Advancements in detection instruments have enabled the real-time acquisition of water information during plant growth; however, the real-time monitoring of freeze–thaw information during plant overwintering remains a challenge. Based on the relationship between the change in the water–ice ratio and branch impedance during freezing, a miniature noninvasive branch volume ice content (BVIC) sensor was developed for monitoring real-time changes in volumetric ice content and the ice freeze-thaw rate of woody plant branches during the overwintering period. The results of the performance analysis of the impedance measurement circuit show that the circuit has a lateral sensitivity range, measurement range, resolution, measurement accuracy, and power consumption of 0–35 mm, 0–100%, 0.05%, ±1.76%, and 0.25 W, respectively. The dynamic response time was 0.296 s. The maximum allowable error by the output voltage fluctuation, owing to the ambient temperature and humidity, was only ±0.635%, which meets the actual use requirements. The calibration curve fit coefficients were >0.98, indicating a significant correlation. The ice content of plant branches under cold stress was measured for indoor and field environments, and the sensors could effectively monitor changes in the branch ice content in plants exposed to cold stress. Additionally, they can differentiate between plants with different cold resistances, indicating the reliability of the BVIC sensor. Cold stress is an atypical biotic stress that woody plants face in temperate and cold climates, and is an important factor effecting plant growth and distribution ,2. It inIn earlier studies, the freeze-thaw condition of plants was usually assessed by observing the morphology of plants under cold stress, and was mainly divided into ecological indices  and morphological indices  ,12. SubsThe TDR, LVDT, and ultrasonic emission methods have the advantage of being nondestructive or microdestructive, and can be used in real-time in the field; however, the cost of TDR and ultrasonic emission testing equipment is prohibitive. The LVDT sensors are more suited to measuring larger diameter plants, but are not suitable for finer branches. By analyzing the impedance changes in plant branches under cold stress, we propose a method based on edge electromagnetic field induction to detect the impedance of branch tissues and thus obtain freeze-thaw information of plant branches. Additionally, a plant BVIC sensor was developed, which is not only capable of dynamic real-time monitoring, but also causes little damage to plants. The sensor is easy to install and measure on small branches, and is inexpensive (costing around USD 20), providing a technical and scientific basis for agricultural and forestry frost prevention and treatment, plant freeze-thaw physiological research, and cold resistance assessment. The main research content in this paper is as follows: (1) analyzing the relationship between the change in water-ice ratio and branch impedance during freezing; (2) developing a miniature noninvasive BVIC sensor to detect branch tissue impedance by edge-based electromagnetic field induction; (3) analyzing the measurement performance of the BVIC sensor; and (4) interpreting the freeze-thaw characteristics of plants under cold stress based on the obtained information of the branch ice content and the ice freeze-thaw rate.Populus L. powder  at a location 10 cm from the first fork point. The Juniperus virginiana Linnaeus was planted on 1 May 2017, the main stem was 2.6 cm at the beginning of the experiment, and the sensor probe was installed at a height of 120 cm from the soil surface on the main stem. Branches were cut from these trees during the nonfreeze-thaw period as isolated live wood branches and used for sensor calibration and testing.Plant materials were divided into simulated and real. The material used for simulating plants was . powder a, which The indoor experimental site was located in the water-saving irrigation control laboratory of Beijing Forestry University, which has an adjustable temperature refrigerator , drying oven , high and low temperature alternating test chamber , precision electronic scale , high frequency vector network analyzer , conductivity meter , oscilloscope , digital multimeter , Shanghai, China, voltage range from 0 to 1000 V, current range from 0 to 10 A), adjustable DC regulated power supply , stainless steel ruler  and pure water , which were used for the experiments.Lagerstroemia indica L. grow in a warm, temperate, semihumid, semi-arid monsoon climate, and where the average temperature in July is 26.0 °C (hottest), whereas the average temperature in January is −5.0 °C (coldest). The other site is in Hohhot, Inner Mongolia, China , where Juniperus virginiana Linnaeus grows, in a midtemperate, continental monsoon climate, where the average temperature in July is 24.0 °C (hottest) and the average temperature in January is −10.0 °C (coldest).The outdoor site is located at Bajia Nursery, Haidian District, Beijing, China , where The dielectric properties of plant branch tissue lie between those of polar and ionic materials, and the branch tissue is viewed as a complex porous dielectric substance consisting of a mixture of gas (g), liquid (l), and solid (s) ,29,30, wDuring overwintering, along with ambient temperature changes, interconversion between liquid water and solid ice occurs within the plant branches. The dielectric constant of ice (approximately 3) is similar to that of solid matter within the branch tissue ,32, whilh tissue ,21,33, wThe measurement principle for detecting the branch tissue impedance based on edge electromagnetic field induction is shown in Because the dielectric constant of ice (approximately 3) is similar to that of the solid material in the branch tissue, while the dielectric constant of liquid water is approximately 81, the impedance of the branch during freezing and melting is principally affected by the percentage of liquid water in the branch. This implies the percentage of liquid water is related to the circuit output voltage Excessively rapid freezing in the plant during freeze-thaw will lead to massive tissue cell death, which in turn will cause irreversible damage to the plant; therefore, the ice freeze-thaw rate of the branches is an important indicator of the freeze-thaw characteristics of plants. The formula is:Lagerstroemia indica L. branches) configured with different volumetric moisture content simulated the branches. The different volumetric ice contents during freezing were measured by the vector network analyzer E5070B to determine the excitation frequency of the impedance measurement circuit, by measuring the changes in the real and imaginary parts of the branch impedance. These corresponded to each moisture gradient under the different signal frequency excitation of the designed probe structure. The simulated branches were made by mixing Populus L. powder uniformly with different amounts of water, and then placing a portion of the sample into a 20 mm diameter test tube and wrapping it with plastic wrap to prevent water loss. The volumetric water content of the branches was 0%, 1.62%, 3.64%, 5.52%, 7.85%, 13.93%, 19.14%, 25.20%, 40.10%, 50.07%, and 74.99%, for the 12 gradients of sample, where 0% is the air in the test tube and 74.99% is the volumetric water content of the sample at saturation. Live wood branches were pruned from healthy Lagerstroemia indica L. plants, were soaked in water for 12 h, and then tested experimentally. After one impedance measurement, the samples were put into the drying chamber , and then removed to enable the branches to return to room temperature. The mass of the samples after drying, and the impedance of the samples at different frequencies, were measured before they were placed in the drying chamber. This process was repeated until the branches of Lagerstroemia indica L. were thoroughly dried, and the change in the mass of water in the samples was obtained by weighing and dividing by the density of water to obtain the volume of water in the samples. The volumetric water content of the samples was further calculated to obtain a total of 20 variants of volumetric water content in isolated live wood branches, which were 0%, 0.38%, 1.28%, 1.33%, 2.99%, 4.30%, 5.66%, 10.67%, 14.66%, 21.84%, 28.26%, 32.56%, 36.33%, 40.45%, 43.58%, 46.22%, 49.03%, 52.69%, 56.01%, and 58.36%.The selection of the appropriate measurement frequency plays an important role in improving measurement accuracy. A related study by Hilhost and Driksen  demonstrPopulus L. powder was selected and a sample of mock branches with a volumetric moisture content of 45% was configured. The impedance measurement probe was mounted on the simulated branches and these were placed whole into the adjustable temperature refrigerator BD-200HEGW. The temperature was set to drop from 20 °C to −16 °C, and then rise to 20 °C when it had reached −16 °C. The process of cold stress was simulated by this cooling and warming, with the temperature adjustment rate set to 3 °C every 10 min. Impedance change in the samples during the freezing and thawing was measured with a vector network analyzer E5070B (measurement frequency 100 MHz). Fresh branches of Lagerstroemia indica L. were pruned and wrapped with cling film immediately after cutting to prevent moisture loss. After the begonia branches were collected, the measurement probe was mounted on them and they were placed in the low-temperature refrigerator (BD-200HEGW). The temperature was set to drop from 20 °C to −20 °C, and then rise to 20 °C when it reached −20 °C. The temperature adjustment rate was set so that every 10 min the temperature changed by 2 °C, and the impedance change of the samples during this freeze-thaw process was measured with a vector network analyzer E5070B (measurement frequency of 100 MHz).The The impedance measurement circuit consists of a detection circuit  and a measurement probe. A schematic diagram of the impedance sensing circuit is shown in After completing the impedance measurement circuit design, performance tests were conducted to ensure the reliability of the measurement results. Static characteristics indicate the relationship between the sensor output and input when the input is constant, or largely unchanged. For application in the scenario of the branch ice content measurement, the static characteristic test focuses on the analysis of the measurement sensitivity range, measurement range, resolution, measurement accuracy, and power consumption. In order to visually characterize the sensor radial sensitivity range changes, this paper uses the energy index Dynamic characteristics indicate the response characteristics of the sensor to input quantity changes with time. By measuring the probe empty value in the air to be its output stability, then quickly placing the probe into a beaker containing 500 mL of water , and by using an oscilloscope to observe the sensor output signal changes, we can establish the dynamic characteristics of the impedance measurement circuit output curve.nφ denotes the value of the change in sensor output, with the equation being:The actual use environment is often complex and harsh, and the plant’s own electrical conductivity may also cause changes in plant tissue characteristics, as shown in The BVIC sensor hardware system consists of an overall hardware circuit system and a mechanical structure. The overall hardware circuit of the BVIC sensor is shown in 2 to 3165.12 mm2 (the surface coverage area is as in Formula (11)), so that it can adapt to different diameters of the branches. The waterproof protective cover of the temperature probe, with a length of 45 mm and an outer diameter of 16.7 mm, is shown in The BVIC sensor mechanical structure contains a bimetallic ring measuring probe, a temperature probe, a waterproof protective cover and sensor housing, and a ring measuring probe, as shown in Pachira glabra Pasq., Lagerstroemia indica L., and Juniperus virginiana Linnaeus plants growing in good conditions, and the volume of the branches was measured by the overflow method. The BVIC sensor was mounted on the branches and they were placed in a drying oven (DHG-9030A) for drying (45 °C), after which the branches were removed and weighed every 6 h to record the voltage value output from the impedance measurement circuit in the BVIC sensor circuit, until the branches had completely dried. The volumetric water content  obtained by the voltage value, and the drying method, was linearly fitted to obtain the calibration coefficients k and b in Equation (6), and was written into the memory of the BVIC sensor. For measuring the branch ice content over winter, by obtaining the output voltage value of the branch impedance measurement circuit in real-time, then the volumetric water content of the branch before the freezing point, and the volumetric water content during the freezing process, can be calculated. The model of Equations (7) and (8) is embedded in the sensor memory through the program code. The BVIC sensor measures in real-time whether the ambient temperature reaches the freezing point during operation, and then calculates the volumetric ice content and ice freeze-thaw rate of the branch at any moment. Calibration improves the consistency of the sensor’s output, allowing measurements to be displayed as volumetric ice content and ice freeze-thaw rate; it also helps to check to exclude defective products from the sensor hardware manufacturing process.The branches were selected from healthy Pachira glabra Pasq. The whole Pachira glabra Pasq. was placed in a low-temperature refrigerator (BD-200HEGW), and the refrigerator temperature was set to −30 °C. After 10 h, the power of the refrigerator was turned off and the refrigerator was opened, allowing the temperature to rise naturally. The ice content and ice freeze-thaw rate of Pachira glabra Pasq. were measured every 1 min to observe the changes in the branch volume ice content and the ice freeze-thaw rate when Pachira glabra Pasq. cells were exposed to cold stress.In the indoor simulated cold stress experiment, the BVIC sensor designed in this study was mounted on living Lagerstroemia indica L. and Juniperus virginiana Linnaeus from 25 October, 2018, to 10 March, 2019. Data were recorded once every 10 min to capture changes in the branch volume ice content and the ice freeze-thaw rate in Lagerstroemia indica L. and Juniperus virginiana Linnaeus during the overwintering period, when they experienced cold stress.In the field cold stress experiment, the BVIC sensors were mounted on Impedance values were measured for simulated branches and live wood branches at different volumetric water contents, using different signal frequencies as excitation. Simulating the environment where the branches encounter cold stress, the impedance changes of the simulated branches and live wood branches during the freeze-thaw cycle are shown in The impedance measurement circuit output voltage and the energy index, and their relationship to the diameter of the branch, are shown in The measurement result of the BVIC sensor in the unloaded state is taken as 0%, and the ideal state of all ice in the branch is regarded as 100% ice content. When the BVIC sensor is installed in a test tube of water and put into the freezer to freeze, the output voltage of the branch impedance circuit first decreases gradually with the increase of ice in the test tube, and continues until the water in the test tube is completely frozen, from where it does not change. This means the BVIC sensor can effectively record the entire change process during water freezing; that is, the BVIC senses the volume ice content over the range from 0% to 100%. During the complete transformation of water from liquid to solid in the test tube, the difference in the voltage change was 1.574 V. It was calculated that each 1 V change in voltage value represents a 63.53% change in ice content, and the sampling resolution of the analog-to-digital conversion module was 0.0008 V. Therefore, the resolution of the BVIC sensor was 0.05% , in order to ensure good measurement results, the resistance component should be a fixed value and only the reactance component is linearly related to the impedance value. However, in practice, the variation of water in the branch will also lead to the variation of the resistance component. We would like, therefore, to try to find a suitable excitation frequency at which the resistance component is small and varies very little , while the reactance component varies as linearly as possible. The results in For the reactance component, when its value is negative, this indicates the object under test is capacitive. When it is positive it indicates that the object under test is inductive, implying the reactance component in the measurement should be negative. Comparing The static characteristics test indicated that the sensitivity range of the impedance measurement circuit is 0–35 mm, the measurement range is 0–100%, the resolution is 0.05%, the measurement accuracy is ±1.76%, and the power consumption is 0.25 W. The dynamic characteristics test obtained a response time of 0.296 s. Compared to similar sensors, the BVIC sensor designed in this study meets practical use requirements ,21,37. FLagerstroemia indica L. and Juniperus virginiana Linnaeus over winter (Lagerstroemia indica L. (a deciduous broadleaf tree) and Juniperus virginiana Linnaeus (an evergreen coniferous tree), shows the branch volume ice content of Juniperus virginiana Linnaeus is less volatile during the overwintering period, and its corresponding ice freeze-thaw rate is small. The standard deviation of the ice freeze-thaw rate is only 0.00405 , leading to plant death. This demonstrated the danger of intracellular ice crystals and rapid cellular freeze-thaw [Lagerstroemia indica L. and Juniperus virginiana Linnaeus over winter, and the return to normal growth in spring, demonstrate the persistence of extracellular ice crystals during winter. This suggests that with proper cold training, plant cells gradually adapt to dehydration caused by extracellular ice crystal formation [When plants experience cold stress, tissue freezing occurs, accompanied by the production of ice crystals, the location of which in plant tissues determines whether the cells die, or not . Intraceeze-thaw ,6,7,8,22ormation , improviormation ,23, thusMany researchers have applied image techniques, TDR sensors, ultrasonic acoustic emission analysis, and LVDT sensors to study plant freeze-thaw, and their performance has been demonstrated ,16,31. HPachira glabra Pasq., Lagerstroemia indica L. and Juniperus virginiana Linnaeus, indicated that the BVIC sensor can effectively monitor changes in the branch ice content. The differences in the volumetric ice content and the branch ice freeze-thaw rate of Lagerstroemia indica L. and Juniperus virginiana Linnaeus over winter demonstrated the BVIC sensor can effectively differentiate characteristics in plants with different cold resistance, and can be used as an effective monitoring tool for plant freeze-thaw studies. As well as image techniques, TDR sensors, ultrasonic acoustic emission analysis, and LVDT sensors, the BVIC sensor designed in this paper is inexpensive, can be applied to the installation of fine branches for measurement, and can be used either alone or integrated into other systems due to the internal collection of wireless communication modules. This study provides a theoretical basis for assessing embolism and plant cold resistance induced by cold stress, as well as a cost-effective tool for monitoring the freeze-thaw information of over wintering plants in the field. The method proposed in this paper can also be combined with plant assessment parameters [In this paper, we developed a miniature noninvasive intelligent sensor (the BVIC sensor) for detecting the volumetric ice content and the ice freeze-thaw rate in the branches of plants during the overwintering period by analyzing the impedance changes of the branches under cold stress. In a series of performance tests, the results showed that the feasible range of excitation frequency sources for impedance measurement circuits is from 100 MHz to 200 MHz, that the static and dynamic characteristics of the BVIC sensor met the requirements for practical use, and that ambient temperature and humidity or branch conductivity had little effect. Measurements were carried out on different plants, demonstrating the application of the method to multiple objectives. Monitoring rameters ,47,48 torameters ,50,51 byrameters ,53 and arameters ,55,56. F


We found three fish species , but the catch was limited to only seven individuals. Euphausiids, amphipods and gelatinous zooplankton dominated the catch weight in the Nansen Basin in the mesopelagic communities. Euphausiids were almost absent in the Amundsen Basin with copepods, amphipods, chaetognaths and gelatinous zooplankton dominating. We postulate asymmetric conditions in the pelagic ecosystems of the western and eastern Eurasian Basin caused by ice and ocean circulation regimes.The rapid ongoing changes in the Central Arctic Ocean call for baseline information on the pelagic fauna. However, sampling for motile organisms which easily escape vertically towed nets is challenging. Here, we report the species composition and catch weight of pelagic fishes and larger zooplankton from 12 trawl hauls conducted in ice covered waters in the Central Arctic Ocean beyond the continental slopes in late summer. Combined trawl catches with acoustics data revealed low amounts of fish and zooplankton from the advective influenced slope region in the Nansen Basin in the south to the ice-covered deep Amundsen Basin in the north. Both arctic and subarctic-boreal species, including the ones considered as Atlantic expatriate species were found all the way to 87.5 Oceanographic changes are also underway, namely amplified warming and increasing salinity in the upper waters of the Eurasian Basin related to upstream conditions2. This will eventually affect the whole ecosystem in various ways4, and dramatic changes are expected in terms of magnitude and spatial distribution of ice-associated and pelagic primary production7. To monitor these changes, it is urgent to establish a baseline mapping of an understudied area in the central Arctic. This is particularly relevant for species potentially subjected to future fisheries8.The Central Arctic Ocean (CAO) becomes increasingly ice-free during the summer due to climate warmingBoreogadus saida) and various ice-associated and pelagic phytoplankton and zooplankton species are present at the underside of the ice and in the upper parts of the water column below the ice12. Vertical zonation of mesozooplankton in the Eurasian Basin has also been characterized and synthesized over the past decades12. Some larger organisms, like armhook squid (Gonatus fabricii) and gelatinous zooplankton and nekton have been found13, as well as single individuals of Atlantic cod (Gadus morhua), glacier lanternfish  and ice cod 13. Faunal densities and biomass for the mapped Eurasian Basin water column are generally low in comparison to Arctic shelves and southern areas14 due to overall low primary production and hence food availability16. However, the presence and distribution of larger and motile organisms like fish and macrozooplankton have been poorly described in the CAO due to severe difficulties in sampling such species in ice covered areas.Earlier expeditions to ice-covered waters in the Eurasian Basin have begun to map biodiversity and faunal densities/biomass patterns. They revealed that polar cod  occur in lower abundances compared to regions further south, and (2) in their taxonomic composition reflect recent physical changes in that subarctic-boreal species present also penetrate the northernmost parts of the Eurasian Basin.Scattering layers in the mesopelagic (200–1000 m) zone are ubiquitous in most world oceansKronprins Haakon covered a transect of ca. 2330 km extending from the Nansen Basin northeast of the Svalbard slope, to 87.5o N at the northern side of the Amundsen Basin just south of the Lomonosov Ridge25  with R/V The trawl and acoustic sampling were conducted in a region spanning strong gradients in sea ice and oceanic conditions Fig. . The seao N, extremely cold (− 1.8 °C < T < − 1.5 °C) and fresh polar waters covered the upper 100 m. A pronounced surface salinity front appeared in the northern Nansen Basin-Gakkel Ridge region  of the Atlantic Water current. Instead, the ADCP data showed rapidly varying horizontal flow near NB1 indicating mesoscale eddies , together with one haul in the Amundsen Basin . The Harstad trawl revealed very small catches at the Gakkel Ridge . The four hauls with the finer-meshed macroplankton trawl generally yielded higher catches (137–696 g nm−1) than the Harstad trawl  contributed most to the catches in the Nansen Basin, while T. libellula , Gadidae (polar cod), and Pleuronectidae . The few fish sampled included one 8 cm  polar cod (at NB1e), one 4 cm Greenland halibut larva (at NB2e), three glacier lanternfishes at NB1m  and one at AB1m (5.0 cm). This is, as far as we know, the first time glacier lanternfish has been caught this far north, although the species has been tentatively identified by video recordings even further to the north in this region13. It is also to our knowledge the northernmost record of Greenland halibut, and the first time it has been observed north of the shelf break in the CAO.The Harstad trawl caught a total of 15 taxa, namely included Ctenophora (44%), Arthropoda (40%), Cnidaria (12%), Chaetognatha (2%), Mollusca (2%) and Teleostei (1%) by wet weight Fig. a. EuphauCalanus hyperboreus and the amphipod Themisto libellula  were found. Notably, more than 90% of the Calanus hyperboreus and Paraeuchaeta spp. recorded were mature females  was also low in the macroplankton trawl catches. Though their biomass was comparatively low, the warm water associated species such as Nematoscelis megalops and Meganyctiphanes norvegica were recorded reaching far north in the Arctic waters. In fact, this is the first record of N. megalops in the study area as far as we know. The macroplankton trawl also caught a single glacier lanternfish (6.5 cm) at AB1.The macroplankton trawl had higher taxon richness 23) and catches consisted of Arthropoda (44%), Ctenophora (38%), Chaetognatha (13%), Cnidaria (4%), Mollusca (0.6%) and Teleostei (0.2%) by wet weight  and hyperiid amphipods (Themisto libellula) were observed along the whole transect. Copepoda is another group that we expect to be represented at all stations, however, this was not evident as the gears we used are not suitable for catching mesozooplankton. In the macroplankton trawl, though, we recorded biomass of the larger Calanus hyperboreus (ca. 7 mm), these are most likely underestimated. Other taxa were taken at either only southern or northern locations. The amphipod Eusirus holmii and the decapod Hymenodora glacialis were found at the northernmost locations only. Polar cod and Greenland halibut were caught at southern locations only, while glacier lanternfish occurred at both southern and northern locations.As expected, the macroplankton trawl had higher catches of the smaller organisms  compared to the Harstad trawl Fig. . Twelve 18/38), and target strength (TS) analysis 50) acoustic scattering layer was present at NB1 at 10–100 m in water with temperatures slightly below zero  weaker than − 55 dB  suggested that organisms containing gas were present. The matching catch from the Harstad trawl (in 60–40 m) showed among other things one 8 cm long polar cod  Figs. a and S16est Fig. . More th38 weaker than − 50 dB, indicating that no larger fish were present. Consistently, the matching trawl catches consisted of a mixture of gelatinous and crustacean plankton and an armhook squid , faint echoes forming a scattering layer could be seen in the depth interval 100–200 m Figs. b and S19id Table . Additioeak Fig. b, but TS dB Fig. .38 values with a mode for − 54 dB  indicated a mixture of organisms with and without gas inclusions, while the catches from the Harstad (50–550 m) and macroplankton (60–537 m) trawls were dominated by arctic hyperiid amphipods  showed a distinct epipelagic layer in 20–40 m depth, in polar waters with temperature below − 1.5 °C Fig. . A sharp dB Fig. . The freods Fig. .38 registrations at depth resembled those at the more southern locations, although with a wider distribution peaking at − 58 dB , both the epi- and mesopelagic scattering was weak , the 25–30 m depth layer had a stronger acoustic signal than elsewhere in the study area, and with a different composition and/or density , and glacier lanternfish and polar cod. The acoustic data revealed a strong echo target  Atlantic cod, or a marine mammal or a big fish without a swim bladder , which is commonly found near the slope in this area56. Juvenile Beaked redfish (Sebastes mentella), haddock (Melanogrammus aeglefinus), capelin , polar cod, Atlantic cod and Greenland halibut can also be found in the meso- and epipelagic layers closer the slope slightly further west, yet some species appear to occur seasonally57. Also, adult redfish and Atlantic cod have been caught in the mesopelagic layer just off the slope slightly further west58. However, these reported catches were taken closer to the Atlantic Water slope current.The epi- and mesopelagic fauna in the southern Nansen Basin was more diverse compared to further north Fig. . It inclget Fig.  with TS359, and such a decline was also evident when moving from the near-slope (NB1) to the central (NB2) Nansen Basin  was sampled near midnight  and low (T < − 1.5 °C) temperatures Fig. a and d, Nematoscelis megalops  were considerably stronger than the previous estimates of about − 62 dB for adult glacier lanternfish66. Since the TS data were filtered to be within 2° of the acoustic axis before analysis, this result should not be biased due to reduced signal-to-noise ratio in the outer parts of the beam. Furthermore, similar (− 55 to 52 dB) modes were evident at all locations south of AB1. If these stronger echoes came from for instance polar cod, our TS38 data imply that polar cod of lengths 6–9 cm51 were present at most locations, although in too low concentrations to be caught by the trawls.The catch in the mesopelagic layer in the Amundsen Basin consisted of a mixture of gelatinous and crustacean plankton and chaetognaths, and the only fish caught were a few individuals of glacier lanternfish in the southern parts of this Basin Figs.  and 5. H11 euphausiid Meganyctiphanes norvegica were observed all the way to 87.5o N in the Amundsen Basin  the catches revealed a strong presence of gelatinous plankton (ctenophores), again including subarctic-boreal species Fig. . Overwinons Fig. , Table 2 the catcp. Table . These c21. Also the acoustic backscattering is two orders of magnitude less than in northern Svalbard waters72, a region which in turn has backscattering an order of magnitude less than the Norwegian Sea21. This confirms our hypothesis that pelagic fishes and large zooplankton in the CAO have lower abundance compared to the south, although the abundances are even lower than we expected. Moreover, recent studies have shown lower densities in the western Nansen and Amundsen Basins as compared to their eastern parts13. Thus, despite being closer to the inflow region in Fram Strait, the density of fish and other pelagic organisms in the deep western Nansen Basin seem to be lower than further east. At the same time, the biogeographic composition reflects connectivity to the Atlantic inflow. It remains unclear if the addition of species not found earlier in the study region is a result of strengthened or more frequent inflow or increased research effort.Our results show that the densities of both fish and their prey organisms in the Nansen and Amundsen Basins are very low compared to regions further south. The observed catch rates are two orders of magnitude lower than in the slope current north of Svalbard53. However, for the off-slope deep Nansen Basin, the largest changes in summer sea ice have occurred in the eastern parts1. Changes in summer sea ice in the deep western Nansen Basin are much smaller due to the Transpolar Drift continuously bringing sea ice westwards into the region and further towards Fram Strait27  down to at least 500 m. The same conclusion was drawn from using 18 kHz acoustic data from a CAO crossing with R/V Oden74, but pelagic fish trawls have so far not been used in the CAO due to its thick ice cover23. Our results show that only modest adaptations of an ordinary pelagic trawl (Harstad trawl) used routinely on Norwegian research vessels for abundance and biomass estimations of fish, functioned well in ice-covered waters. We used ice trawl gallows to move the tow blocks into the centre of the stern of the vessel, thereby closing the trawl before it reached the surface during retrieval and before sinking beneath the surface during setting. In addition, the trawl was slightly modified by reducing the flotation and adding weight to the cod-end. This made the trawl hang almost vertically from the stern of the vessel during deployment and retrieval, which further reduced the problem with sea ice entering the trawl. For the smaller macroplankton trawl, the only modification made was adding 40 kg to the cod-end.Pelagic trawling in ice-covered waters with ordinary equipment is challenging because ice floes floating behind the vessel can easily destroy the net during deployment or retrieval. Under-ice trawl samples have previously been obtained with a Surface and Under Ice Trawl3 of water. Nevertheless, with its vertical opening of 10–12 m , there are uncertainties related to whether the catch composition reflects the actual composition of organisms in the sea. The different catchabilities of the Harstad and macroplankton trawls add further complexity to this issue. Using a larger trawl would undoubtedly be a more efficient approach for catching fish but would also result in severe complications regarding operations in ice-covered waters. For catching smaller organisms , smaller nets like WP2 or Multinet with 180 µm mesh are more efficient.Horizontal towing of a trawl has clear advantages compared to other sampling methods , particularly for extremely low densities as in the CAO. Towing one to two nautical miles, as during this survey, the Harstad trawl filtered approximately 500.000–1.000.000 mwww.fao.org/faolex/results/details/en/c/LEX-FAOC199323). In addition to preventing fisheries in the high seas of the CAO at least up to 2037, the agreement calls for the establishment of a Joint Program of Scientific Research and Monitoring with the aim of improving the understanding of the ecosystems. A draft plan for such a program emphasised the need for baseline mapping, monitoring and research8.In 2021, a CAO Fisheries Agreement entered into force  was used with modified rigging. The trawl (~ 250 m2 opening area) is graded from 200 mm mesh in the front of the trawl to 60 mm before the cod-end. The cod-end has an 8-mm mesh net. The modified rigging included wire instead of spectra sweeps (both 60 m long), reducing the total buoyancy of the trawl net (from 1047 to 50 kg), and applying 150 kg weights in front of each lower wing and a 40 kg weight at the end of the cod-end. The ice-modified Harstad trawl had an average opening height of between 9.6 and 12.4 m and an average door spread of 44.7–71.5 m  in northern areas  from the front of the trawl to the cod-end.The macroplankton 92 m trawl . When the doors were at approximately 4–6 m depth, the vessel increased its speed and the warps were deployed as during ordinary pelagic trawling. The Harstad trawl was towed at 1.5 ms−1 while the macroplankton trawl was towed at 1.0 ms−1 (speed over ground—SOG) for ~ 30 min at different depths  samples using a 1 mm sieve. Representative subsamples of 100 g were preserved in 4% hexamine-buffered formalin solution in 500 ml plastic bottles. The preserved samples were species identified length measured, when possible, at the IMR laboratory. Institute of Marine Research fully adheres to Norwegian laws relevant to Ethics in Science as well as Animal Welfare. Institute of Marine Research is a governmental research institute with given permission to perform research cruises including fish samplings by the Norwegian Government. All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines. No experiments were carried out on live organisms.https://kvalitet.hi.no/docs/pub/DOK06839.pdf) and UiT The Arctic University of Norway leading the Nansen Legacy project (https://doi.org/10.7557/nlrs.5882).The sampling protocols were approved by Institute of Marine Research . The 38 kHz frequency was used to determine backscatter intensity. Analysis was done in the postprocessing tool LSSS v. 2.11.0 (ref.79). The backscattering data output were in the form of sA, and all definitions and notations of the acoustic quantities were according to Maclennan50  when feasible.Various types of organisms of the same size often have different acoustic properties at different frequencies, and the distribution of types of scattering organisms was assessed using frequency response in sWe identified type of organisms present by measurements of TS of individual scatterers. We mainly considered TS measurement from the 38 kHz, but TS from 18 kHz were also assessed when available. The higher frequencies were only considered when we recorded extraordinary registrations (like at NB1). The TS measurements were identified in LSSS using the following settings: Detector type SED (Single echo detector), Min TS − 70 dB, Pulse length determination Level: 6 dB, Minimum echo length: 0.5 (relative to pulse length), Maximum echo length 2.0 (relative to pulse length), Max gain compensation 6 dB, Phase Deviation Check: On. Max phase deviation: 8 steps. Thereafter, we filtered the data restricting them to be within 2° off axis to avoid TS distributions being biased towards higher values due to reduced signal-to-noise ratio in the outer parts of the beam.https://osi-saf.eumetsat.int/products/sea-ice-products). Daily sea ice concentration from passive microwave satellite data is from the interim climate data record (OSI-430-b80) and is provided daily on a grid with a resolution of 25 km. Sea ice drift velocities are from the low-resolution sea ice drift product (OSI-405-c) and are daily at a resolution of 62.5 km.Both sea ice concentration and drift during the cruise period were obtained from the EUMETSAT Ocean and Sea Ice Satellite Application Facility (OSI SAF), in collaboration with ESA CCI , and then averaged to obtain a vertical profile of velocity at the CTD station. This returned velocity along a vector with 10 km horizontal resolution up to 82.5o N and the resolution of the CTD stations to the north of this.Observations of ocean currents were obtained from the vessel mounted RDI 150 kHz Acoustic Doppler Current Profiler (ADCP) mounted behind the ice window under the hull. Post-processing of ADCP data was performed using the Common Oceanographic Data Access SystemTemperature and salinity were measured using a Seabird 911plus CTD at and between the trawl stations Figs. d, e. TheSupplementary Information 1.Supplementary Tables.


The ultimate load and the estimated mean survival until failure were higher for the interference screw . Graft displacement at ultimate load reached values of up to 7.2 (interference screw) and 11.3 mm . The allograft screw for implant fixation in ACL reconstruction demonstrated comparable behavior in terms of graft slippage to the interference screw but underperformed in terms of ultimate load. However, the ultimate load, occurring at progressive graft slippage, may not be considered a direct indicator of clinical failure.The purpose of this study was to compare the biomechanical stability, especially graft slippage of an allograft screw and a conventional interference screw for tibial implant fixation in ACL reconstruction. Twenty-four paired human proximal tibia specimens underwent ACL reconstruction, with the graft in one specimen of each pair fixed using the allograft screw and the other using the conventional interference screw. Specimens were subjected to cyclic tensile loading until failure. The two fixation methods did not show any statistical difference in load at graft slippage ( Bioabsorbable and metal interference screws have been used for implant fixation in the anterior cruciate ligament (ACL) reconstruction with good midterm results . BioabsoThe recently introduced commercially available allograft osteosynthesis system, the Shark Screw , has already been applied in foot, and hand surgery . Shark SNumerous aspects must be considered for physiologically relevant biomechanical testing of ACL fixations, but not all have been carefully addressed in previous studies. Most investigations on this topic have evaluated biomechanical properties in terms of stiffness and ultimate load of ACL graft fixation methods in animal bone, predominantly porcine. However, two studies  demonstrin vitro study  in an ro study . A relatTherefore, the aim of this study was to compare the biomechanical stability, especially the onset of slippage of the Shark Screw ACL for tibial implant fixation in ACL reconstruction compared to a commonly used bioabsorbable interference screw in human anatomic specimens. The authors hypothesise that the two fixation devices will demonstrate no significant differences in terms of fixation strength.Twenty-four paired anatomic specimens of human proximal tibiae with preserved semitendinosus and gracilis tendons were obtained for this study from three female and nine male donors aged 72.7 ± 5.6 years  at the Medical University of Vienna . The donors had given their written consent during lifetime for their body to be used for research and education. The study was approved by the Ethics Committee of the Medical University of Vienna (2144/2020). The donor age was limited between 18 and 80 years. Furthermore, all specimens were inspected for bone damage, tendon degeneration or previous surgical treatment. The specimens were thawed at room temperature for 24 h, kept moist with 0.9% saline solution during the preparation and tested at room temperature. The proximal part of each tibia was cut at a length of 110 mm using an oscillating saw and carefully stripped of soft tissues. Following the surgical procedure (described below), the distal 30 mm of each specimen was embedded in polymethylmethacrylate . Projected line laser beams ensured that both the graft tunnel and the graft itself were in line with the test system axis, i.e. in the planned loading direction, representing the mechanical worst-case scenario for pullout failure.BMD was assessed from quantitative computed tomography (QCT) scans for correlation with the mechanical parameters, aiming to account for the high donor age and the associated potentially lower bone quality. A 128-slice multi-detector computed tomography (MDCT) scanner  was used with the following settings: 120 kV tube voltage, 90 mAs tube current-time-product, 128 × 0.6 mm collimation, 1 s rotation time and 0.8 pitch. The specimens were placed pairwise in longitudinal direction on the scanner bed together with a calibration phantom . Images were reconstructed using the following parameters: 512 × 512 matrix size, 1 mm slice thickness, 0.8 mm slice increment and Br60 reconstruction kernel. The reconstructed field-of-view (FOV) was limited to the size of a single specimen.The BMD assessment was performed using Mimics 22 . After importing the QCT image series, a Hounsfield units (HU) histogram was created for the most proximal 100 mm of each specimen and exported using the 3D Histogram function. Bone tissue was assumed in the range of 226–3071 HU, as defined in Mimics using global thresholds segmentation function for bone. Volumetric BMD (mg/cm³) was then converted from the HU by multiplying with a calibration factor. The latter was determined as the mean HU value of the bone compartment of the reference body, divided by 200 mg Calcium Hydroxyapatite per ml, normalized with a unique scaling factor. The BMD range was limited between 100 and 1,400 mg CaHA/cm³ to restrict the effect of residual air and to minimize artefacts.The semitendinosus and gracilis tendons were harvested in their full length, quadrupled and, when necessary, longitudinally trimmed to match an 8 mm sizing sleeve. The distal 30–40 mm of the graft were then sutured in a running whipstitch manner using two No. 1 Polysorb sutures  and pretensioned at 50 N, based on previous pilot tests using animal tissue. A guidewire was placed anatomically in lateral to medial direction through the origin of the original ACL with an angle of 65°, followed by incremental drilling of the tibial tunnel with a 5 mm and an 8 mm diameter drill. The graft was inserted into the distal tunnel end and subsequently pulled into proximal direction. In the interference screw group, a 1.0 mm guidewire was placed laterally in the interface between the tendon graft and the tunnel, followed by placement of a fully threaded 8 × 28 mm BioComposite Interference Screw   over theThe insertion of the Shark Screw ACL  was perfThe embedded specimens were mounted in an electrodynamic test system  . A tensiIt has to be noted that some data of fourteen specimens used in this study has been previously reported in a paper concerning general aspects in measurement of structural characteristics and mechanical behavior of ACL implant fixations .Outcome parameters of interest were tested for normality of distribution within each group using the Shapiro-Wilk test. Independent-Samples t-test and Wilcoxon Signed-Rank test were performed to screen for significant differences between the two fixation groups in terms of BMD, insertion torque, load at slippage onset, number of cycles at slippage onset, ultimate load, and number of cycles at slippage onset. The relation of construct failure to loading cycles was assessed by use of Kaplan-Meier survival analysis and Mantel-Cox test for screening of differences between the implant groups. The Pearson product-moment correlation coefficient was computed to investigate linear correlations between 1) BMD of the two groups, 2) BMD and insertion torque, 3) BMD and load at slippage, 4) BMD and ultimate load, 5) load at slippage onset and ultimate load, and 6) insertion torque and load at slippage onset. To investigate if the inter-individual characteristics have an effect on the outcome variables, further correlation analyses between the specimen pairs were performed for 7) load at slippage onset, 8) number of cycles at slippage onset, 9) ultimate load, and 10) number of cycles at slippage onset. Level of significance was set at 0.05 for all statistical tests. The analysis was performed using IBM SPSS Statistics 26 . Descriptive values were calculated in terms of either mean and standard deviation , or median and interquartile range .p = 0.004), which was also the case for the cumulative survival based on the ultimate loads (p = 0.018)  .p = 0.021).The Kaplan-Meier survivorship results for the two implant groups revealed no significant difference in estimated mean survival until slippage onset. The interference screws  were significantly more resistant to cyclic loading until catastrophic construct failure than the Shark Screw ACL   suggesting similar material properties in paired specimens. There was no significant correlation between BMD and insertion torque, BMD and load at slippage onset, BMD and ultimate load, and ultimate load and load at slippage onset . In the interference screw group, insertion torque correlated significantly with load at slippage onset  and non-significantly with ultimate load  , and between insertion torque and ultimate load . There was no significant correlation between the two groups with regard to the load at slippage onset, number of cycles at slippage onset, ultimate load, and the number of cycles at slippage onset (0.057 < R2 < 0.480).There was a strong correlation between the BMD of the two groups ( = 0.18) . In the The ACL graft was pulled out together with the screw in one specimen from the Shark Screw ACL group. This specimen reached the highest ultimate load (375.8 N). The failure mode in all other specimens was graft pullout.The present study aimed to compare the biomechanical stability of an allograft fixation system, Shark Screw ACL for graft fixation in ACL reconstruction compared to a conventional interference screw under consideration of slippage onset, local bone density, and intraoperative insertion torque. The data demonstrated that the biomechanical stability during cyclic testing, i.e. slippage, of the Shark Screw ACL was comparable to a standard interference screw. However, the Shark Screw ACL underperformed in ultimate load testing.To the best knowledge of the authors, this is the first study quantifying the true slippage in different fixation devices by tracking the absolute displacement of the suture firmly attached to the distal end of the ACL graft. Previous investigations have used machine displacement to draw conclusions on fixation stability. Since any ACL graft must be considered as hyperelastic, the slippage of the graft and corresponding stiffness of the fixation are potentially different from the values generated by the displacement of the load frame. A recent study  has showThe Shark Screw ACL reached only 63% of the ultimate load and 65% of the estimated mean survival load based on the ultimate load compared to the interference screw. Both findings were significantly different and relevant considering the accompanying confidence intervals. The used interference screw has a significantly higher thread length. The association between implant length and ultimate load is intuitive, however, it could not be confirmed consistently. Testing 28 mm vs. 35 mm tapered screws in sixteen anatomic specimens of tibiae (mean donor age: 38.5 years) revealed significantly higher ultimate loads in the longer screw  . In contThe ultimate load of the interference screw has been evaluated in numerous studies. . The autin vivo loads and strains in the ACL. Li et al. reported the highest in situ force in the ACL to be 45 N at 15° knee flexion after applying 200 N quadriceps and 80 N antagonistic hamstrings load  are the same and questioned to establish the age of 40 years as a barrier to successful ACL reconstruction (in vitro studies should also not be limited to specimens of young donors. While it is difficult to obtain anatomic specimens from younger donors, the authors took this limitation into account by limiting the maximum age to 80 years and by considering the specimen-specific BMD in data analysis. However, the BMD did not correlate with the biomechanical outcomes. Also, due to limited availability of anatomic specimens, sex distribution in the present study – in favor of the male sex – does not fully reflect the injury incidence which is higher in female athletes when practicing the same sport (in vivo loading, which remains unknown. The testing methodology limits the application of the findings in routine clinical practice. Simulation of accurate in vivo conditions in biomechanical studies is difficult and deviates from simplified models with simulated pathologies. Third, the small sample size resulted in a large deviation of data, however with some detected significance between the groups. Overall, it can be concluded that the Shark Screw ACL allows for sufficient initial fixation stability. Finally, the presented model describes the primary stability of the fixation immediately after surgery and does not consider any biological effects occurring during the weeks and months after surgery. This is important to consider since ACL re-ruptures occur 3.5 (This study was performed on a large number of paired human specimens under consideration of true slippage, BMD and insertion torque. Nevertheless, it has several limitations. First, the quality of cancellous bone in younger, more active patients, who normally undergo an ACL reconstruction surgery, is more robust than of that in fresh-frozen anatomic bone specimens from older donors available for truction . For theme sport . Second,ccur 3.5  to 53 (Bccur 3.5  months aIn conclusion, the Shark Screw ACL used for implant fixation in ACL reconstruction demonstrates similar primary stability in terms of graft slippage compared to the interference screw but remains inferior in terms of ultimate load. However, the ultimate load may not be considered as a direct indicator of clinical failure. The clinical relevance of these findings, as well as the anticipated advantage of the Shark Screw ACL for achieving secondary stability, should be further investigated in clinical studies.


Girardia tigrina planarian tissues. The results indicate that both hormones influence the recovery rate of the regenerating tissue in either a stimulatory or inhibitory way depending on the hormone, its concentration and the regenerating body part of the worm. We suggest that exogenous glucocorticoids can influence endogenous mechanisms of endocrine regeneration, including hormone-receptor interaction. The revealed features of the effects of hydrocortisone and methylprednisolone may be associated with the formation of specific hormone–receptor complexes with each of them, as well as with the dependence of the effects of methylprednisolone on the type of interaction with hydrocortisone receptors i.e., as an agonist or as an antagonist.We have studied the effect of two glucocorticoid hormones: hydrocortisone and its synthetic analogue methylprednisolone on regeneration activity of Girardia tigrina planarian. The regeneration activity was studied in head and tail blastema formed after resection by means of lifetime computer morphometry and immunohistochemical labeling of neoblasts. The search for orthologous proteins—glucocorticoid receptors (hydrocortisone) was performed using the SmedGD database of the Schmidtea mediterranea planarian. The results indicate that both hormones influence the recovery rate of the regenerating head and tail blastema. The worms with regenerating tail blastema have less sensitivity to the hormones’ treatment compared to the ones with regenerating head blastema. Hydrocortisone at a high concentration (10−3 M) suppressed the regeneration rate, while stimulating it at lower concentrations (10−4–10−6 M). The same concentrations of methylprednisolone inhibited the regeneration of head blastema, but did not affect the tail blastema regeneration. The two hormones acted differently: while hydrocortisone stimulated the proliferation of neoblasts in the periwound region, methylprednisolone reduced the mitotic activity, mainly on the tail zone furthest from the wound surface. We suggest that exogenous glucocorticoids can influence endogenous mechanisms of hormone-dependent regeneration.We have studied the effect of two glucocorticoid hormones: hydrocortisone and its synthetic analogue methylprednisolone on the regeneration activity of head and tail blastema of the  Drosophila melanogaster or Caenorhabditis elegans [Regeneration is the ability of living organisms to restore missing structures over time after damage and injury. In the last decade, planarians have been considered a promising model for regenerative medicine and stem cell biology in particular due to an extremely high content up to 30%) of pluripotent neoblast stem cells in their body . In addi0% of plu elegans . All thiMicrostomus lineare flatworms [The research of humoral regulation mechanisms in the study of regeneration has a great importance in this field. In flatworms, the neurosecretory part of the endocrine system has been studied in the most detail. In their nervous system the neurons are known to be responsible for synthesis of the main mediators—serotonin 5-HT), FMRF-like peptides  and NPF-like peptides, as well as a family of pancreatic polypeptides  which are localized both in the central nervous system (brain ganglia and nerve trunks) and in the gonadal plexus -HT, FMRF,7,8. Thelatworms .1-2 and morphogen of hydra stimulate planarian regeneration. AKTH4-10 showed weak stimulating effect regeneration. LHRH9-10 and dalargin acts as a powerful inhibitor of planarian regeneration while delta sleep peptide did not have any effect on planarian regeneration [The possibility of the participation of neuropeptides in the regulation of the regeneration process in different types of planaria is confirmed by the influence of certain vertebrate neuropeptides on regeneration, such as dalargin, somatostatin and vasopressin. Additionally, NPF and FMRF-like peptides can accelerate the recovery of the regenerating pharynx function, and FMRF can in certain cases, slow down this process . LHRH1-2In addition, representatives of free-living and parasitic flatworms are supposed to have another level of endocrine regulation—steroid hormones . In diffThe physiological significance of these hormones has not been sufficiently studied; however, a number of data indicate the possibility of their inclusion in the regeneration regulation process. It was shown that certain reproductive and glucocorticoid hormones such as prednisone, progesterone and testosterone, are involved in the planarian regeneration regulation process in a specific way . The effThe data on the morphogenetic activity of glucocorticoids are fragmentary and need more detailed study.To study the functional activity of a certain hormone, the antagonists and/or agonists thereof are often used to see whether they are able to compete with the studied hormones for binding to their receptors. Some of these synthetic analogues are capable of acting both as antagonists and agonists of the studied hormones depending on the concentration. In this case, their ways of interacting with hormone receptors change, and this leads to the development of different physiological effects. A well-known concentration dependent agonist and antagonist is methylprednisolone . A compaSteroid hormones typically act through nuclear receptors. The family of nuclear steroid hormone receptors includes androgen-, estrogen-, and gluco-mineralocorticoid receptors. The manifestation of a specific hormonal effect is determined by the hormone-receptor interaction of a single hormone with its corresponding nuclear receptor, as well as the nature of the acceptor—the target organ cells . The preGirardia tigrina planarians. To achieve this goal, we set the following tasks:(1)−3 M to 10−6 M and methylprednisolone in the concentration range from 10−4 M to 10−6 M on the reparative regeneration process of the head and tail planarian blastemas (new unpigmented tissue formed at the regeneration site);To study the effect of hydrocortisone in the concentration range from 10(2)To study the effect of exogenous steroid hormones hydrocortisone and methylprednisolone on the proliferation of planarian neoblast stem cells;(3)Schmidtea mediterranea, with fully sequenced and annotated genome.To search for homologs of glucocorticoid receptor proteins in a closely related species The aim of our research was to study the morphogenetic effects of the steroid hormones: hydrocortisone and its synthetic analogue methylprednisolone, capable of acting as an agonist and antagonist of hydrocortisone, in regenerating Girardia tigrina  was used as a model organism for the experiments. Planarians were kept in a darkened room at a temperature of 20–23 °C. Feeding was performed once a week by larvae of chironomids . In the experiments, planarians with a body length of 9–11 mm were used. Previously, they were kept without feeding for 7 days.The laboratory culture of the asexual planarian species The effect of two steroid hormones of the glucocorticoid group on the reparative regeneration process was studied: hydrocortisone and a synthetic hormone of this group—methylprednisolone, a well-known agonist of glucocorticoid receptors and to a lesser extent of mineralocorticoids .−1 M or methylprednisolone  in DMSO in concentration of 10−1 M was added into a 50 mL glass with planarians in an amount sufficient to achieve the needed concentration.The hydrocortisone stock  in DMSO  at a concentration of 10The control and experimental group were kept in 50 mL of a solution of tap and distilled water (in a 2:1 ratio). In each glass, 30 individuals were kept for each experimental and control group. The test compound solutions were added to the experimental groups, to achieve the needed concentrations. The experiments were conducted in three independent series.The regeneration results were evaluated using the method of lifetime computer morphometry . The metThe decapitation and tail removal of planarians was performed as follows. The animals were placed upon a cooled (+6 °C) platform, stationed under a binocular microscope, . At a low temperature the planarians stop moving, which facilitates the operation. The operation was performed with a scalpel under 20× magnification.−3 M, 10−4 M, 10−5 M and 10−6 M, and three concentrations of methylprednisolone: 10−4 M, 10−5 M and 10−6 M were used in the experiments. The experiment lasted for 6 days. Measurements were conducted every 24 h for 4 days in the period from the 3rd to the 6th day of the experiment. The recovery rate of the blastema formed as a result of either decapitation (anterior blastema) or tail area removal (posterior blastema) was estimated. As a quantitative criterion for the development of the blastema and the assessment of the dynamics of the recovery of the original proportions of the animal body, the regeneration coefficient (RC) calculated by the formula was used:Four concentrations of hydrocortisone: 10The method of immunohistochemical labeling of neoblasts was used to identify mitotic cells in the planarian body . The plaAfter washing the planarians of secondary antibodies, they were placed in a Vectashield immunofluorescence medium  and visualized using an AxioScope A1 microscope  equipped with an AxioCamMRc camera . The calculation of the number of dividing cells and the planarian body area was performed using the ImageJ program. 2.The mitotic index (MI) was used as a criterion of neoblasts’ mitotic activity. It was calculated by the formula:Only the decapitated worms with regenerating head blastema  were useG. tigrina genome has not been sequenced yet, we have performed a search for orthologous proteins of glucocorticoid receptors (hydrocortisone) in the genome of a closely relates species, namely Schmidtea mediterrana, using the SmedGD genomic database . We used the BLAST algorithm for this, which was also used previously to search for amino acid sequences based on similarity with the target protein receptors in humans (Homo sapiens), house mouse (Mus musculus), African clawed frog (Xenopus laevis), and fruit fly (Drosophila melanogaster). The search was performed both by the complete amino acid sequence and on the basis of ligand-binding domains.As the www.ebi.ac.uk/, accessed on 21 October 2022).The homologues found in the planarian genome were also compared in the GenBank database and the most similar sequences were selected. Domain analysis in the protein structure was determined using the NCBI Conserved domains database. Next, multiple alignment of amino acid sequences was performed with the ClustalW algorithm using an online service EMBL .Phylogenetic comparisons of amino acid sequences were performed using an online service, EMBL  involved in the regeneration regulation process started from 4 days after amputation  had a pronounced stimulating effect on the course of the regeneration process in head blastema (−6), the differences in the blastema size in experimental and control animals in this solution on the fifth day of the experiment were not significant (The three lower concentrations of hydrocortisone (10blastema a. The dunificant a.−3)  b. The hi−4 M had practically no effect on the regeneration processes of the tail blastema. The effect of the hormone at a concentration of 10−6 M had a significant stimulating effect only during the second day of the experiment. Only in a solution containing hydrocortisone at concentration of 10−5 M, was the stimulating effect of the hormone observed, and it persisted for five days of the experiment. On the sixth day, the values of RC in the control and experimental groups of planaria were represented by similar values , (−5 and 10−4 M) also stimulated the proliferative activity of neoblasts in this zone, but the intensity of their effect on this process was much lower than in the first case,  24 h after decapitation. The maximum values of MI were recorded with the lowest hydrocortisone concentration of (10−6 M), a. Higherst case, b,c.The tail zone farthest from the amputation site (zone III) turned out to be the least sensitive to the hormone. The studied concentrations did not significantly affect the proliferative activity of the neoblasts located here a–c.−6 M practically did not affect the number of stem cells: the values of MI in animals were similar to those in the control group, (−5 and 10−4 M changed to the opposite: both concentrations reduced the rate of mitosis. A particularly noticeable inhibitory effect of the hormone was registered under the influence of the hormone at a concentration of 10−5 M (A day after decapitation (48 h), the nature of the hormone’s effect on the proliferative activity of neoblasts changed. In the decapitation zone (zone I), the hormone at a concentration of 10l group, d. The hyf 10−5 M e.−6 M had a significant inhibitory effect on the mitotic activity of neoblasts in the near-pharyngeal zone (zone II)  to the medium did not affect the change in the number of dividing cells (The hormone at a concentration of 10zone II) d. An inc hormone e. The adng cells f.−6 M and 10−4 M) inhibited the regeneration process of the head blastema (−5 M) did not affect the growth activity of the blastema. This type of hormone exposure persisted for five days of the experiment. It should be noted that the effect of the highest concentration of methylprednisolone (10−4 M) on the fourth day of the experiment was not statistically significant. By the sixth day, all the experimental concentrations of the hormone did not have a significant effect on the regeneration process course: the RC values in animals from the experimental groups were close to the control ones (In the first days after surgery (day 3), the lowest and highest concentrations (10blastema a. The avrol ones a.−4 M) changed the rate of blastema growth. Its effect was inhibitory, and the next day (day 4) changed to the opposite; the hormone stimulated the growth of the blastema. The lowest hormone concentration was involved in the regeneration regulation process only by the fifth experimental day. An increase in the regeneration rate was observed in experimental media. As a result, the RC indicators in planarians significantly exceeded the control values. At the final stage of the experiment (day 6), none of the concentrations affected the recovery rate of the tail blastema (The recovery of the regenerating tail blastema was low depending on methylprednisolone b. On theblastema b.−4 M) had a pronounced inhibitory effect a day after amputation (24 h). The MI values in all the studied zones of the worm body in the experiment were significantly lower than in the control. Particularly low values of neoblast mitotic activity were recorded in zone I and zone III (The highest concentration of the hormone (10zone III c.−5 M) was not practically involved in cell division regulation in any of the studied sites on the regenerating fragment. The differences in the MI in experimental and control animals were not consistent  and near-pharyngeal zones (zone II). In the tail zone (zone III), the hormone significantly inhibited this process a.None of the methylprednisolone concentrations had a statistically significant effect on the mitogenic activity of neoblasts 48 h after amputation d–f.Schmidtea mediterranea. It also has a high degree of homology to another nuclear receptor—nuclear liver factor 4.An orthologous protein of the glucocorticoid receptor was detected using a BLAST algorithm search in the SmedGD genomic database of S. mediterranea genome consists of 665 amino acids .The glucocorticoid receptor discovered in the no acids . Analysino acids . MoreoveThe multiple alignment of the amino acid sequence of the glucocorticoid receptor planarian orthologue demonstrated that this protein has a 57.8% homology with the consensus sequence of the human glucocorticoid receptor protein .Drosophila, are quite far from the group of vertebrate glucocorticoid receptor orthologs but they descend from a common ancestor. The results are consistent with the data on the taxa origin, since the cladogram traces the origin of the glucocorticoid receptor from a common ancestor in amphibians, as well as the later isolation of the avian and mammalian receptor .The identified inhibitory effect of hydrocortisone is confirmed by the data of classical endocrinology, where the hormone is described mainly as an anti-growth one . CurrentIt was shown previously, that the nature of hydrocortisone action may vary depending on its concentration. In vitro experiments demonstrated that lower hormone concentrations can stimulate growth and anabolic processes in the connective tissue structures of vertebrates, and higher ones can suppress the same processes ,25. This−4 to 10−6 M). Unlike hydrocortisone, they had different effects on the degree and nature of the responses of the planarian regenerating blastema. In medical practice, methylprednisolone and its derivatives are known as synthetic steroids widely used as anti-inflammatory drugs or immunosuppressants [The effect of methylprednisolone on regeneration activity was studied before only in a range of low concentrations  had an inhibitory effect, as opposed to hydrocortisone. The concentration of 10−5 M turned out to be inactive.While studying growth of the regenerating head blastema in methylprednisolone solutions, it was found that two of the concentrations (10−5 M) was inactive, in contrast to the stimulating effect of hydrocortisone. The nature of the effect of the highest concentration of methylprednisolone (10−4 M) at the beginning of the experiment changed from negative to positive over time, while hydrocortisone was not involved in the regulation of planarian recovery during this period.For the regenerating head blastema less sensitive to the introduction of the hormone, there is not enough data for general conclusions about the nature of its effect on the regeneration rate. In cases where a comparison of similar concentrations of two hormones was possible, it was found that one of the concentrations of methylprednisolone . Based on the results, it can be proposed that flatworms have nuclear receptors for glucocorticoid hormones. All of them have rather conservative domains that characterize the performed functions. The specific effects of each hormone that we have identified may be associated with the formation of specific hormone–receptor complexes. They include a cascade of biochemical reactions characteristic only for such a given complex.We found an orthologous protein of the glucocorticoid receptor in the SmedGD genomic database of the The differences we found in the response to the added hormones in the anterior and posterior regenerants may be related to differences in their morphogenetic patterns. Their formation in all planarian tissues normally and during regeneration processes is ensured by the work of “positional control genes” encoding such signaling pathways as: Wnt, FGF, BMP and Hedgehog (Hh). The Wnt signaling pathway controls the correct restoration of the anterior and posterior poles during planarian regeneration and is activated immediately after injury. At the same time, the main proteins of the Wnt pathway are expressed at the posterior pole, and the Wnt antagonist protein notum is expressed only at the anterior pole .Steroid hormones can be included in the expression regulation of different genes at the level of nuclear receptors, thereby changing the work of the corresponding signaling pathways in the regenerating planarian blastema. This may be related to the identified features of the studied steroids’ effects on the recovery of the anterior and posterior structures of planaria. Another reason for the different sensitivity to hormones of recovering areas may be the features of the disto–proximal distribution of receptors in the planarian body ,33.Girardia tigrina is confirmed by data obtained from other planarian species. The treatment of the Dugesia lugubris planarian by prednisone—the dehydrated analogue of hydrocortisone had a stimulating effect on their growth which lasted until the complete finish of regeneration. The effect was more pronounced in the anterior regenerants than in the posterior ones [The morphogenetic effect of glucocorticoid group steroids indicated by us in ior ones .Daphnia magna crustaceans, it had a stimulating effect on somatic growth and reproductive activity [Sarcophagidae, a stimulating effect of this hormone was found [The data on the effect of glucocorticoids on a range of physiological processes in other invertebrate taxa could be another confirmation of the possibility of this group of hormones to be involved in the regulation of the growth process in planarians. For example, in activity . In bivaas found . Girardia tigrina planaria also influenced the course of regeneration in the head and tail blastema. The anterior regenerants were more sensitive to these hormones, they retained the head ganglion after amputation. The effect of these hormones on the regeneration process rate differed: estradiol had a stimulating effect, and testosterone, on the contrary, had an inhibitory effect on the recovery process [The addition of two sex steroids—estradiol and testosterone—into the media with  process .Such sensitivity to steroid hormones can also be explained by the presence of a presumably two-level endocrine system in flatworms. It includes two ways of humoral regulation of various body functions: (1) with the help of neurosecretory compounds synthesized in different parts of the nervous system and 2) using different groups of steroid hormones  using di,7,35. ThPlathelminthes, the neurosecretory system is most well studied, in parasitic species—the steroid profile. The steroid hormones, namely, hydrocortisone and testosterone, were detected and quantified in the Fasciola hepatica parasitic flatworm in concentrations of 0.001223 ug and 0.01193 ug per g of body weight, respectively [In free-living ectively .Clonorchis sinensis, Opisthorchis viverrini and the blood fluke Schistosoma haematobium, the steroid compounds have been found by means of HPLC and mass spectrometry: namely the oxysterols and catechol estrogens [In another three parasitic flatworm species, strogens .Cestoda) possess their own systems of steroidogenesis. So, the Taenia crassiceps worms are able to transform androstendione into testosterone in in vitro experiments, which was proven by recrystallization studies. In vitro experiments have also shown that steroidogenesis blocking compounds stop the steroid synthesis in flatworms, which confirms the presence of enzymatic pathways responsible for sex steroid synthesis in these animals [The flatworms  suppressed the division of neoblasts in the postblastemic region.The ability of the hormones to influence regeneration processes is confirmed by the data we have obtained on their ability to influence the activity of neoblast proliferation. During the first mitotic peak, hydrocortisone significantly increased the rate of mitosis in the periwound zone. Methylprednisolone, on the contrary, mainly suppressed the proliferation of neoblasts and did so in the tail zone where normally their number is already small. The second peak is characterized by a complete absence of mitotic activity of methylprednisolone and a change in the nature of the hydrocortisone effect. Two hormone concentrations (10Immunohistochemical labeling of neoblasts was performed using antibodies to phospho-histone H3. This histone is a marker of the transition from the G2 phase to the mitosis phase in the cell cycle. Obviously, hydrocortisone is able to both shorten and increase the duration of this transition. Methylprednisolone acted only as an inhibitor in this process.Girardia tigrina planarian. There is a dependence of the nature and exposure degree of hormones on their qualitative specificity, the concentration and the regenerating fragment; the head or tail area. The anterior regenerants have less sensitivity to the studied hormones.Hydrocortisone and its synthetic analogue methylprednisolone are able to influence the course of the morphogenetic process in the The nature of the hydrocortisone effect on the regeneration process differs from its synthetic analogue methylprednisolone which can act as an antagonist and an agonist of hydrocortisone. A high concentration of hydrocortisone suppresses the recovery of the amputated area, and low ones stimulate it. The nature of the methylprednisolone effect in the range of concentrations similar to the hydrocortisone ones, was more dependent on the specific concentration of the hormone and was mainly inhibitory.Both hormones change the mitotic activity of neoblasts during the first peak of proliferation. Their effects are the opposite: hydrocortisone stimulates the process in the periwound zone, and methylprednisolone, on the contrary, inhibits it mainly in the zone furthest from the wound. The second wave of mitotic activity does not depend on methylprednisolone, and hydrocortisone inhibits the proliferation of neoblasts.Schmidtea mediterranea planaria. Based on the obtained results, it can be proposed that flatworms have nuclear receptors for glucocorticoid steroid hormones. All of them have conservative domains that characterize the performed functions and probably play a role in many biological processes in planarians.An orthologous protein of the glucocorticoid receptor was found in the SmedGD genomic database of the It can be assumed as a working hypothesis that exogenous hormones from the glucocorticoid group affect the endogenous mechanisms of endocrine regeneration, including through hormone–receptor interaction. The revealed features of the effects of hydrocortisone and methylprednisolone may be associated with the formation of specific hormone–receptor complexes, as well as with the dependence of the methylprednisolone effects on the type of interaction; as an agonist or an antagonist.


Nuclear trafficking that accompanies ubiquitination and phosphorylation at critical residues plays essential roles in the activation of PKCβII, a representative member of the conventional PKC isoforms. PKCβII, a conventional PKC family member, plays critical roles in the regulation of a variety of cellular functions. Here, we employed loss-of-function approaches and mutants of PKCβII with altered phosphorylation and protein interaction behaviors to identify the cellular mechanisms underlying the activation of PKCβII. Our results show that 3-phosphoinositide–dependent protein kinase-1 (PDK1)–mediated constitutive phosphorylation of PKCβII at the activation loop (T500) is required for phorbol ester–induced nuclear entry and subsequent Mdm2-mediated ubiquitination of PKCβII, whereas ubiquitination of PKCβII is required for the PDK1-mediated inducible phosphorylation of PKCβII at T500 in the nucleus. After moving out of the nucleus, PKCβII interacts with actin, undergoes inducible mTORC2-mediated phosphorylation at the turn motif (T641), interacts with clathrin, and then translocates to the plasma membrane. This overall cascade of cellular events intertwined with the phosphorylation at critical residues and Mdm2-mediated ubiquitination in the nucleus and along with interactions with actin and clathrin plays roles that encompass the core processes of PKC activation. PKC is a family of protein kinases that exert functional control over other proteins through the phosphorylation of serine and threonine residues . Based o2+, which bind to the C1 and C2 region  has long been proposed : F-actinBased on this experimental evidence, in the current study, we set out to clarify the molecular mechanisms involved in the activation of PKCβII. To that end, we determined the mechanistic and functional relationships between ubiquitination and constitutive or inducible phosphorylation at the three major motifs, focusing on interactions with actin and clathrin.3 receptor (D3R)  see , which iD3R) see . These fD3R) see . In partD3R) see . In agreD3R) see .3R, cells were pretreated with latrunculin A (LatA), which sequesters G-actin to suppress actin polymerization (To further characterize the roles of actin in PKCβII-mediated endocytosis of Drization . As show3R was significantly inhibited when the endogenous clathrin heavy chain (CHC) was knocked down; in contrast, knockdown of cellular caveolin1 (Cav1) did not affect it, suggesting that D3R endocytosis occurred via CME. The inhibitory activities of LatA on the D3R endocytosis were abolished in CHC-KD cells but remained intact in control knockdown (Con-KD) cells and Cav1-KD cells  (3R endocytosis (Because actin is involved in the CME of DLSQE281)  CHCX-PKCLSQE281)  nor did LSQE281)  and failocytosis . These rA recent study showed that Mdm2-mediated ubiquitination of PKCβII, whose original role is the degradation of target protein, is required for the PMA-induced translocation to the plasma membrane . BecauseLike other isotypes of PKCs that belong to conventional and novel families, PKCβII is functionally regulated by specific phosphorylations, including those at three critical sites located in the activation loop (T500), TM (T641), and HM (S660) . BecauseThese results suggest that PDK1-mediated constitutive phosphorylation of PKCβII at T500 is likely to be involved in the ubiquitination of PKCβII; in turn, ubiquitination occurs at upstream levels of actin or clathrin, allowing PKCβII to translocate to the plasma membrane.3R was enhanced by co-expression of WT- or T641A-PKCβII but not T500A-PKCβII , which inhibits exportin1, the major karyopherin receptor that mediates the nuclear export of a wide range of proteins. PMA-induced interactions between PDK1 and PKCβII were enhanced at 1 min , but theBecause Mdm2-mediated ubiquitination and interaction with actin and clathrin are needed for the translocation of PKCβII to the plasma membrane, we assessed the roles of ubiquitination in the inducible phosphorylation of PKCβII. As shown in According to our results, the constitutive phosphorylation of PKCβII at T500 that occurs before PKCβII enters the nucleus is a prerequisite for the PMA-induced nuclear entry, which is required for Mdm2-mediated ubiquitination. Ubiquitination in turn determines the inducible phosphorylation of PKCβII at T500 and T641. Thus, we determined how the inducible phosphorylation of PKCβII at T500 and T641 is functionally connected to the interaction with actin.As reported previously , PKCβII Next, we examined whether PKCβII phosphorylation at T641 occurs before or after it binds with actin. As shown in Finally, we determined whether the interaction with actin occurs in the nucleus or after PKCβII moves out of the nucleus. As shown in These results suggest that ubiquitinated PKCβII, which can be inducibly phosphorylated at T500 , moves oAs for actin, we determined whether PKCβII interacts with clathrin in the nucleus or cytosol. We found that pretreatment with LMB blocked the PMA-induced interaction between PKCβII and clathrin , suggest2+ levels, PKCβII is recruited to underneath the plasma membrane where DAG and phosphatidylserine bind to the C1 domain. Thereafter, PKCβII undergoes conformational changes, accompanied by the dissociation of pseudosubstrate from the C4 domain, leading to the catalytic activation of PKCβII  and stays in the cytosol in the “primed” state. Even though PKCβII is inactive in the primed state because the pseudosubstrate is still bound to the substrate binding site, it retains its competency for activation by cellular stimuli . With thf PKCβII .Current studies are showing that the phosphorylation of PKCβII at three major motifs plays critical roles in its activation . The phoIn this study, we determined the molecular mechanisms involved in the activation of PKCβII based on its phosphorylation at three conserved phosphorylation sites; its Mdm2-mediated ubiquitination; and its interactions with Mdm2, actin, and clathrin.According to the results obtained in this study, the role of phosphorylation differs depending on whether it occurs in a constitutive or an inducible manner. For PKC activation, PKC needs to enter the nucleus to be ubiquitinated by Mdm2 and inducibly phosphorylated by PDK1 in the A-loop. Actin and clathrin are needed for the subsequent phosphorylation at TM and the translocation to the plasma membrane, respectively.Our study shows that phosphorylation of PKCβII at T500 and T641 occurs in two different ways—constitutive and induced—and that they have different functional meanings. Constitutive phosphorylation of PKCβII at T500, which occurs in the cytosol in a basal state, is required for the nuclear entry of PKCβII in response to PMA treatment and the subsequent ubiquitination in the nucleus. In contrast, inducible phosphorylation occurs after PKCβII enters the nucleus and plays a key role in mediating subsequent PKCβII activation processes that include binding with actin, phosphorylation at T641 by mTORC2, and translocation to the plasma membrane after binding with clathrin.The fact that constitutive phosphorylation is required for ubiquitination and that, in turn, ubiquitination is necessary for inducible phosphorylation is demonstrated by several experimental results of the current study. For example, PMA treatment increased the interactions between PDK1 and PKCβII at 1–2 min after PMA treatment and the phosphorylation of PKCβII at T500, and both were abolished by mutations in the ubiquitination sites (2KR-PKCβII) or by knockdown of cellular Mdm2 . The basVarious functional interactions between actin filament and CME have been reported. For example, the interaction between actin microfilaments and clathrin-coated structures  and requIn conclusion, our study reveals a sequence of cascades involved in PMA-induced PKCβII activation. (1) PDK1-mediated constitutive phosphorylation of PKCβII at T500 in the cytosol plays permissive roles in PKCβII activation by allowing the nuclear entry of PKCβII in response to PMA treatment. (2) PKCβII undergoes K63-linked Mdm2-mediated ubiquitination in the nucleus, which is needed for the interaction with PDK1, resulting in the inducible phosphorylation of PKCβII at T500. (3) Inducible phosphorylation of PKCβII at T500 allows the nuclear export of the ubiquitinated PKCβII and interactions with actin in the cytosol. (4) Interactions with actin lead to the dissociation of ubiquitinated PKCβII from PDK1, which is needed for the mTORC2-mediated phosphorylation of PKCβII at T641. (5) Ubiquitination and inducible phosphorylation of PKCβII at T641 allow the interaction with clathrin and the translocation to the plasma membrane.3H]-sulpiride was purchased from Perkin Elmer Life Sciences.PMA, leptomycin A (LMB), agarose beads coated with monoclonal antibodies against FLAG epitope, rabbit anti-FLAG M2 antibodies (AB_439687), rabbit antibodies against GFP (AB_439690), and HA antibodies (AB_2610070) were purchased from Sigma-Aldrich Chemical Co. Latrunculin A (LatA) was obtained from Cayman Chemical. Mouse monoclonal antibodies against CHC (AB_397865) were obtained from BD Bioscience. Rabbit antibodies against rictor (AB_2179961), phosphor-PKCα/βII (Thr638/641) (AB_2284224), K48-linkage specific polyubiquitin (AB_10557239), and K63-linkage specific polyubiquitin rabbit mAb (HRP conjugate) antibody (AB_2798064) were purchased from Cell Signaling Technology. Anti-rabbit HRP-conjugated secondary antibodies (AB_2533967) and phospho-PKCβ1&2 (Thr500) (AB_2533805) were obtained from Thermo Fisher Scientific, and anti-mouse HRP-conjugated secondary antibodies (AB_10015289) were purchased from Jackson ImmunoResearch Laboratories, Inc. Mdm2 E3 ligase inhibitor and antibodies against Mdm2 (AB_627920) were obtained from Santa Cruz Biotechnology and torin1 from Selleck Chemicals. Alexa Fluor 594–conjugated anti-rabbit (AB_142057) and anti-mouse (AB_141593) antibodies were from Molecular Probes. -sulpiride (3R were seeded (1.5 × 105 cells/well) 1 d after transfection on 24-well plates. The following day, the cells were rinsed once and pre-incubated for 15 min with 0.5 ml pre-warmed serum-free medium containing 10 mM Hepes (pH 7.4) at 37°C. The cells were stimulated with 100 nM PMA or 1 μM isoproterenol for 30 min. They were then incubated with 250 μl of [3H]-sulpiride (7.2 nM) at 4°C for 150 min in the presence or absence of unlabeled competitive inhibitor . The cells were washed thrice with the same medium, and 1% SDS was added. Samples were mixed with 2 ml Lefko-Fluor scintillation fluid and counted on a liquid scintillation analyzer.Endocytosis of Dulpiride . HEK-293t tests to compare two-group or one-way ANOVA with Tukey’s post hoc test for multiple group comparisons. A P-value < 0.05 was considered significant.All data are expressed as the mean ± SD. Statistical significance was analyzed using paired two-tailed Data and the materials used in this study are available to any qualified researcher upon reasonable request addressed to K-M Kim.