  was used to investigate the relationship between wind anomalies and seal foraging activity. We found that there was a positive relationship  were observed for years of increased sea ice concentrations and earlier sea ice advance. We hypothesize that the early development and advance of sea ice in autumn would enhance primary production within the ice54826282530313233Female SESs demonstrate a more than 40% increase in their hunting times when foraging in years of earlier sea ice advance. This was associated with increased dive duration but slightly fewer dives per day . This reThe linkages between sea ice and animal foraging activity are complex and dependent upon the regional setting and the spatio-temporal variability of the sea ice cover1325In contrast to females, male hunting times increased in years with lowest sea ice concentration, and the timing of sea ice advance had a weak effect on their foraging activity. In previous studies, we showed that males remain deeper in the sea ice zone and are able to forage on the Antarctic shelf and slope front region probably due to the presence of recurrent and persistent coastal polynyas and leads20Lobodon carcinophagus)Both wind-driven dynamics and thermodynamic processes have played an important role in determining the regional complexity and variability of sea ice changes since 1979527st century, becoming less katabatic in nature which may effect the “sea icescape”, prey availability and access for air breathing predators through the persistence and timing of polynya opening, sea ice expansion and thinning. Although highly speculative, it is interesting to put these predicted changes in the context of the results presented in this study. Weakening of katabatic winds probably inducing later sea ice advance and decreased sea ice concentration might affect predator foraging success since Antarctic ecosystems are not only adapted to sea ice presence but also to its seasonal rhythms and propertiesWe observed in the present study that both local anomalies of sea ice concentration and advance are (at least partly) controlled by local near-surface winds. Reference Our study describes for the first time the significant combined effects of the inter-annual variability of near-surface winds as they affect sea ice coverage on the foraging activity of a predator (based upon an 11-year time series). It has also proposed mechanisms by which climate forcing affects both abiotic and biotic components of the Antarctic marine ecosystem. Understanding responses to environmental change is particularly important in the case of predators, which play crucial roles in regulating ecosystemsWe use location and dive depth data from 43 post-moulting SESs  that were instrumented with CTD-SRDLs  between December and February in 2004, 2008–2009 and 2011–2014 on the Kerguelen Islands  . These a−1 was calculated. This time was termed hunting time per dive and was used as a proxy for foraging activity.Foraging activity of each seal was analysed at the dive scale using the methodology developed by ref. SSM/IS daily sea ice concentration (resolution 25 km) provided a continuous time-series for the years of the study. The mean seasonal cycle of sea ice concentration was produced by averaging daily maps corresponding to the same day of year, over the 11 years of the study. Once the seasonal cycle was computed from this time series, we then removed this signal from the time series of sea ice concentration, to create an anomaly from the local seasonal cycle.i.e. independent of collocated sea ice concentration anomalies. We then compared the distribution of the 10,000 differences in hunting time from the 10,000 random pairs of groups, to the difference of hunting time from the two groups based on sea ice concentration anomalies. To take into account individual variability, we computed two more tests, presented in the In order to test relationships between daily sea ice concentration anomalies and seal hunting times, we grouped all seal hunting times corresponding to the location of anomalously negative and positive sea ice concentrations . For this calculation, we only considered seals inside the sea ice region  and from March onward, as previously defined. We then compared the two distributions of hunting times using a permutation test Surface wind anomalies).Finally, SSMI/S monthly sea ice concentration (resolution 25 km) were used to compute monthly sea ice concentration anomalies to perform the correlation with monthly wind anomalies (see section http://nsidc.org/data/nsidc-0051.html) with a resolution of 25 km. Following ref. The day at which sea ice advances in the season (hereafter referred to as sea ice advance) was derived following ref. The goal of computing sea ice advance anomalies was to determine any possible influence of relatively early or late sea ice advance on seal foraging behaviour. The period during which seal hunting behaviour is likely to be affected by an earlier or later sea ice advance would be around the time of year at which sea ice usually advances. We therefore only selected seals’ positions during the seasonal advance of sea ice from March to Junehttp://apps.ecmwf.int/datasets/) with a spatial resolution of approximately 80 km. We computed meridional wind anomalies  by removing the local climatological seasonal cycle computed over the 11 years of the study. The relationship between monthly meridional wind anomalies and monthly sea ice concentration anomalies was likely to be non-linear, so the correlation for each longitude/latitude pixel over the 11 year time period was performed using a Spearman correlation. For both variables, the periodic inter-annual variability was taken into account and the first trend in the anomalies was removed prior to correlation. The relationship between monthly meridional wind anomalies and sea ice advance anomalies was processed in three steps: (i) for each 5° longitude bin, monthly ERA-Interim 10 m wind anomalies were averaged within the minimum and maximum latitude band of average day of advance from 2004 to 2014; then (ii) the resulting averaged winds per month for each 5° longitude were averaged from March to June to obtain one-yearly data per bin of longitude; and finally (iii) a Spearman correlation between sea ice advance anomalies and averaged wind anomalies for each 5° bin of longitude was computed.Surface zonal and meridional winds were extracted from monthly ERA-Interim 10 m atmospheric reanalysis  using restricted maximum likelihood. Outliers in the variables were checked. Sex was included in the model as an interaction factor variable. We first determined the optimal structure by assessing if individual seals as a random intercept term and if monthly 10 m wind  as a random slope term contributed to the model fit. The final model was then fitted using restricted maximum likelihood (REML). Model validation was checked by plotting Pearson residuals against fitted values, and against the explanatory variable, to verify homogeneity and normality of residualsMuMIn .A Gaussian additive mixed effects model (GAMM) was first fitted to examine the statistical relationships between seal foraging activity (expressed by the hunting time per dive) and the 10 m wind anomaly meridional component data. Because the estimated relationship with a GAMM was linear, we then fitted a linear mixed effects model (LMM). Monthly ERA-Interim 10 m wind anomalies were collocated at each seal position and time. A subset of the data was extracted to only focus on parts of the tracks influenced by sea ice; for this, only positions inside the sea ice and from March .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


ATM kinase is a master regulator of the DNA damage response (DDR). A recently published report from the d’Adda di Fagagna laboratory ATM belongs to PI3K-like kinase family that consists of ATR, DNA-PKcs, mTOR, TRRAP and SMG1 kinases, which share similar structures and ability to phosphorylate their substrates on Serine and Threonine residuesUpon recruitment to DSBs, ATM dimer undergoes a complicated process of activation. First, it is acetylated by lysine acetyltransferase 5 (KAT5/TIP60) at Lysine 3016In recent work, Adamowicz et al., show that ATM localizes to the DSBs in a protein complex together with FOXO3a and KAT5/Tip60 (from now on referred to as AAC (ATM Activation Complex)). Formation of this complex is dependent on FOXO3a bridging an interaction between ATM and KAT5 . Formatiin vitro kinase assay ATM S1981A mutant can phosphorylate CHK2 and p53 in response to linear dsDNA to the same extent as the wild-type ATMin vitroin vitro ATM kinase assay in the presence of NOTCH1 and mutated ATM (S1981A).ATM phosphorylation on Serine 1981 is commonly used as a marker for activated ATM. ATM phosphorylation was first observed almost 20 years agoIt has been suggested that ATM autophosphorylation is necessary for ATM retention at the DSBsXenopus laevis egg extract that NOTCH1 blocked a substantial amount of ATM-mediated phosphorylationWe have repeatedly observed that NOTCH1 inhibits ATM activation by blocking its autophosphorylation (Ser1981) and phosphorylation of downstream substrates such as KAP1, SMC1, p53, DNA-PKcs or CHK2It has been suggested that there is redundancy between PI3K-like kinases in terms of H2AX phosphorylation. DNA-PKcs or ATR kinases have been shown to phosphorylate H2AXThese results suggest that H2AX can be phosphorylated by protein kinases other than ATM, ATR and DNA-PKcs. Indeed, it has been reported that JNK and p38 can phosphorylate H2AX in response of UV light irradiation or starvation respectivelyBecause NOTCH1, unlike small molecule ATM inhibitors, cannot directly inhibit ATM kinase activity we can speculate that NOTCH1 binding to ATM could strongly impair ATM substrate recognition, resulting in an inhibition of phosphorylation of some substrates such as p53 or CHK2, but not H2AX. It has been already reported that NOTCH1 can bind and hence modulate the substrate recognition of LSD1 demethylaseNOTCH1 was very early connected to the tumorogenesis and marked as an oncogene due to its ability to induce tumour growthNeural stem cells (NSC) are known to express moderate levels of activated NOTCH1, which is necessary for their proliferationIn summary, I would like to propose that the physiological role of NOTCH1 is not to inhibit fully ATM activation, but rather to induce its mild impairment, to modulate a balance between the amount of DNA damage and DDR signalling. This would result in the suppression of DNA damage induced apoptosis or differentiation, giving time for necessary repair.Formation of AAC is necessary for ATM activation at DSBs and DDRWe tend to think about ATM through its role in DDR regulation, although ATM has been described to be involved in many more cellular processes like: stress responseIt is thought-provoking to picture ATM in different complexes that differentially regulate its activity and substrate recognition. The identification of different active ATM complexes opens new and exciting areas of research and raises even more fascinating questions. For example, how is ATM activation in those complexes stimulated, and how is ATM substrate recognition and kinase activity regulated? Hopefully, in the near future we will know the answers.


Most previous phosphorylation studies were conducted using a range of phosphorylation site-specific antibodies. These studies describe relative changes of different phosphorylation sites, however, it is hard to estimate total, absolute and collective changes in phosphorylation. To overcome these problems, we have recently applied the Phos-Tag technique to the analysis of tau phosphorylation in vitro and in vivo. This method separates tau into many bands during SDS-PAGE depending on its phosphorylation states, creating a bar code appearance. We propose calling this banding pattern of tau the “phospho-tau bar code.” In this review article, we describe what is newly discovered regarding tau phosphorylation through the use of the Phos-Tag. We would like to propose its use for the postmortem diagnosis of tauopathy which is presently done by immunostaining diseased brains with anti-phospho-antibodies. While Phos-tag SDS-PAGE, like other biochemical assays, will lose morphological information, it could provide other types of valuable information such as disease-specific phosphorylation.Tau is a microtubule-associated protein which regulates the assembly and stability of microtubules in the axons of neurons. Tau is also a major component of neurofibrillary tangles (NFTs), a pathological hallmark in Alzheimer's disease (AD). A characteristic of AD tau is hyperphosphorylation with more than 40 phosphorylation sites. Aggregates of hyperphosphorylated tau are also found in other neurodegenerative diseases which are collectively called tauopathies. Although a large number of studies have been performed on the phosphorylation of AD tau, it is not known if there is disease-specific phosphorylation among tauopathies. This is due to the lack of a proper method for analyzing tau phosphorylation  MAPT indicates that tau may be a causative factor for other tauopathies in addition to FTDP-17 -associated protein (MAP) predominantly expressed in the axons of neurons   Figure . While tPhosphorylation is the balance of protein kinase and protein phosphatase activity. Several tau kinases such as PKA, CaMKII, PKC, and MAPKs are transiently activated by external or internal stimuli, and their target sites should also be phosphorylated only transiently in healthy neurons and brains. In contrast, several Ser/Thr-Pro {(S/T)P} phosphorylation sites in tau are constitutively phosphorylated which suggests that they are phosphorylated by house-keeping kinase–protein kinases that are active in resting cells  toxicity;  site, (S/T)xx(x)p(S/T), by a priming kinase. There are 25 such sequences in tau, and 12 sites are indeed phosphorylated, even though not all of them have the primary phosphorylation Figure . This diin vivo phosphorylation of tau at Ser202 properly with that antibody.The third complication in tau phosphorylation is problems in the specificity or reactivity of anti-phospho-specific antibodies. Some phospho-specific antibodies are generated against a phosphopeptide with a single phosphorylation at a site of interest. However, several phosphorylation sites in tau are located very close together and, in extreme cases, are right next to each other. The reactivity of the phospho-specific antibody would be affected by any other phosphorylated sites nearby. We recently encountered such a case where the reactivity of anti-pSer202 varied depending of phosphorylation at Thr205  If it is known that the protein is phosphorylated, then a phosphorylation site(s) can be readily determined by the loss of the upward shift with a nonphosphorylation Ala mutant at candidate sites. We have newly identified the phosphorylation sites of several proteins, ataxin 2, drebrin and GRAB, by applying this method  The relative ratio of each phosphoisotype can be measured as a percent ratio of the total by densitometric scanning of the blots. We have determined the ratio of phosphoisotypes of p35 Cdk5 activator in vivo and found it changes with brain development  The total phosphorylation profile of the particular protein is seen at a glance , AT180 (Thr231 and Ser235) and PHF1 (Ser396 and Ser404) Figure . Since eTauopathy neurodegenerative disorders including AD are characterized by intracellular inclusions composed of hyperphosphorylated tau. Immunocytochemical detection of tau inclusions with phospho-specific tau antibodies is usually used as a postmortem diagnosis of diseases. The different staining patterns depending on disease type suggest disease-specific phosphorylation but it is not yet clear. It is desirable to use a method which examines the total phosphorylation profiles of tau simply. We recently applied the Phos-tag technique to analyze tau phosphorylation in mouse and human brains, and found that tau is separated into many bands, resembling a bar code of many different phosphoisotypes. Here, we name such a banding pattern of tau on Phos-tag SDS-PAGE the “phospho-tau bar code” and propose its use for the diagnosis of tauopathies.TK and SH wrote the whole manuscript. GS and KI made figures and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Metformin (500 mg/kg/day) was administered via gastric gavage for three weeks during the pregnancy period. Combined maternal HFR plus post-weaning HFA induced hypertension in male adult offspring, which prenatal metformin therapy prevented. The protective effects of prenatal metformin therapy on HFR/HFA-induced hypertension, including downregulation of the renin-angiotensin system, decrease in uric acid level, and reduction of oxidative stress. Our results highlighted that the programming effects of metformin administered prenatally might be different from those reported in adults, and that deserves further elucidation.Widespread consumption of a Western diet, comprised of highly refined carbohydrates and fat, may play a role in the epidemic of hypertension. Hypertension can take origin from early life. Metformin is the preferred treatment for type 2 diabetes. We examined whether prenatal metformin therapy can prevent maternal high-fructose plus post-weaning high-fat diets-induced hypertension of developmental origins via regulation of nutrient sensing signals, uric acid, oxidative stress, and the nitric oxide (NO) pathway. Gestating Sprague–Dawley rats received regular chow (ND) or chow supplemented with 60% fructose diet (HFR) throughout pregnancy and lactation. Male offspring were onto either the ND or high-fat diet (HFA) from weaning to 12 weeks of age. A total of 40 male offspring were assigned to five groups ( Metformin  is the favorite first-line oral blood glucose-lowering treatment for type 2 diabetes . CurrentAdulthood hypertension can take origin from early-life insults, referred to the developmental origins of health and disease (DOHaD) . The WesSince BP is regulated by a complex process that includes major contributions from the kidney, and that developing kidney is vulnerable to adverse early-life insults, several mechanisms occurring in the kidney have been proposed to interpret HFR diet-induced renal programming and hypertension. These mechanisms include nutrient sensing signals, uric acid, oxidative stress, and nitric oxide (NO) pathway . MaternaMetformin and ADMA are structural analogs; they have opposite effects at multiple signaling pathways . AdditioMaternal high-fructose diet had no effect on litter sizes . HFD and metformin did not affect the survival of male pups . The ND/As shown in Sirt1, Prkag2 (encoding for AMPKγ2), and Ppargc1a (encoding for PGC-1α) in HFR/HFA rats were lower, while Prkaa2 (encoding for AMPKα2) and Ppara (encoding for PPARα) were higher than those in ND/ND rats. HFR/HFA+M group had the lowest mRNA expression of Sirt4, Prkaa2, Prkag2, Ppara, Pparb (encoding for PPARβ), Pparg (encoding for PPARγ), and Ppargc1a among the five groups. Consistent with the change in mRNA level, We first analyzed genes involved in the nutrient sensing pathway. As shown in We next assessed oxidative stress by immunostaining of 8-OHdG. 8-OHdG is a metabolite of oxidative damage to leukocyte DNA, which has been identified as a marker of oxidative stress. Both cytoplasmic and nuclear staining for 8-OHdG in the glomeruli and renal tubules showed little staining in the ND/ND (10 ± 3 positive cells) and HFR/HFA+M (19 ± 10 positive cells) groups, an intermediate level of staining in the HFR/ND (90 ± 19 positive cells) and ND/HFA (95 ± 28 positive cells) groups, and intense staining in the HFR/HFA group (205 ± 32 positive cells) .l-arginine levels and l-arginine-to-ADMA ratios in ND/HFA and HFR/HFA rats. HFR/HFA+M group had the lowest plasma l-citrulline and l-arginine levels and l-arginine-to-ADMA ratios among the five groups. In contrast, plasma levels of ADMA and SDMA were highest in HFR/HFA+M group.Since the interplay between oxidative stress and ADMA-NO pathway involved in the pathogenesis of programmed hypertension ,20, we hRen, Atp6ap2 (encoded for prorenin receptor), Agt, and Agtr1a (encoded for angiotensin II type I receptor) were higher in ND/HFA group than in those of ND/ND group  combined maternal high-fructose plus post-weaning high-fat diets induced hypertension in adult male offspring, which prenatal metformin therapy prevented; (2) HFR/HFA induced hypertension relates to alterations in the nutrient sensing signals, uric acid, oxidative stress, NO pathway, and RAS; (3) in HFR/HFA-induced programmed hypertension, downregulation of the RAS, decreases in uric acid level, and reduction of oxidative stress were mediated by prenatal metformin, to prevent the development of hypertension.Despite clear cardiovascular benefits , limitedSo far, some particular mechanisms contributing to protective effects of metformin have been suggested, such as AMPK activation and its antioxidant property ,2,22. Wel-arginine-to-ADMA ratios. Given that both ADMA and SDMA are inhibitors of NO synthase [l-arginine-to-ADMA ratios represent NO bioavailability, signals formed by NO pathway are supposed to cause vasoconstriction instead of the expected vasodilatation. Thus, our findings suggest that prenatal and postnatal administration of metformin might have opposite effects on nutrient sensing signals and NO pathway, although this remains speculative.Another reported protective effect of metformin is mediating NO system ,2. Our psynthase , and theRen at 1 day of age, is relevant to programmed hypertension in a maternal HFR-induced hypertension model [Ren, Atp6ap2, Agt, and Agtr1, which promote vasoconstriction and rise BP. Consistent with previous reports showing that cardiovascular protection of metformin is through blockade of the RAS [In this work, prevention of programmed hypertension with prenatal metformin therapy in HFR/HFA offspring might be, at least in part, due to blocking the RAS, decreasing uric acid, and reducing oxidative stress. The activation of the RAS, and production of uric acid and oxidative stress, play a key role in the pathogenesis of fructose-induced hypertension ,15. Firson model . This da the RAS , we obse the RAS ,32. This the RAS . Last, o the RAS ,19,20. WOur study has a few limitations. First, the anti-hypertensive effects of prenatal metformin therapy may be derived from other organs that control BP. Additional studies are required to examine the effects of prenatal metformin exposure on other organs responsible for BP regulation. Also, the beneficial effects of prenatal metformin therapy in other hypertensive models of developmental origins await further evaluation. Another limitation is that we did not conduct a ND/ND+M group because metformin is currently used during pregnancy with a good safety profile . Neverthn = 4) or chow supplemented with 60% fructose  throughout pregnancy and lactation periods [n = 8 for each group): ND/ND, HFR/ND, ND/HFA, HFR/HFA, and HFR/HFA+M. Male offspring rats were administered either a normal diet with regular chow (ND) or a high-fat hypercaloric diet  plus high sucrose (25% carbohydrate)) from weaning to 3 months of age. In addition to HFR/HFA diets, mother rats in the HFR/HFA+M group received metformin 500 mg/kg/day via gastric gavage for 3 weeks during pregnancy period. The dose of metformin used here was based on our previous study conducted with rats [The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Kaohsiung Chang Gung Memorial Hospital and are in close agreement with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health . Virgin Sprague–Dawley (SD) rats (12–16 weeks old) were obtained from BioLASCO Taiwan Co., Ltd. . Animals were maintained in an AAALAC-accredited animal facility, housed in a 12-h light-dark cycle. Male SD rats were housed with individual females until mating was confirmed by the examination of a vaginal plug. Pregnant SD rats received regular chow  according to methods described previously . The lev reagent . StandarSirt1), sirtuin-4 (Sirt4), protein kinase, AMP-activated, subunit-α2 (Prkaa2), -β2 (Prkab2), and -γ2 (Prkag2), peroxisome proliferator-activated receptor (PPAR)-α (Ppara), -β (Pparb), and -γ (Pparg), and PPARγ coactivator 1-α (encoded by Ppargc1a). Additionally, several components of the RAS analyzed in this study included renin (Ren), (pro)renin receptor (Atp6ap2), angiotensinogen (Agt), angiotensin converting enzyme-1 (Ace), and angiotensin II type I receptor (Agtr1a). We used 18S rRNA gene (Rn18s) as a reference gene. Primer sequences are provided in Ct) values were used for all manipulations and were first normalized to average Rn18s value by calculating the ΔCt for each sample. Values were then calculated relative to control to generate a ΔΔCt value. The fold-increase of the experimental sample relative to the control was calculated using the formula 2Ct−ΔΔ.RNA was extracted from the kidney cortex according to previously described methods . SeveralSamples were subjected to electrophoresis, western blot, and antibodies icubation using the methods published previously . BrieflyParaffin-embedded tissue sectioned at 3-µm thickness was deparaffinized in xylene and rehydrated in a graded ethanol series to phosphate-buffered saline. 8-Hydroxydeoxyguanosine (8-OHdG) is a DNA oxidation product that was measured to assess DNA damage. Following blocking with immunoblock , the sections were incubated for 2 h at room temperature with an anti-8-hydroxydeoxyguanosine (8-OHdG) antibody . Immunoreactivity was revealed using the polymer-horseradish peroxidase (HRP) labelling kit (BIOTnA Biotech) and 3,3’-diaminobenzidine (DAB) as the chromogen. A negative control of identical staining omitting incubation with a primary antibody was used. 8-OHdG-positive cells in high-power fields (400×) in the renal sections were quantitatively analyzed as previously described .p-value of 0.05 was accepted as statistical significance. All analyses were performed using the Statistical Package for the Social Sciences software .Values given in figures and tables represent mean ± standard error of the mean. Statistical significance was determined using one-way ANOVA with Tukey post hoc test for multiple comparisons. Significance of BP data were determined using two-way repeated-measures ANOVA and Tukey post hoc test. In all cases, a In conclusion, prenatal metformin therapy reduced BP in adult male offspring rats that was induced by maternal high-fructose and post-weaning high-fat consumption. Some important mechanisms involved in the beneficial action of prenatal metformin therapy on HFR/HFA-treated offspring kidneys, including blockade of the RAS, reduction of oxidative stress, and decreases in uric acid level. Unlike common mechanisms of metformin reported in adults ,2, our s


Dysregulated transforming growth factor β (TGFβ) signaling is implicated in osteoarthritis development, making normalizing TGFβ signaling a possible therapy. Theoretically, this can be achieved with small molecule inhibitors specifically targeting the various TGFβ receptors and downstream mediators. In this study we explore in primary chondrocytes the use of small molecule inhibitors to target TGFβ-induced pSmad1/5/9-, pSmad2/3- and TGFβ-activated kinase 1 (TAK1)-dependent signaling.Primary bovine chondrocytes and explants were isolated from metacarpophalangeal joints. To modulate TGFβ signaling the activin receptor-like kinase (ALK)1/2/3/6 inhibitor LDN-193189, the ALK4/5/7 inhibitor SB-505124 and the TAK1 inhibitor (5Z)-7-Oxozeaenol were used. pSmad1/5 and pSmad2 were measured using western blot analysis and TGFβ1-induced gene expression was measured using quantitative real time PCR (qPCR).In chondrocytes, TGFβ1 strongly induced both pSmad1/5 and pSmad2. Remarkably, LDN-193189 did not inhibit TGFβ-induced pSmad1/5. In contrast, SB-505124 did inhibit both TGFβ-induced Smad2 and Smad1/5 phosphorylation. Furthermore, (5Z)-7-Oxozeaenol also profoundly inhibited TGFβ-induced pSmad2 and pSmad1/5. Importantly, both SB-505124 and (5Z)-7-Oxozeaenol did not significantly inhibit constitutively active ALK1, making an off-target effect unlikely. Additionally, LDN-193189 was able to potently inhibit BMP2/7/9-induced pSmad1/5, showing its functionality. On gene expression, LDN-193189 did not affect TGFβ1-induced regulation, whereas both SB-505124 and (5Z)-7-Oxozeaenol did. Similar results were obtained in cartilage explants, although pSmad1/5 was not strongly induced by addition of TGFβ1.Our data suggest that ALK5 kinase activity plays a central role in both TGFβ-induced Smad1/5 and Smad2/3 phosphorylation, making it difficult to separate both pathways with the use of currently available small molecule inhibitors. Furthermore, our data regarding (5Z)-7-Oxozeaenol suggest that TAK1 facilitates Smad-dependent signaling.The online version of this article (doi:10.1186/s13075-017-1302-4) contains supplementary material, which is available to authorized users. Transforming growth factor β (TGFβ) plays a crucial role in regulation of tissue homeostasis via its control over diverse cellular processes such as proliferation, differentiation and matrix formation. By binding to different receptors of the activin-receptor like kinase (ALK) family, TGFβ induces intracellular carboxy-terminal  phosphorylation of receptor-regulated Smad (R-Smad) proteins. Phosphorylated R-Smads form complexes with the common-mediator Smad; Smad4, and these complexes translocate to the nucleus where they bind DNA to regulate gene transcription via recruitment of transcription factors , 2. Use In many tissues, e.g. in cartilage, phosphorylated Smad2/3 and Smad1/5 have opposing functions , 7. In cInhibiting Smad phosphorylation can be achieved by blocking the kinase domain of ALK receptors. Small molecules have been developed that specifically inhibit the kinase activity of ALK4, ALK5 and ALK7, the receptors that phosphorylate Smad2 and Smad3, or ALK1, ALK2, ALK3 and ALK6, the receptors that phosphorylate Smad1, Smad5 and Smad9 , 15. In In this study we show that inhibition of ALK4/5/7 kinase activity prevented TGFβ1-induced C-terminal phosphorylation of both Smad2 and Smad1/5. Furthermore, treatment with (5Z)-7-Oxozeaenol, a selective TAK1 kinase inhibitor, attenuated phosphorylation of both Smad2/3 and Smad1/5 and expression of Smad target genes. Use of the ALK1/2/3 inhibitor LDN-193189 did not affect TGFβ1-induced R-Smad phosphorylation or gene expression. In conclusion, our data show that in cartilage the activity of the ALK5 kinase domain is essential for TGFβ1 signaling, whereas ALK1 kinase activity is not. Furthermore, our results obtained with (5Z)-7-Oxozeaenol underline that TAK1 kinase activity is facilitating R-Smad phosphorylation. Our study indicates that TGFβ1-induced Smad1/5 and Smad2/3 phosphorylation cannot be selectively targeted by using the small molecule inhibitors SB-505124 and LDN-193189.5 cells per cm2 in 6-well plates  for protein studies, or in 24-well plates  for mRNA experiments. Cells were cultured for 1 week at 37 °C and 5% CO2 before the start of the experiments. For explant studies, four 7-mm2 explants per condition were pooled in 1 ml of medium. Both monolayer and explant studies were repeated in different donors.Primary bovine chondrocytes were isolated from the metacarpophalangeal joint of cows (2–5 years old) obtained from a slaughterhouse within 3 hours post mortem. Chondrocytes were obtained by incubating cartilage slices overnight in Collagenase B (for details see ). Chondr12-luciferace reporter construct [Adenoviruses were used to induce expression of constitutively active (ca)ALK1 , constitonstruct  in prima50), 0.8 nM for ALK1, but far below its IC50 for ALK5 of 350 nM [Before stimulation, cells were deprived of serum for 24 h and thereafter stimulated with 1 ng/ml recombinant human TGFβ1  for 2 or 24 h. In experiments where inhibitors were used, DMSO was used as vehicle control. To block TAK1 activity, we used (5Z)-7-Oxozeaenol   containing a protease inhibitor cocktail  and subsequently sonicated on ice using a Bioruptor . With a BCA-assay  protein concentration was measured and after addition of Laemmli sample buffer, samples were boiled for 5 minutes at 99 °C.2 explants were homogenized using a Mikro-dismembrator  and dissolved in 1 ml ice cold radioimmunoprecipitation assay (RIPA) buffer with added 1 mM Na3VO4 and protease inhibitor cocktail . After 1 h incubation on a roller bench at 4 °C, samples were spun down for 3 minutes at 104 × g and the pellet was discarded. Cetylpyridinium chloride was added up to a concentration of 1% (m/v) and samples were incubated on a roller bench for 1 h at 4 °C. Hereafter, samples were spun down twice for 15 minutes at 104 × g at 4 °C and the pellet was discarded. Using 10 kDa centrifugal filter units  the supernatant was concentrated to a volume of 50 μl. Subsequently, proteins were precipitated by addition of 950 μl of 20% m/v trichloroacetic acid and 0.1% m/v dithiothreitol in aceton at -20 °C and spinning samples down for 15 minutes at 6700 × g at 4 °C. Next, the pellets were washed three times with 0.1% m/v dithiothreitol in aceton at -20 °C. Finally, the pellets were dried under vacuum for 20 minutes and dissolved in 100 μl 1% m/v sodium dodecyl sulfate in 100 mM tris(hydroxymethyl)aminomethane in H2O pH 9.0.To isolate proteins from explants, four 7-mmProteins were seperated on a 7.5% Bisacrylamide gel, and transferred to a nitrocellulose membrane using wet transfer . After overnight incubation at 4 °C with 1:1000 polyclonal Rabbit anti P-Smad1/5 (S463/465)/Smad8 (S426/428)  or anti P-Smad2 (S465/467) , membranes were incubated with 1:1500 polyclonal Goat anti Rabbit labeled with horseradish peroxidase (HRP)  for 1 h. Hereafter, enhanced chemiluminescence using ECL plus  was used to visualize the proteins. To visualize overexpression of constitutively active ALKs, a rabbit polyclonal antibody directed against their internal HA tag was used: HA-probe Antibody (Y-11)  (1:1000). As loading control either Gapdh was stained with an anti-Gapdh mouse mAb (1G5)  (1:10 000) in combination with IRDye 680RD Donkey anti mouse (1:10 000)  using the Odyssey detection system  or Vinculin with a rabbit pAb (H300)  (1:1000) in combination with HRP-labeled Goat-anti Rabbit (1:2000)  using ECL. Finally, blots were quantified using ImageJ.glyceraldehyde 3-phosphate dehydrogenase and ribosomal protein S14.TRI-reagent  was used for RNA isolation according to the manufacturer’s protocol (for details see ). RNA coAll quantitative data are expressed as a mean of multiple repeats ± SD. For every analysis data were checked for normality using the Shapiro-Wilk test. One-way analysis of variance (ANOVA) with Tukey multiple comparison post-hoc test was used to determine the significance. The statistical analyses were performed using Graphpad Prism 5.0 software.. ALK1, ALK2 and ALK3 [To begin, we confirmed in primary bovine chondrocytes the expression of the receptors that TGFβ can use to induce Smad2/3 phosphorylation i.e. ALK5 or Smad1/5 phosphorylation, i.eand ALK3 . All fouSubsequently, we analyzed the effects of the ALK4/5/7-kinase inhibitor SB-505124, the ALK1/2/3-kinase inhibitor LDN-193189 and the TAK1-kinase inhibitor (5Z)-7-Oxozeaenol on TGFβ1-induced R-Smad phosphorylation Fig. . StrikinTo investigate if the striking effect of SB-505124 on Smad1/5 phosphorylation was a dose-dependent effect, we performed a dose-response experiment Fig. . This exTo investigate if the lack of effect of LDN-193189 on TGFβ1-induced Smad1/5 was maybe dose-dependent, we added a 10 times higher dose of LDN-193189 (0.5 μM) to the cells, but were still unable to inhibit TGFβ1-induced Smad1/5 phosphorylation Fig. . TherefoIn conclusion, in primary chondrocytes, TGFβ1-induced Smad1/5 phosphorylation is unaffected by LDN-193189, whereas both Smad2/3 and Smad1/5 phosphorylation are inhibited by SB-505124 and (5Z)-7-Oxozeaenol.bId1 and bSerpine1 were used as markers of pSmad1/5 and Smad3 signaling, respectively [bId1 was profoundly upregulated by stimulation with 1 ng/ml of TGFβ1 -7-Oxozeaenol, and was unaffected by LDN-193189. Expression of bSerpine1 was upregulated by 1 ng/ml of TGFβ1 and fully inhibited by SB-505124, but was only partly inibited by (5Z)-7-Oxozeaenol -7-Oxozeaenol and SB-505124, showing that pSmad3-signaling is indeed lowered in these conditions. When the effect of the inhibitors on multiple TGFβ1-induced -7-Oxozeaenol, which significantly lowered bNgf, bTgfb1 and bMmp3 expression -7-Oxozeaenol -7-Oxozeaenol inhibited TGFβ1-induced expression of bFn1, bTgfb1, whereas no effect of LDN-193189 was observed. Therefore, SB-505124 also inhibits both TGFβ1-induced Smad1/5 and Smad2 phosphorylation and gene expression in intact cartilage, whereas LDN-193189 does not. Furthermore, (5Z)-7-Oxozeaenol partly inhibited both TGFβ1-induced Smad1/5 and Smad2 phosphorylation.As TGFβ1 signaling is known to be affected by cellular context, we sought to validate our observations in monolayer chondrocytes in intact cartilage ex vivo. Therefore we stimulated cartilage explants with 1 ng/ml TGFβ1 with or without inhibitors, and investigated Smad phosphorylation. Explants were stimulated for 2 h to allow for diffusion of TGFβ1 through the cartilage matrix. In explants, 1 ng/ml TGFβ1 induced pSmad2, but Smad1/5 phosphorylation was more difficult to detect Fig. , in contnol Fig. . In explof TGFβ1 , and furTGFβ1 signaling is a crucial regulator of chondrocyte homeostasis. We investigated the impact of small molecule inhibitors on TGFβ1 signaling in primary chondrocytes both in vitro and ex vivo. We report that inhibition of ALK4/5/7 kinase with SB-505124 lowered both TGFβ1-induced Smad1/5 and Smad2/3 phosphorylation and downstream gene expression, whereas inhibition of ALK1/2/3/6 kinase with LDN-193189 did not affect TGFβ1-induced Smad1/5 phosphorylation or transcriptional activity. In addition, we showed that treatment with (5Z)-7-Oxozeaenol, a selective TAK1 kinase inhibitor, attenuated TGFβ1-induced R-Smad phosphorylation and downstream gene expression.50 of this inhibitor for ALK1 (0.8 nM), ALK2 (0.8 nM) and ALK3 (5.3 nM) [In many tissues, including cartilage, TGFβ1 induces pSmad1/5 and pSmad2/3 , 7. ALK5(5.3 nM) . Additio(5.3 nM) , 23, 33.(5.3 nM) , 25. Fur(5.3 nM) , 35.Fig.Based on structural analyses of R-Smad-ALK interactions, the ability to phosphorylate Smad1/5 is thought to be limited to BMP type I receptors , 37. OurApart from R-Smad phosphorylation, TGFβ1 also induces TAK1 activation, a key component of Smad-independent TGFβ signaling . NoteworLinker modification of R-Smads is an essential aspect of TGFβ1-signaling . ThA limitation of our study is the possibly imperfect specificity of the used compounds, making it possible that off-target effects can explain our observations and interfere with our conclusions. However, in the experiments with the caALKs, we did not observe a significant effect of SB-505124 or (5Z)-7-Oxozeaenol on caALK1-induced pSmad1/5, or an effect of (5Z)-7-Oxozeaenol on caALK5-induced pSmad2. Therefore we do not think that these compounds have off-target effects on ALK1. Furthermore, a dose-response curve showed that SB-505124 inhibited TGFβ-induced Smad1/5 phosphorylation more potently than Smad2/3 phosphorylation, demonstrating that this effect of SB-505124 on TGFβ signaling is not an off-target effect obtained at high dosage, and this compound therefore cannot be used to specifically inhibit TGFβ-induced Smad2 in chondrocytes.Another limitation of our study is that we did not include in vivo data. Unfortunately, the in vivo use of both SB-505124 and LDN-193189 is difficult because of their unfavorable pharmacokinetic properties, resulting in the need for daily re-administation via injection . Up to nIn conclusion, with the use of the small molecule inhibitors SB-505124 and LDN-193189 we were unable to direct TGFβ1-signaling towards either Smad1/5 or Smad2/3 phosphorylation in cartilage. Our data suggest that in cartilage, ALK5 plays a central role in both TGFβ-induced Smad1/5 and Smad2/3 phosphorylation, making it difficult to separate the Smad pathways with the use of currently available intracellular small molecule inhibitors of the ALK receptors. Furthermore, we showed that treatment with the TAK1 inhibitor (5Z)-7-Oxozeaenol inhibits TGFβ-induced C-terminal phosphorylation of both Smad1/5 and Smad2/3, suggesting a link between the non-canonical and the canonical TGFβ pathway in cartilage.Additional file 1: Figure S1.a With the use of qPCR, expression of ALK1, ALK2, ALK3 and ALK5 was measured in both freshly isolated cartilage explants and in primary chondrocytes after 1 week of cell culture in DMEM/F12 supplemented with 10% non-heat-inactivated FCS without passage. All four ALKs were readily detected in both groups, but expression of all the receptors was higher in freshly isolated tissue. For calculations of the -ΔCt, two reference genes were used: bGapdh and bRps14. (PDF 2065 kb)Expression of ALK1, ALK2, ALK3 and ALK5 mRNA in primary bovine cartilage and chondrocytes. Additional file 2: Figure S3.Quantification of TGFβ-induced pSmad2 and pSmad1/5 in cartilage explants. Quantification of the western blot as shown in Fig. Additional file 3: Figure S2.ID1 expression in cartilage explants. Primary chondrocytes were incubated with LDN-193189 for 2 h in a dose of 0.05 μM and bId1 expression was measured using qPCR. LDN-193189 significantly inhibited bId1 expression showing the bioactivity of this compound in cartilage explants. (PDF 2122 kb)LDN-193189 in a concentration of 0.05 μM inhibits basal


Monodon monoceros) echolocation signals were made using a linear 16 hydrophone array in the pack ice of Baffin Bay, West Greenland in 2013 at eleven sites. An average -3 dB beam width of 5.0° makes the narwhal click the most directional biosonar signal reported for any species to date. The beam shows a dorsal-ventral asymmetry with a narrower beam above the beam axis. This may be an evolutionary advantage for toothed whales to reduce echoes from the water surface or sea ice surface. Source level measurements show narwhal click intensities of up to 222 dB pp re 1 μPa, with a mean apparent source level of 215 dB pp re 1 μPa. During ascents and descents the narwhals perform scanning in the vertical plane with their sonar beam. This study provides valuable information for reference sonar parameters of narwhals and for the use of acoustic monitoring in the Arctic.Recordings of narwhal ( Cetacean species in the Arctic ecosystem are subject to broad-scale ecological changes as a result of sea ice loss and climate warming . Arctic The additional burdens from increased anthropogenic activities are very likely to amplify the already negative impacts from Arctic ecosystem change. In particular increases in anthropogenic sound sources will likely interfere with important biological functions for cetaceans such as foraging, migration, communication, and predation avoidance . TherefoNarwhals are one of only two species of toothed whales that inhabit waters above the Arctic Circle (67°N) year round. Narwhals are an important representative species for understanding increasing noise in the Arctic with loss of sea ice and how these items will have potential behavioral and ecological effects. Most of the world’s narwhals inhabit the Baffin Bay/Davis Strait pack ice in winter. They make extensive annual migrations from high Arctic summering grounds in West Greenland and high Arctic Canada to offshore wintering grounds, where >80,000 narwhals (over 80% of the world’s population) occupy dense pack ice between November and April –8. NarwhNarwhals inhabit a complex environment where hearing and processing sounds serve critical biological functions related to communication, foraging, reproduction, navigation and predator-avoidance . Our stust and March 31st 2013 based out of Niaqornat, West Greenland. On clear weather days the helicopter was flown 100 to 150 km offshore in the pack ice using strategically placed fuel depots while observers searched for narwhals in the vicinity of the leads and cracks in the sea ice  on seven days between March 21 sea ice . When whAn array of 16 Reson TC-4013-5 hydrophones  frequency response between 1 and 150 kHz) was used to record narwhal echolocation signals. The 16 receivers were oriented vertically in a linear vertical array, attached to a line (diameter 2 mm) and spaced 1 m apart. The topmost receiver was 3 m below the water surface, the lowest receiver at 18 m depth. The hydrophone array was kept vertical by 4 kg of weight attached to the end of the line.The hydrophone signals were amplified by 38 dB using a custom made 16 channel amplifier, no high pass filter was used and the hydrophones served as a low pass filter . Each hydrophone was calibrated, and the resulting frequency response of each channel known. Simultaneous analog to digital conversion was performed by National Instrument A/D converter with 16 bit resolution at a sampling rate of 500 kHz per channel . The clipping level of the entire recording chain was 206 dB pp re 1 μPa at 100 kHz and the frequency response between 1 and 150 was flat (+/-2 dB).Multiple channels were visualized during the recordings by the custom made software MALTA . On six days the array was deployed at eleven independent locations to record narwhals and recordings were made continuously for up to 2:31 h per location. In total, 10:32 hours (550 GB of data) were recorded. Recordings were chunked (loss-less) to 5 s long wav-files for optimal post-processing. The narwhal data reported in this study were selected from two locations (sites 13_10 and 13_11), for a total of 1:39 h of recordings. We only report data from these two sites in this study for all parameters for sake of consistency. Recordings made at a third site (site 13_16) were used to measure the localization accuracy.Each recording was screened visually for the presence of narwhal clicks. Only recordings with clicks present were chosen for subsequent analysis. For each click recorded with a received level (RL) of 146 dB pp re 1 μPa, i.e. with sufficient signal to noise ratio SNR >12 dB), the position of the source at click emission was computed by measuring the time of arrival difference (TOAD). First the cross correlations of all 120 possible receiver pairs were performed. The receiver resulting in the best cross correlation with the 15 other receivers was chosen as a template for subsequent processing. The TOADs between the template channel and each of the remaining 15 channels was computed using the cross correlations of short signal sections containing the clicks. Based on these 15 TOADs, the overdetermined source position was calculated based on a least square error optimization  mak(-DI/20)  having a(-DI/20) , 42–44, W of 8.3° Recent s earlier , 34, 37.Pseudorca crassidens) [Tursiops spp.) [Mesoplodon densirostris) [The echolocation beam is not symmetric in the vertical plane. Both, the analysis of the composite beam and single click beam measurements indicate a wider beam shape below the beam axis compared to the beam shape above the beam axis. Most studies on the directionality of toothed whale or bat echolocation signals assume a symmetrical echolocation beam. Few studies have investigated horizontal asymmetry and discussed it in respect to the mechanisms of click production , 46, 47.ssidens) , bottlenps spp.) –50 and tps spp.) . In all rostris)  and the rostris)  are direBats and toothed whales use highly directional signals to increase the detection distance in the forward direction and reduce clutter from the periphery. To sufficiently sample the volume ahead of them, the moving animals direct their clicks or calls in different directions for subsequent signals. This scanning behavior has been studied in captive bats , 53, batNarwhals emit very broadband clicks containing considerable energy at frequencies above 200 kHz. These high frequencies have only recently been reported for narwhals  scanningBaffin Bay, West Greenland is an abyss up to 2,300 m and essentially an “acoustic bowl” where narwhals have developed highly specialized acoustic sensory systems to find their prey at depths of >1500 m. Narwhals have very high site fidelity to winter feeding areas in this area and over 80% of the world’s narwhals spend the winter in this area. Their summering refuge for some of these whales  is the site of a future year-round shipping route that is expected to be operational as a consequence of sea ice loss (Northwest Passage). Information reported in this study can inform future work on the use of sound by narwhals and the potential impacts of increasing anthropogenic noise in the Arctic. Finley et al.  reported