Proteasome inhibitors are useful for biochemical research and clinical treatment. In our previous study, we reported that the 4N-coordinated platinum complexes with anthracenyl ring and heterocycle exhibited proteasome-inhibitory activity. In the present study, the structure–activity relationships and characterization of these complexes were determined for the elucidation of the role of aromatic ligands. Lineweaver–Burk analysis revealed that the chemical structure of heterocycles affects the binding mode of platinum complexes. Platinum complexes with anthracenyl ring and pyridine showed competitive inhibition, although platinum complexes with anthracenyl ring and phenanthroline showed non-competitive inhibition. The structure–activity relationships demonstrated that anthracenyl moiety plays a crucial role in proteasome-inhibitory activity. The platinum complexes with naphthyl or phenyl rings exhibited lower inhibitory activities than the platinum complex with anthracenyl ring. The reactivity with N-acetylcysteine varied according to the chemical structure of complexes. In particular, non-covalent interactions involving aromatic rings (e.g. aromatic–aromatic interactions and hydrophobic interactions) have many characteristics and are very interesting. Aromatic rings play crucial roles in the structure and function of proteins , C 40.71%; H 4.75%; N 8.26%. 1H NMR (D2O), 8.55 , 8.34 , 8.16–8.01 , 7.97 (m 1H), 7.88 , 7.82–7.73 , 7.75–7.69 , 7.70–7.60 , 7.57 , 7.49 , 7.37 , 6.91 , 4.69 , 4.36 , 3.44 , 3.16 , 3.00 . ESI-MS (m/z): 588.02 [M-Cl]+Complex 4.2.2.2Cl2  in H2O (20 ml), N-benzyl-1,2-ethanediamine  was added. The mixture was stirred at 80°C for 16 h. The solution was filtered immediately, and the filtrate was concentrated under vacuum. Acetone was added, and the pale-yellow powder was obtained. The powder was reprecipitated from water/acetone. Yield: 190 mg (62%). Elemental analysis: found, C 37.75%; H 4.80%; N 8.79%. Calcd for [C19H24N4Cl2Pt · 2H2O], C 37.38%; H 4.62%; N 9.18%.1H NMR (D2O), δ: 8.71 , 8.82–8.53 , 8.55 , 8.07–7.88 (m 1H), 7.70–7.46 , 7.63–7.47 , 7.47–7.37 , 7.37–7.25 , 4.01, 3.05, 2.84, 2.62. ESI-MS (m/z): 538.14 [M-Cl]+To a suspension of Pt(py)4.3.λex, 365 nm) was measured by using a fluorescence microplate reader. The inhibition against PGPH activity of the 20S proteasome was determined as described above except that Z-LLE-AMC was used as the substrate. For the assay of inhibition against the trypsin-like activity of complex 2, the SDS was omitted from the assay buffer and Boc-LRR-AMC was used as the substrate. The assay of inhibition against the trypsin-like activity of complexes 1 and 3 was carried out by the Screening Committee of Anticancer Drugs .For the proteasome inhibition assays, the peptide-7-amino-4-methylcoumarin (AMC) substrates (25 µM Suc-LLVY-AMC) and inhibitors were added to assay solutions. The assay buffer had the following composition: 25 mM HEPES, 0.5 mM EDTA, 0.03% SDS (pH 8.0). Human erythrocyte 20S proteasome (100 ng) was added to the assay buffer containing substrates and inhibitors at a final volume of 100 µl. After incubation at 37°C for 1 h, the fluorescence emission spectrum at 450 nm , enzyme activity was measured at various concentrations of Suc-LLVY-AMC .4.5.Cultures of colon26 were grown at 37°C in RPMI1640 supplemented with 10% fetal bovine serum. Whole-cell lysates were prepared according to the method in the literature .4.6.λex = 365 nm) by using a fluorescence microplate reader.For the determination of inhibitory activity in whole-cell lysates, peptide-AMC substrates (50 µM Suc-LLVY-AMC), inhibitors and the whole-cell lysate (15 µg) were added to the assay solutions. The following assay buffer was used: 25 mM HEPES, 0.5 mM EDTA, 0.03% SDS (pH 8.0). After incubation at 37°C for 4 h, the fluorescence emission was measured at 450 nm , 10 mM platinum complexes were incubated with 10 mM N-acetylcysteine at 25°C. The products were analysed by


Ranunculus glacialis grows and reproduces successfully, although the snow-free time period is short (2–3 months) and night frosts are frequent. At a nival site (3185 m a.s.l.), we disentangled the interplay between the atmospheric temperature, leaf temperatures, and leaf freezing frequency to assess the actual strain. For a comprehensive understanding, the freezing behavior from the whole plant to the leaf and cellular level and its physiological after-effects as well as cell wall chemistry were studied. The atmospheric temperatures did not mirror the leaf temperatures, which could be 9.3 °C lower. Leaf freezing occurred even when the air temperature was above 0 °C. Ice nucleation at on average −2.6 °C started usually independently in each leaf, as the shoot is deep-seated in unfrozen soil. All the mesophyll cells were subjected to freezing cytorrhysis. Huge ice masses formed in the intercellular spaces of the spongy parenchyma. After thawing, photosynthesis was unaffected regardless of whether ice had formed. The cell walls were pectin-rich and triglycerides occurred, particularly in the spongy parenchyma. At high elevations, atmospheric temperatures fail to predict plant freezing. Shoot burial prevents ice spreading, specific tissue architecture enables ice management, and the flexibility of cell walls allows recurrent freezing cytorrhysis. The peculiar patterning of triglycerides close to ice rewards further investigation. Ranunculus glacialis, night frosts were recorded on 68% of 74 snow-free nights [In the nival life zone, the snow-free time slot is short, not exceeding 2–3 months ,2. Durine nights ,6,7. Howe nights . In the R. glacialis is one of the nival herbs of the European Alps that can grow at the highest elevations  of R. glacialis leaves from lower elevations (2600–2800 m a.s.l.) ranged between −8.2 °C and −9.4 °C [R. glacialis down to −13.0 °C (LT50) [50; [iss Alps ). The spiss Alps . In earl −9.4 °C . In a reC (LT50) . In cont2 gas diffusion and photosynthesis, also the after-effects of leaf freezing are well known. Depending on their severity, freezing temperatures and ice formation in leaves can implicate significant photosynthetic depressions for several consecutive days [R. glacialis is possible and how the challenge of frequent night frosts is managed at a physiological level.Night frosts and ice formation in leaves sensitively affect photosynthetic performance. While ice in the intercellular space per se inhibits COive days ; on the ive days . Given tPachysandra terminalis [Sphagnum capillifolium [Once ice forms in ice-tolerant plant tissues, cells are exposed to mechanical stress by extracellular ice formation and freeze dehydration. These processes can result in the deformation (“caving-in”) of cell walls, so-called freezing cytorrhysis ,16. Strurminalis ; Sphagnulifolium ). Freezilifolium . A prerelifolium . Furtherlifolium ,22. Stillifolium ). AlthouR. glacialis, we aimed to assess: (1) How frequently ice forms in the leaves within the short snow-free period and how whole plants freeze under natural (radiative) night frosts. Freezing exotherms and whole plant freezing were monitored on site and compared to the results of infrared differential thermal analysis (IDTA) obtained under controlled freezing conditions. (2) The effect of ice formation on the “after-freezing physiological performance” was determined by measurements of the chlorophyll fluorescence and photosynthetic gas exchange. (3) In frozen leaves, ice masses were localized with cryo-microscopy in reflected polarized light  of  , and thIndeed, the in situ leaf temperatures deviated greatly from the air temperature; the daily minimum and maximum air temperatures were in a narrow range between −7.2 and +16.2 °C within the growth periods of 2018 and 2019 (blue ribbon in n = 77)  values were assessed four times in the growing seasons of 2018 and 2019, and were between −9.0 °C and −13.0 °C  was unaffected by the freezing treatment and time. These findings additionally apply to the efficiency of PS II (Fv/Fm). However, measurements revealed that the treatment (frozen vs. unfrozen) was almost significantly influencing Fv/Fm (p = 0.050). Before the treatments, the Fv/Fm was 0.79/0.79 (unfrozen/frozen), 1 h after the treatments it was 0.79/0.76, and 1 day after it was 0.77/0.75. The overall Fv/Fm values were not significantly different; consequently, ice in the leaves of R. glacialis does not affect the photosystem II efficiency.Under the experimental conditions, neither 1 h after the treatments (frozen at −3 °C vs. unfrozen at −1 °C) nor 1 day afterwards did the detached leaves show a significant difference in gas exchange  . A/GH2O rpl) revealed that, particularly in the spongy parenchyma, huge ice masses are accommodated  and spongy tissue layers with a thickness of 309 ± 67 µm (n = 30). The palisade cells are tightly packed and close together, with apparently only narrow intercellular spaces. Cells in the spongy parenchyma tissue are heterogeneously shaped and have much larger intercellular spaces than the palisade cells. The intercellular spaces occupied 13% ± 3% (n = 3) of the total area of the leaf cross sections. With CMrpl in frozen leaves, no major ice masses could be detected in the palisade tissue. Ice masses were exclusively found in the intercellular spaces of the spongy parenchyma tissue  was reduced to 54% ± 15% of the initial value, whereas the spongy parenchyma CA (n = 15) was reduced to 48% ± 15% of the untreated controls; the CA of the spongy parenchyma cells was more pronouncedly reduced than that of the palisade cells, but not significantly (p = 0.06). Upon extracellular freezing, the palisade cells showed an intensive reduction in diameter, but not in cell length  and 62% ± 4% (2019)) exposed to nocturnal freezing temperatures. In contrast, during the unusually warm summer of 2018, the air temperature dropped only once, during a cold snap with snowfalls, severely below 0 °C. When the leaf temperature drops below 0 °C, two scenarios are possible: either the leaf tissues remain unfrozen or upon ice nucleation extracellular ice forms. Our results show that, in the nival habitat, ice nucleation occurred in the leaves of R. glacialis at −2.6 ± 0.9 °C. This corresponds to the ice nucleation temperatures measured in nature in various species in different environments .R. glacialis never froze at once. Independent ice nucleation events were necessary. Ice propagation from one leaf into another is thermally prevented, as the connecting shoot is buried at a depth of several centimeters in the unfrozen soil. Already, Körner [R. glacialis can remain unfrozen during night frosts. Staying unfrozen as long as possible may be assigned to a sort of transient ice avoidance strategy as a first line of defense. In case of ice nucleation, a second line of defense by reversible freezing cytorrhysis has to be assembled. To minimize the duration of exposure to the peculiar strains upon freezing cytorrhysis, transient ice avoidance may be supportive. Still, the effects of low temperature exposure on photosynthesis were similarly independent from the kind of freezing process, neither unfrozen nor extracellular ice formation and freezing cytorrhysis had a specific negative impact on the photosynthetic gas exchange and photosystem II functioning. These results corroborate earlier findings from an afroalpine plant [In the field as well as in the lab experiments, individuals of , Körner  suggestene plant .R. glacialis are well adapted to the low-temperature conditions of the nival zone. Exhibiting such a high freezing resistance even during the growing period [The minimum leaf temperatures during the observation periods in 2018 and 2019 never dropped below the maximum freezing resistance capacity; hence, the leaves of g period ,37 in thThe tremendous cellular changes observed upon ice nucleation and freezing cytorrhysis accompanied by severe freeze dehydration may emphasize the importance of the ice avoidance strategy. However, the accommodation of ice masses and enduring the resulting strains is key for survival and reproduction in the nival zone ,7. Cryo-Sphagnum capillifolium showed a cell diameter decrease down to 43% of the initial value during freezing cytorrhysis at −5 °C—i.e., an approximated volume reduction of 82% [R. glacialis are very irregularly shaped, quantifying the decrease in cell volume is difficult. For the palisade cells, by approximating a cylindrical shape the volume decrease would be 73%. Indeed, despite morphologic and phylogenetic differences, the quantity of freezing-induced dehydration seems quite similar among the three species.At the cellular level, severe freezing cytorrhysis was detected in the palisade cells  as well as the spongy parenchyma cells . In comparison, the peat moss n of 82% . The dehAs illustrated by the FM 1-43 staining of palisade cells, mostly the cell diameter was reduced, but not the cell length. This may suggest structural differences within the cell wall of palisade parenchyma cells, or else that the tissue architecture of the tightly packed palisade cells only allows changes in diameter.R. glacialis by FM 1-43 clearly allowed to determine the changes between the untreated and the frozen cells. Although diffuse changes in a blurred plasma membrane were captured during freezing, the distinct formation of FIVs could not be demonstrated, but their existence cannot be excluded.Whether the plasma membranes change during freezing is more difficult to judge even when stained by FM 1-43. A study by Yamazaki, Kawamura and Uemura  has showrpl nicely correlates with the observation of an even stronger freezing cytorrhysis within this tissue, which, however, was not significant. The freeze dehydration of palisade parenchyma cells must occur via the spongy parenchyma cells. It seems to be the cell walls of the spongy parenchyma cells where the intracellular water is transferred to the extracellular ice. This indicates that the determination of where ice forms may be decided by predetermined local cell wall patches through which water is transferred from the supercooled cells to the apoplast. Additionally, this might explain why, immediately after thawing, the CA of the spongy cells was higher than that of the palisade cells , and the lipid droplets (triglyceride) prominently occurring in the spongy tissue. The ice barrier tissue below buds of Norway spruce that transfers water from the bud to the extraorgan ice masses in the shoot below was also characterized by pectin-rich cell walls [Raman spectroscopy demonstrates peculiar molecular components in the cell walls but also in the lumen of cells of the mesophyll of ll walls ,45. Pectll walls ,47,48. TRanunculus glacialis L. was studied at 3185 m a.s.l. at Mt. Kleiner Isidor (N 46°58.424 E 11°6.436 E) in the Austrian Central Alps. The site is located at the upper distributional vertical range of the species—i.e., 2300 to 3200 m a.s.l.—but individuals can occasionally be found at even higher elevations [Ranunculus glacialis predominantly grows on limestone-free and nutrient-poor soils, mostly on moraines or screes [evations . Ranuncur screes .Samples for the investigations were taken either from Mt. Kleiner Isidor (in 2018 and 2019) or, as in 2011, from the location Eisgrat . In 2011, individuals were excavated and potted in a substrate mixture (2:1) of nutrient-poor and limestone-free soil and Vermiculite. Potted individuals were transferred to the Alpine Garden of the University of Innsbruck  on Mt. Patscherkofel. From these cultivated plants leaf samples could be collected for cryo-microscopic investigations. In 2018 and 2019, whole individuals were dug out and potted immediately on site into natural soil. Plants were transported in a chilled thermal bag to the lab. For the Infrared Differential Thermal Analysis (IDTA), they were stored in darkness at +4 °C until the beginning of the experiments. For the gas exchange measurements, the potted plants were transferred into a growth chamber  for 3 days to eliminate potential physiological limitations due to prior frost nights. All the other experiments  were performed in 2018/2019 with single leaves, which were randomly collected from various individuals on Mt. Kleiner Isidor, stored in plastic bags on moist paper towels, transported to the laboratory, and stored in darkness at +4 °C. The transportation time from the Alpine Garden of the University of Innsbruck was 1 h, and from Mt. Kleiner Isidor it was 2 h to the lab in Innsbruck.TM  to the abaxial leaf surface. The logged data were uploaded automatically every 6 h to a server, which allowed online data monitoring. This was particularly useful after the winter, as the development of minor daily amplitudes of the soil surface temperature did forecast the loss of snow cover and hence the beginning of the growing season. Additionally, standardized air temperature data were provided by the Avalanche Warning Service Tyrol from the nearby weather station “Stubaier Gletscher-Schaufeljoch” .On Mt. Kleiner Isidor, microclimatic data were collected during the growing seasons of 2018 and 2019. Leaf temperatures and photosynthetic active photon flux density (PPFD) were recorded with a climate station . In 2018, the leaf temperature of 7 individuals was monitored with 25 thermocouples; in 2019, the temperature of 4 individuals was monitored with 12 thermocouples. In 2019, additionally the air temperature at the average canopy height (10 cm) and soil temperature at a depth of 5 cm were recorded. For the air temperature and soil temperature, assessment copper constantan thermocouples  were used; the air temperature sensor was protected by a solar radiation shield . For leaf temperature records, copper constantan thermocouples  were attached with Transpore−1). This allowed the detection of leaf-freezing-events, as, by the phase transition from liquid water to ice, crystallization heat is released, so-called freezing exotherms. A characteristic and sudden leaf temperature increase, which was not detectable in the air temperature, indicated the freezing exotherms. Exotherms were detected with computer aid by searching leaf temperature increases To estimate the incidence of leaf-freezing-events, the temperature data were monitored at a high frequency . With tR. glacialis was assessed by the simulation of artificial frost nights with different severities in the temperature-controlled commercial chest freezers. The experimental protocol is described precisely but briefly: Leaves were laid out on wet paper towels inside of a sealable plastic bag. To avoid the artificial supercooling of the samples, ice nucleation was triggered by the application of a few droplets of an ice nucleation active (INA) bacteria suspension (Pseudomonas syringae van HALL 1902) to the wet paper towels. For each treatment temperature, 10 randomly selected leaves were chosen. Treatment temperatures were set in a sequence with at maximum 3 °C difference. After controlled cooling (−3 °C h−1) to the target temperature, the leaves were exposed to the target temperature for 4 h, followed by controlled thawing (+3 °C h−1). After the treatment, the samples were stored at +20 °C under moderate light conditions until the frost damage became visible. Damage was assessed by the chlorophyll fluorescence. The chlorophyll fluorescence (Fv/Fm) was measured either with the MINI PAM  or the IMAGING-PAM M-Series .The freezing resistance of leaves of 50 values and standard deviation are presented.R. glacialis individuals, potted plants were monitored during freezing treatments down to −14 °C by Infrared Differential Thermal Analysis (IDTA). Infrared Differential Thermal Analysis spots ice nucleation, monitors ice propagation, and detects also supercooling. For IDTA, a digital infrared camera  was placed on a custom-made lid on top of a commercial chest freezer. The lid has an aperture for the optic of the infrared camera, which allowed monitoring the samples throughout the freezing treatment. The digital infrared camera recorded images at a frequency of 15 frames s−1. The IDTA subtraction of previous images was processed by Researcher Pro , as described previously in detail [For spatio-temporal analysis of the freezing behavior of n detail .To test whether the ice nucleation in leaves alters the leaf photosynthetic performance, gas exchange measurements were performed before, one hour after, and one day after a controlled freezing temperature treatment.n = 6) and placed in a vial filled with tap water. The chlorophyll fluorescence (Fv/Fm) and gas exchange parameters  were continuously recorded during a period of 1 h , to allow the gas exchange parameters to come into a steady state.Gas exchange measurements were started using two gas exchange measurement systems  in parallel. One fully developed leaf was randomly selected and excised from each individual  to control the leaf temperature during the following exposure to freezing temperatures. Two different experimental approaches were followed:(1)−1 down to −1 °C. After a 4 h exposure to −1 °C, the leaves were rewarmed (+5 °C h−1) to +4 °C until further use.Unfrozen leaves: Six leaves were cooled at a rate of −3 °C h(2)−1 down to −3 °C. After a 4 h lasting exposure to −3 °C, the leaves were rewarmed (+5 °C h−1) to +4 °C until further use. To ensure ice nucleation and extracellular ice formation in the leaf mesophyll, INA-bacteria (ice nucleation temperature approx. −2.3 °C) were added to the water one day before the freezing treatment.Frozen leaves: Six leaves were cooled at a rate of −3 °C hFine wire thermocouple sensors  were mounted to the leaf blade by adhesive tape Transpored were divided by the diffusive conductance GH2O.Gas exchange measurements of each individual leaf from both experimental approaches were determined 1 h and 1 d after the end of the freezing exposure treatments. To minimize the effect of different stomatal opening, the net assimilation rate A as well as the dark respiration rate Rrpl; [rpl, it is possible to visualize and localize ice crystals unambiguously in plant tissues. Briefly, a reflected light microscope  was placed in a fully temperature-controlled environment. Inside the controlled environment, entire leaves were frozen to sublethal temperatures; ice nucleation was ensured by INA bacteria. At the target temperature (−7 °C), leaves were sectioned transversally with a razorblade and the cut surface was immediately inspected with the CMrpl. With crossed polarization filters, the visibility of ice is enhanced, while the liquid water and surface reflections disappear.How ice is accommodated in viable leaves at sublethal temperatures was assessed by the recently developed method: cryo-microscopy in reflected polarized light . By CMrR. glacialis was studied on leaf cross sections exposed to a controlled freezing treatment under the microscope. Temperature control was performed by a specifically developed temperature-controlled chamber for the stage of an inverted light microscope . Cross The cellular reactions throughout the freezing treatment were monitored by a Zeiss Axiovert 200M inverted microscope; microscope control was provided by the software AxioVision Rel. 4.7, and for image acquisition an Axiocam Mrc5 camera was used . For plasma membrane staining, the amphiphilic fluorescent styryl dye FM 1–43 was applied. Cross sections were exposed to a 20 µM Film Tracer FM 1–43  for 10 to 15 min .To investigate the changes in the plasma membrane, improve the visibility of individual cells, and facilitate the tracking of changes in cell size, confocal laser scanning microscopy (CLSM) was used with LSM 5 PASCAL (Carl Zeiss). Samples were excited by an argon laser beam (488 nm). Emission was collected by two separate channels: (1) band-pass filter  at 505–550 nm, and (2) long-pass filter  at 560 nm. For details regarding confocal laser scanning microscopy, see Holzinger, Lütz and Karsten .The acquired image series in the course of the freezing treatments were analyzed as follows: As the 3D-shape of spongy parenchyma cells is very heterogeneous, we therefore opted for 2D analysis and measured the maximum cell area (CA) at the middle focal plane by outlining cells with the freehand selection tool in ImageJ . For eacR. glacialis leaves in context with the microstructure and a spatial resolution of 0.4 µm [−1 grating, UHTS 300, WITec GmbH, Germany), a CCD camera  and a 100× oil immersion objective  (Carl Zeiss), hyperspectral maps were acquired of the regions of interest. In some cases, the section quality was good enough to map across the entire leaf section  and spread through the mesophyll. Indeed, ice from a single leaf did not necessarily spread into the other leaves, suggesting the soil acting as a thermal ice barrier. During the second line of defense, after ice nucleation, ice is accommodated in the intercellular spaces of the spongy parenchyma and all mesophyll cells undergo severe, reversible freezing cytorrhysis. No photosynthetic limitations after ice formation could be observed. The mesophyll is characterized by huge intercellular spaces and a high cell wall flexibility. The pectin richness may be responsible for the high flexibility and water permeability during the freeze dehydration of cells. The mapping of triglyceride in the spongy parenchyma, where the majority of ice masses are located during freezing, may be the starting point for future research.At our investigation site in the nival zone, atmospheric temperatures insufficiently describe the environmental strain of leaves of


Eristena mangalis, with 1,724 individuals (relative abundance of 18.78%), followed by Monolepta sp. with 1,649 individuals (relative abundance of 17.96%). These are the phytophagous insects associated with mangrove leaves. This study concluded that the older mangrove ecosystem have a denser canopy that supports insect life.Mangrove restoration in Trenggalek, East Java has resulted an age variation of mangrove ecosystem. Diverse species of insects predominantly found in mangroves were collected using yellow pan traps, swipe nets and by direct picking from three different sites. This research was conducted from April until August 2015. There are 9,181 individual insects associated with mangroves comprised of 42 species from 31 families and eight orders. The first site or the 15 years old mangrove (66.22% canopy cover) indicated the highest Shannon diversity index at 2.54, Evenness index of 0.32 and Margalef richness index of 4.84. The lowest diversity was recorded in the third site or the five years old mangrove (19.65% canopy cover), with the Shannon diversity index at 2.28, Evenness index at 0.26 and Margalef richness index at 4.59. The most abundant species located was the  Three areas of mangrove restoration have different tree height and canopy cover, the older mangrove has the highest tree and denser canopy significantly compared to young mangrove.Eristena mangalis , Monolepta sp. (Coleoptera: Chrysomelidae) and Ryparida wallacei (Coleoptera: Chrysomelidae) were dominant insects in mangrove ecosystem.The structure community of insects in the denser canopy was higher compared to young mangrove.Since mangrove destruction started, efforts are being made to conserve mangrove as they are valuable for social life and extremely beneficial for ecosystems . MangrovInsects can be permanent residents or temporary visitors to the mangrove ecosystem and might play as a pest . FurtherInsects play an important role in nutrient flow and biochemical energy in the mangrove ecosystem. The high abundance of insects in mangroves confirms mangrove litter detritus formation and ecosystem function . FurtherMangrove insects have been studied at some locations in Southeast Asia  which is under the management of Marine and Fishery Office of Trenggalek, East Java, Indonesia. There were three different mangrove ages, 15 years old (first site), eight years old (second site) and five years old (third site) . Tree heagalocha .A total of 15 plots (10 m × 10 m for each plot) were assigned in each site. The highest tree from each plot will be used as a centre tree. The average distance among plots was 50 m. These plots are placed by using purposive random sampling and arranged to cover each site. The tree height, canopy density and insect sampling will also be measured in the same site.Thirty (30) trees selected from each site (two trees from each plot) will be used for tree height and canopy measurement. The tree height is measured using indirect techniques. An indirect technique is the most commonly used for standing trees b = the distance to the tree baseC = the tree heightThe canopy is measured using densiometer for 30 trees (the same tree used for tree height measurement). Four observations are made for each tree and the accurate duplication of the observer procedure must be assured  are installed into each plot by 50 cm from ground (15 pan traps per site). Each pan trap filled by water and detergent to break the water surface. All pan traps are modified with a netting on the site to prevent overflow during heavy rainfall. The traps installation started at 07:00 am and collected three days later. Insects explorations are also conducted by using swipe net every 3 h (started 07:00 until 16:00) in the same plot (15 plots 10 m × 10 m per site explored by swipe net and direct picking). Sometimes the plant material needed to be sampled by cutting the stems and branches with plant cutters because some insects such as wood-borers insect can only be found in the stems or branches . Insect diversity for each site was analysed using Shannon index (H′), Evenness index (E′) and Richness index (R) and calculated using Margalef Index in R statistical software. The estimated curve to visualise the collected species number is calculated using PAST version 3 software.2, 87 = 646.74, P < 0.001) and denser canopy compared to the youngest mangrove . Mangrove in the first site (15 years old) has the highest tree (8.36 m) and denser canopy (66.22%) followed by the second site or 8 years old mangrove with 4.68 m tree height and 30.51% for canopy cover and 5 years old mangrove has the lowest tree height (2.26 m) and canopy cover (19.65%).Mangrove ecosystem in Trenggalek has different tree height and canopy cover based on mangrove age . In thisEristena mangalis  with 1,724 individuals (18.78%), followed by Monolepta sp. (Coleoptera: Chrysomelidae) with 1,649 insects (17.96%) and Rhyparida wallacei (Coleoptera: Chrysomelidae) with 1,262 insects (13.75%).This study found 9,181 individual of insects associated with mangrove, consisting of 42 species from 31 families and 8 orders . LepidopThe number of species and individual that were found in the first site  were higher compared to the second  and the third site . The three sites showed high insect diversity (H′ > 2.0); first site (H′ = 2.54), second site (H′ = 2.34), and third site (H′ = 2.29). Additionally, the first site accounts for the highest value for Shannon diversity index (H′ = 2.54), Evenness index (E′ = 0.32), and Margalef richness index (R′ = 4.84) . The estThe mangroves ecosystem provides habitat and resource that supports a large number of insects at different trophic levels  has higher canopy cover percentage (66.22%) that resulting height insect diversity compared to second site (8 years old) and third site (5 years old). It becomes the evidence that mangrove ecosystems have a relatively old age and dense canopy that can support more variety of insects.


Recent rapid Arctic sea-ice reduction has been well documented in observations, reconstructions and model simulations. However, the rate of sea ice loss is highly variable in both time and space. The western Arctic has seen the fastest sea-ice decline, with substantial interannual and decadal variability, but the underlying mechanism remains unclear. Here we demonstrate, through both observations and model simulations, that the Pacific North American (PNA) pattern is an important driver of western Arctic sea-ice variability, accounting for more than 25% of the interannual variance. Our results suggest that the recent persistent positive PNA pattern has led to increased heat and moisture fluxes from local processes and from advection of North Pacific airmasses into the western Arctic. These changes have increased lower-tropospheric temperature, humidity and downwelling longwave radiation in the western Arctic, accelerating sea-ice decline. Our results indicate that the PNA pattern is important for projections of Arctic climate changes, and that greenhouse warming and the resultant persistent positive PNA trend is likely to increase Arctic sea-ice loss. The fastest sea-ice decline has been observed in the western Arctic, but the underlying mechanisms are still unclear. Here, the authors show that the Pacific North American pattern plays an important role in western Arctic sea-ice variability. This decline is strongest in late summer  with large interannual variability7. The long-term decline in Arctic sea ice has been attributed, in large part, to increasing anthropogenic greenhouse warming and is expected to result in an ice-free Arctic summer within a few decades8. Arctic sea ice loss is not spatially uniform, with the strongest decline and interannual-to-decadal variability occurring in the western Arctic10 /North Atlantic Oscillation (NAO)22, Arctic Dipole (AD)23, Arctic cyclones7, and anticyclones24 have been linked to sea ice decline in the region. In particular, an enhanced anticyclonic circulation over Greenland (GL-Z200) has been found to be the main driver of interannual variability and long-term trends in western Arctic sea ice since 197920. This anticyclonic circulation is largely generated by tropical Pacific sea surface temperature (SST) forcing through atmospheric teleconnections27. The link between the tropical Pacific and the Arctic has been referred to as the Pacific–Arctic teleconnection and features enhanced warming and sea ice loss in response to La Niña-like Pacific SST anomaly29 . Some other modes of decadal climate variability, such as the Interdecadal Pacific Oscillation (IPO), the Pacific Decadal Oscillation (PDO), and the Atlantic Multidecadal Variability (AMV) have also been shown to drive low-frequency Arctic sea ice fluctuations35.Despite their widespread impacts, the mechanisms that drive western Arctic sea ice decline and interannual-to-decadal variability are not well established. Many recent studies have suggested an important role for internal variability of both the atmosphere and ocean36 found that the record-breaking 2007 summer western Arctic sea ice decline was accompanied by a strong positive Pacific North American37 (PNA) pattern, expressed as an anomalous anticyclone over the western Arctic, and argued for an important role of the PNA pattern in driving sea ice loss in the region. However, such strong western Arctic anticyclones are not unprecedented in the instrumental record. For example, a stronger western Arctic anticyclone occurred during the summer of 1987 but was not associated with strong sea ice loss38. Although more recent work found a statistical link between western Arctic sea ice and PNA trends21, the physical mechanisms behind this link are not well established. Furthermore, it remains unknown whether the PNA pattern plays a role in the year-to-year variability in western Arctic ice. As a result, it is still an open question whether and how the PNA pattern is systematically linked with interannual to decadal variability in western Arctic sea ice.In contrast to Arctic atmospheric circulation patterns and Pacific SST forcing, the influence of extratropical teleconnection patterns on western Arctic sea ice has received less attention. Ref. In this study, we examine the long-term variability in summer western Arctic sea ice concentration (SIC) in the context of PNA changes to provide a more comprehensive picture of how the PNA pattern affects western Arctic sea ice on interannual-to-decadal timescales. We combine observational satellite data of Arctic SIC, ERA-Interim atmospheric reanalysis, and numerical simulations (see “Methods” section) to demonstrate the importance of the PNA as a driver of western Arctic sea ice loss on both interannual and decadal timescales, and diagnose the associated physical mechanisms.37. Although strongest in winter, the PNA also has an atmospheric presence during summer36, featuring a wave train shifted poleward of its wintertime location and a persistent anticyclonic circulation over the western Arctic36 (see “Methods” section). A positive summer PNA pattern has been associated with enhanced poleward transport of heat and moisture from the North Pacific into the western Arctic36. The PNA pattern has had a positive trend throughout the satellite era  superimposed on strong interannual-to-decadal variability, largely in parallel with the evolution of the observed PNA index (see “Methods” section)  of shared variance. This SIC-PNA correlation is still robust  when the long-term trends are removed  Fig. . Lead-laon) Fig. , suggestr = −0.34, p < 0.05), the AO , and the AD . This is in agreement with the previous studies23, but the strength of these correlations is substantially weaker than that with the PNA pattern. The evolution of western Arctic sea ice also shows significant coherence with low-frequency SST variabilities such as the PDO37  and the AMV24 . The AMV, which exhibits the strongest correlation, has previously been shown to drive multidecadal fluctuations in Arctic sea ice39. The recent trends in western Arctic sea ice may also be associated with the transition of AMV from a negative phase to a positive phase in the mid-1990s. Considering the potential influence of such long-term trends on correlation, we repeat our analysis by linearly detrending the original time series, which results in weak and insignificant correlations between these climate indices and western Arctic SIC . The pattern of Z500 regressed onto the SIC index bears a striking resemblance to the observed Z500 secular trend over the same period  onto the SIC index reveals a strong anticyclonic anomaly over the western Arctic and a meridional dipole pattern over the North Pacific with a weak cyclonic anomaly over the Aleutians and anticyclonic anomaly over mid-latitudes Fig. . This ciiod Fig. , both ofiod Fig. . This is36. Warm and moist Pacific air is advected into the western Arctic along the west coast of North America, leading to significant increases in lower-tropospheric temperature and humidity in the region  and they are also significantly correlated with both the PNA index  anomalies over the western Arctic during the positive PNA phase , contributing to sea ice declineuds Fig. . This deuds Fig.  and 10d.uds Fig. , but exh46, with a magnitude that seems unprecedented over the past millennium47. This trend is particularly strong for the summertime PNA during the period 1979–2016 , with the pattern shifting from a negative phase in the 1980s through the mid-1990s to a strong positive phase after the mid-1990s , humidity (r = 0.61), and DLR (r = 0.61), and between these parameters and the SIC index .Given the strong correlation and mechanistic association between interannual PNA variability and SIC, it seems likely that longer-term changes in the PNA pattern may contribute to trends in western Arctic sea ice. The PNA, both in winter and summer, has exhibited a significant positive trend since the satellite era90s Fig. . These c90s Fig.  and thus90s Fig. . The cli48 coupled with a slab ocean sea-ice model49. We first run the LMDZ nudged towards observed winds over the period 1979–2016 (see “Methods” section), but with imposed climatological sea surface temperature (SST) and sea ice conditions, as well as fixed CO2 (labeled ‘forced-nudged simulation’). Then, in a second simulation, we include these same forcings, but couple the LMDZ to a slab ocean sea-ice model (labeled ‘slab-nudged simulation’) with a flux correction (see “Methods” section).To complement the observational results and test the plausibility of the proposed SIC-PNA link, we perform two numerical simulations using the Laboratoire de Météorologie Dynamique Zoom (LMDZ) atmospheric general circulation modelr = 0.86 and 0.82 for forced-nudged and slab-nudged simulations, respectively; Fig. Both simulations show Z500 and lower-tropospheric temperature responses to interannual PNA variability that are comparable to the observed patterns Fig. . The modr = −0.51, p < 0.01) . The CMIP5 multi-model ensemble mean (MMEM) captures the observed declining trend in sea ice across the entire Arctic, but the magnitude is largely underestimated4, especially in the western Arctic 36 and shows that the PNA pattern is more generally an important driver of western Arctic sea ice variability and trends. Our results from both observations and simulations suggest that recent intensification of the positive PNA pattern has contributed to the accelerated decline of western Arctic sea ice through enhanced poleward heat and moisture transport and thus increased DLR in the region.This work builds on the previous recognition that an extreme positive PNA circulation pattern contributed to the record-breaking 2007 summer Arctic sea ice decline52 and ice drift53, which are missing in this simple slab ocean model, and anthropogenic warming54, which was not included in our experiments. Second, the PNA-driven western Arctic ice decline may be also amplified by local feedbacks between the sea ice and atmosphere55. To isolate the importance of the PNA-like atmospheric forcing from local feedbacks, we compare atmospheric circulation and lower-tropospheric temperature changes between the forced- and slab-nudged simulations. Similar results between the two experiments  for the period 1979–2016 at a global 1° × 1° spatial resolution. For comparison, we also analyze the outputs from 12 climate models  archived the Coupled Model Intercomparison Project Phase 5 (CMIP5)50. We focus our analysis on the period 1979–2016 by combining historical simulations (1979–2005) and Representative Concentration Pathway (RCP) 8.5 simulations (2006–2016). In this study, both observed and simulated anomalies are referenced to climatological means for 1979–2016.The observed monthly SIC is obtained from the National Snow and Ice Data Center (NSIDC) satellite data64 atmospheric general circulation model (AGCM), developed at the Laboratoire de Météorologie Dynamique (LMD). LMDZ5A is the atmospheric component of the Institute Pierre Simon Laplace (IPSL) coupled model IPSL-CM5A48 that takes part in the Coupled Model Intercomparison Project 5 (CMIP5) program50. The model is run with a horizontal resolution of 1.875°in latitude and 3.75°in longitude, with 19 hybrid layers in the vertical.Model simulations are performed with the LMDZ5A64. To realistically reproduce the Arctic sea-ice variability, a simple thermodynamic sea ice model49, of comparable complexity to the slab ocean, is used. In the model, over each grid point, the sea ice is represented by a uniform layer of thickness Hi and fractional area fi. It may be covered by a layer of snow. The temperature at the bottom of the ice layer is always equal to the freezing temperature of seawater To, while the top temperature Ts on top . The temperature profile within the snow or ice layer is assumed to be linear so that the mean ice temperature is ci are the volumic mass and specific heat capacity of the ice, and The slab ocean is used as the surface model coupled to LMDZ5ATo, and the resulting energy difference is used to build ice mass and, if ice is already present, to bring the new ice to temperature Ti. A set of rules is used to determine how the new mass is divided between extending the fractional area and thickening. If the ocean temperature becomes above freezing, sea ice is melted and the ocean temperature brought back to To in a reverse process. If the surface temperature of the snow or ice becomes positive through surface heat fluxes, it is similarly brought back to freezing level, and the energy difference is used to first melt the snow mass, if present, then part of the ice mass. Any energy left after all the ice is melted is used to warm the ocean, ensuring energy conservation. In practice, once the sea ice is present the slab temperature is set to To and the different heat fluxes into the ocean  are used to create or melt ice.As soon as the temperature of the slab ocean layer falls below freezing, its temperature is set back to L the latent heat for freezing, SIV the sea ice volume, Fsurf is the surface heat fluxes  and OHT is the convergence of energy by the ocean circulation. The Q-flux is then computed by taking the climatology of these different terms (except OHT) from an atmosphere-only simulation using the same model, with prescribed observed SST and sea ice. The seasonal Q-flux is then:The slab model needs an additional, seasonally-varying flux correction, usually called “Q-flux”, to accurately reproduce the observed pattern of SST and sea ice. The Q-flux represents the convergence of heating by oceanic motion . To estimate the Q-flux, we use the energy budget for the slab of the ocean and the overlying sea ice:65 averaged over the 1979–2008 period, as well as constant CO2. To ensure a realistic representation of large-scale atmospheric circulation, horizontal winds fields are nudged by the European Centre for Medium-Range Weather Forecasts (ECMWF) reanalyses66, in which, the simulated winds are relaxed in each grid point from surface to the top of the atmosphere toward the corresponding reanalysis winds with a relaxation time of 1 h67. The simulation is run for 40 years (1977–2016), with the first two years considered as spin-up and thus discarded from the analysis. Here we call this experiment ‘forced-nudged simulation’.Two model experiments are conducted to test the observed link between the PNA and western Arctic sea ice and the associated causal relationship. For the first experiment, the LMDZ5A is run forced by a monthly-mean climatology of SST and sea ice based on the Atmospheric Model Intercomparison Project (AMIP) protocolThe second model experiment is a slab-ocean simulation, in which the above LMDZ5A is coupled to a slab ocean model with a flux correction (referred to as the Q-flux) so that SST and sea ice can respond to the atmospheric forcing through thermodynamic processes. The Q-flux is derived from the forced-nudged simulation described above, with conditions averaged over the 1979–2016 period. The simulation is run from 1979 to 2016, with a 104-year (1875–1978) spin-up simulation that was run until no more trend in SST or sea ice was noticed neither at the global nor at the regional scale. This spin-up simulation was initialized from the forced-nudged simulation and ran with 13 loops of 8 years  nudged by winds from the 1981-1988 period. We call this experiment ‘slab-nudged simulation’.37, has an atmospheric presence throughout the entire year with a varying wave train in location and intensity across seasons36. The summertime PNA features a wave train that is shifted north of its winter position with a ridge over the western Arctic36.The PNA pattern, although most pronounced during boreal winter37. The second one is based on a rotated empirical orthogonal function68 (REOF) or EOF69 analysis of Z500 anomaly poleward of 20° latitude for the Northern Hemisphere. It has been suggested that the REOF or EOF method is superior to the pointwise-based analyses because the teleconnection patterns are based on the whole flow field and not just anomalies at a few selected locations36. Therefore, we adopt the second method for our analysis. The observed PNA index is obtained from the NCEP Climate Prediction Center . For simulated PNA index, we first apply EOF to the summer Z500 anomaly field and then regress summer Z500 onto the principle component (PC) time series to obtain the regression patterns. We compare such patterns with the regression pattern of the observed Z500 against the PNA index time series provided by the CPC. As shown in Supplementary Fig. r = 0.57, p < 0.01).The PNA index can be computed using two different methods. The first one is a linear combination of the standardized 500 hPa geopotential height (Z500) anomalies at four centers of actionSupplementary informationPeer Review File


Adaptation to hypoxia is a common feature in solid tumors orchestrated by oxygen-dependent and independent upregulation of the hypoxia-inducible factor-1α (HIF-1α). We unveiled that G protein-coupled receptor kinase (GRK2), known to be overexpressed in certain tumors, fosters this hypoxic pathway via phosphorylation of the mRNA-binding protein HuR, a central HIF-1α modulator. GRK2-mediated HuR phosphorylation increases the total levels and cytoplasmic shuttling of HuR in response to hypoxia, and GRK2-phosphodefective HuR mutants show defective cytosolic accumulation and lower binding to HIF-1α mRNA in hypoxic Hela cells. Interestingly, enhanced GRK2 and HuR expression correlate in luminal breast cancer patients. GRK2 also promotes the HuR/HIF-1α axis and VEGF-C accumulation in normoxic MCF7 breast luminal cancer cells and is required for the induction of HuR/HIF1-α in response to adrenergic stress. Our results point to a relevant role of the GRK2/HuR/HIF-1α module in the adaptation of malignant cells to tumor microenvironment-related stresses. Oxygen is a key factor in embryonic development and in normal tissue homeostasis, and its shortage triggers adaptive mechanisms required for cell survival ,2. HypoxInterestingly, in cancerous cells, the HIF signaling pathway can also be regulated by other environmental and intracellular signaling factors in an oxygen-independent manner, via modulation of protein stability, gene transcription, or mRNA binding proteins such as YB-1 and HuR (Human antigen R), which are often up-regulated in tumor contexts thereby leading to enhanced translation of HIF-1α mRNA in normoxic and hypoxic conditions ,8,9. HIFDifferent G protein-coupled receptors (GPCRs) have been associated with the progression of several types of tumors ,14. Of nIn this report we unveil that GRK2 is a relevant modulator of the hypoxic pathway in transformed epithelial cells via phosphorylation-dependent modulation of the mRNA-binding protein HuR, leading to subsequent nuclear accumulation of the HIF-1α protein. Stimulation of GRK2 upon hypoxia or adrenergic receptor activation emerges as a novel mechanism of regulation of HIF signaling in physiological or pathological processes, such as cancer progression.Our previous results pointed to GRK2 as a common regulatory and transducing effector of diverse pathways that are altered in luminal and basal breast cancer ,22. RemaWe analyzed the steady-state levels of HuR protein under different GRK2 expression and activation status contexts in Hela cells, a cellular model widely used in the functional characterization of HuR. The protein levels of HuR directly correlated with GRK2 abundance, being significantly upregulated in cells over-expressing the wild-type GRK2 (Hela-WT5), but down-modulated upon stable GRK2 silencing (Hela-shGRK2) A. InterePurified GST-HuR was efficiently phosphorylated by recombinant GRK2  on the GRK2-phosphorylated GST-HuR, by using proteomic approaches . Single Hypoxia is a well-characterized stress known to upregulate HuR protein levels, in order to foster HuR actions . Interes2 exposure, a condition mimicking hypoxic stress [HuR is mainly localized in the cell nucleus in basal conditions, whereas its activity in stress conditions is tightly linked to protein cytosolic shuttling ,36. Akinc stress . We obsec stress , and enhOverall, these results indicated that GRK2-mediated phosphorylation at the residues located in the different domains of HuR can modulate the cellular levels and the cytoplasmic localization of HuR protein, in response to hypoxia.Central to cellular hypoxia is the upregulation of HIF-1α, which is in turn required for the transcriptional induction of genes involved in metabolism, angiogenesis, or survival ,6,41. InThese results suggested that GRK2-phosphorylated HuR could be more efficient in binding to HIF-1α mRNA or in regulating its decay or translation rates.Then, we performed RNA immunoprecipitation (RIP) assays to detect HuR-HIF-1α mRNA complexes in Hela cells that were overexpressing HA-tagged constructs of HuR. As shown in It has been proposed that stress-induced HuR might not bind with identical affinities to its mRNA targets , suggestELAVL1 (HuR) and ADRBK1 (GRK2) genes showed a direct correlation in breast cancer patients inspected with the web tool CANCERTOOL -positive and luminal A human breast tumors engineered to express different dosages of GRK2 , and furThis effect depended on the GRK2-dependent phosphorylation of HuR, as the inducible over-expression of GRK2-S670A did not trigger a reduction in nuclear HuR levels, whereas a kinase mutant mimicking the positive regulatory S670 phosphorylation event (GRK2-S670D) decreased the nuclear HuR levels akin to the wild-type GRK2 protein C.We next investigated whether GRK2-dependent HuR modulation in MCF7 cells might affect HIF-1α nuclear distribution. As expected in tumor cells , normoxiA main pro-carcinogenic effect of the HIF-1α factor is the induction of angiogenesis and lymphangiogenesis through the activation of transcription of the VEGF family members ,45. Of nThe effects of GRK2 on the HuR/HIF1-α axis in MCF7 cells suggested that stressful or microenvironment conditions other than hypoxia might also contribute to foster this tumorigenic pathway. In this context, it is known that adrenergic stimulation promotes ERK-mediated phosphorylation of GRK2 on Ser670 in different cellular systems  and alsoTherefore, we addressed whether chronic β2-adrenergic receptor stimulation promoted HuR regulation by GRK2 in MCF7 breast cancer cells. Isoproterenol challenge upregulated total HuR protein levels in both parental and MCF7-GRK2 cells, but not in GRK2-silenced cells A. IsoproOverall, our results point that the GRK2/HuR/HIF-1α axis is potentiated in response to hypoxic and adrenergic stresses, thus emerging as a relevant pro-tumorigenic module.In this paper, we identified GRK2 as a modulator of the HuR/HIF-1α axis in relevant pathological settings. We uncovered that HuR is a GRK2 substrate, requiring prior phosphorylation of GRK2 at its Ser670 regulatory site. Phosphorylation by GRK2 enhances the HuR levels and the cytosolic shuttling of HuR upon hypoxic conditions, favoring its binding to HIF-1α mRNA and increased nuclear accumulation of this central modulator of hypoxia-driven pathways. In the MCF7 cells, a cellular model of luminal A breast cancer in which upregulation of GRK2 and HuR frequently concur, GRK2 increased the total and basal cytosolic HuR protein levels, which correlated with a higher nuclear distribution of HIF-1α and enhanced secretion of the lymphoangiogenic factor VEGF-C in normoxic conditions. Moreover, chronic adrenergic stimulation fosters the HuR/HIF1-α module in MCF7 cells through GRK2 , which cSer670 phosphorylation is an emerging regulatory switcher in GRK2 substrate specificity and partner association . ContextWe have identified residues within the hinge region (S197) and the RRM2 domain (T142/T143) as the main targets of GRK2-mediated HuR phosphorylation, and mutations preventing such modifications specifically impair hypoxia-induced HuR cytoplasmic shuttling in HeLa cells, without altering its basal localization. Moreover, the triple HuR-T142/143/197A mutant also displays reduced binding to HIF-1α mRNA in hypoxic conditions.Several mechanisms might underlie the observed effects of GRK2 on HuR functionality. Enhanced cytosolic levels of GRK2-phosphorylated HuR might be a consequence of increased nuclear export/decreased nuclear import or fostered cytoplasmic retention. The long hinge region (aa 187–243) includes the HuR Nucleocytoplasmic Shuttling (HNS) sequence (aa 205–237) containing both nuclear localization and nuclear export determinants ,32,33,56GRK2-mediated phosphorylation at T142/143 within the RRM2, the second HuR RNA binding domain also fosters HuR cytoplasmic localization. It has been reported that interaction of HuR with nuclear importin TNR2 is reduced by binding of ARE-containing RNA to HuR . Since GThe subcellular location in which the phosphorylation of HuR by GRK2 takes place could shed light on the GRK2-mediated modulation of HuR trafficking. HuR and GRK2 display opposite canonical subcellular distribution at the basal conditions. Although GRK2 was initially described as a cytoplasmic protein, its nuclear presence has also been reported , thus alhttp://rna.tbi.univie.ac.at/AREsite), HIF1α mRNA could fit in the group of mRNAs with greater dependence on HuR conformations. Therefore, it is tempting to suggest that GRK2 might favor recognition of a subset of mRNAs displaying lower intrinsic affinities for HuR.Preventing HuR modification by GRK2 also results in a defective interaction with HIF1-α mRNA. HuR phosphorylation has a notable impact on the binding of HuR to mRNA targets . ConformFurthermore, although we have not analyzed in-depth the mechanism whereby HuR phosphorylation by GRK2 impacts its total protein levels, it is worth noting that phosphorylation of RRM2 residues has been linked to HuR stabilization. Phosphorylation of Thr118 by CHK2 protects HuR from proteasomal degradation upon heat shock . ModificThe role of HuR in breast cancer development is well-documented in model cell lines, in vivo animal models, and clinical studies ,25. The HuR can modulate HIF1α mRNA via the 3′UTR region for transcript stabilization or via 5′UTR for translation, depending on the cellular context ,68. It sAdrenergic stress is emerging as a relevant microenvironmental player in cancer progression ,50. ChroIn sum, our data put forward a relevant role for the GRK2/HuR/HIF1α module in cancer cells and in response to the adrenergic overdrive, regardless of the oxygen status. Enhanced GRK2 expression and S670 phosphorylation status in the given tumor contexts would lead to enhanced HuR functionality, thus, counteracting the canonical HIF protein degradation driven by prolyl hydroxylases  and reca2) was achieved in an Invivo2 400 hypoxia Workstation . Isoproterenol (50 μM) was supplemented with 1 mM ascorbic acid and 20 mM HEPES pH 7.5.Cells were maintained in DMEM supplemented with 10% fetal bovine serum. Stable Hela (ATCC) cells were previously described . MCF7luc® Lightning site-directed mutagenesis kit . HeLa cells (70%–80% confluent monolayers in 60 or 100 mm dishes) were transiently transfected using the lipofectamine/plus method.Plasmids encoding HA-HuR WT were previously described . HA-HuR-3V04). Membranes were incubated with the indicated primary antibodies—α-Actin , GAPDH , , GRK2 , HuR , Nucleolin , p21 , and α-Tubulin . Blots were developed using a chemiluminescent method . Band density was quantitated by laser densitometric analysis.Whole cellular lysates were prepared in RIPA buffer , as described . For theVEGF-C secretion was determined in cell-conditioned media of MCF7 cells incubated in DMEM 1% FCS for 48 h, by using a Human VEGF-C Quantikine ELISA Kit . Measurements in duplicates were normalized to the amount of the total protein in cell lysates quantified by the Lowry method.As described in , 4% PFA-In vitro kinase assays with wild-type, S670A or K220R GRK2 full-length (50 nM); and WT, T142/143A or S197A GST-tagged HuR recombinant proteins  were performed, as described . The MicGRK2 (50 nM) and GST-HuR (100 nM) were incubated in the presence of cold ATP, as detailed above and resolved by SDS–PAGE. The band corresponding to full-length GST-HuR was digested by trypsin and chymotrypsin, and the fragments were analyzed using LC–MS/MS in the ‘CBMSO PROTEIN CHEMISTRY FACILITY’ that belonged to the ProteoRed network, PRB2-ISCIII, supported by the grant PT13/0001.RNA Immunoprecipitation (RIP) assays were performed, as previously described . Whole-cELAVL1 (HuR protein) and ADRBK1 (GRK2 protein) levels in breast tumors was calculated and represented using the CANCERTOOL webtool [p-values were adjusted using the Benjamini–Hochberg method. Datasets with a correlation coefficient greater than 20% (−0.2 < R < 0.2) and a p-value lower than 0.05 were considered.Pairwise correlation of gene expression  webtool . Pearsont-test, as indicated in the figure legends. All results are expressed as mean ± SEM.Data analysis was performed using the GraphPad Prism for Windows. Means between groups were compared by two-way or one-way ANOVA with Bonferroni’s or Tukey’s post-hoc test, or with unpaired Student’s An active GRK2/HuR/ HIF-1α module, particularly in estrogen-positive luminal A breast tumors could facilitate survival of malignant cells, even before the expanding tumor mass becomes hypoxic, and also improves the adaptation of tumor cells to the hypoxic environment of lymphatic vessels and to other tumor-related stresses, such as chronic over-activation of the adrenergic system, in order to foster tumor progression.


Synaptic plasticity is the cellular and molecular counterpart of learning and memory and, since its first discovery, the analysis of the mechanisms underlying long-term changes of synaptic strength has been almost exclusively focused on excitatory connections. Conversely, inhibition was considered as a fixed controller of circuit excitability. Only recently, inhibitory networks were shown to be finely regulated by a wide number of mechanisms residing in their synaptic connections. Here, we review recent findings on the forms of inhibitory plasticity (IP) that have been discovered and characterized in different brain areas. In particular, we focus our attention on the molecular pathways involved in the induction and expression mechanisms leading to changes in synaptic efficacy, and we discuss, from the computational perspective, how IP can contribute to the emergence of functional properties of brain circuits. The emergence of brain functions, from motion control to cognition and abstract thinking, is tightly bound to the ability of brain circuits to adjust synaptic connections . For a lIn this article, we discuss how the various forms of IP shape brain circuits architectures and functions with particular attention on neuronal computation. We begin with a panoramic view on the variety of GABAergic cell types and circuits to continue with an analysis of the induction and expression mechanisms of inhibitory LTP and LTD. We then proceed with discussing the functional consequences of the aforementioned mechanisms, including computational models proposed to predict the effects of IP. Finally, we analyze the potential involvement of inhibitory plasticity in the emergence of pathological disorders to end with a few suggestions regarding the implementation of synaptic learning rules into artificial circuits. Although it is widely accepted that inhibitory neurons are actively engaged in network computation by providing global stability to network dynamics, by controlling the degree of circuit synchronization and by controlling the timing of neuronal firing , it is sOne of the major obstacles in identifying the way inhibitory plasticity tunes the activity of brain circuits comes from the diversity of inhibitory interneurons in the CNS, which still leaves these cells the capacity to provide inhibition to a large variety of excitatory input classes . Among nSeveral attempts have been indeed made to classify cortical interneurons, for instance, the so-called “Petilla terminology” , by collWhen considering the functional differences between GABAergic classes, it is evident that the shown diversity can be one of the main causes of the inhibitory capability to stabilize and finely regulate circuits activity . FurtherA receptors (GABAARs) activity or in mixed forms [B receptors activation [Given the diversity of inhibitory classes, it can be envisaged that inhibitory plasticity presents heterogeneous molecular and functional characteristics accordingly. Interestingly, as observed for excitatory synapses, IP was shown to occur as changes in the presynaptic release, in postsynaptic GABAed forms . Modifieed forms , whereased forms . The moded forms ,46 from ed forms . Alternaed forms . In the ed forms . The fired forms , throughed forms . Similared forms . In the ed forms , lamina ed forms , neonataed forms  and basoed forms . Interesed forms . As in ted forms , the poted forms ,46 and ted forms ,59. The ed forms . The BDNed forms . It binded forms . The BDNed forms  and GABAtivation . In an ativation . Similartivation . It shoutivation . As in the case of i-LTP, the identified forms of i-LTD presynaptically expressed depend either on the direct NMDA-Rs activation in the GABAergic terminals  Figure.D. These Inhibitory plasticity can also be induced through mechanisms requiring the direct activity of GABAergic afferents. One of these homosynaptic mechanisms has been described in the primary visual cortex, where the firing of a presynaptic neuron paired with the activation of a postsynaptic pyramidal neuron can induce inhibitory plasticity ,78. The A receptors, in response to specific patterns of induction requiring calcium influx following postsynaptic firing activity, can be phosphorylated by different kinases  [A receptors gephyrin [A-Rs dynamics. Moreover, the continuous cycling of GABAA-Rs insertion and removal, together with movements of lateral diffusion at synaptic surface regulates synaptic functionality [A-Rs may change in response to receptor trafficking regulation. Inhibitory responses can be, therefore, bidirectionally modulated by alternatively acting on the exocytosis and endocytosis cycling [Changes in inhibitory strength can also be associated with purely postsynaptic expression through a large variety of mechanisms  , as it hand PKA)   , which cand PKA) . A similand PKA) . Interesand PKA) . Anothergephyrin . The avagephyrin  and is rionality . In addi cycling . Finally cycling . A-Rs has been well characterized and described. Conversely, the functional alternative to the action of ionotropic GABAA-Rs is the slow inhibition mediated by the metabotropic GABAB receptors. Although the biochemical signaling engaged by GABAB-Rs is characterized in detail as well as their role in shaping neuronal activity, it is not much understood whether they can undergo plastic changes. Nevertheless, reports of persistent changes in GABAB-Rs activity have been shown to occur in the hippocampus [+ (GIRK) channels has been shown to induce LTP in cultured hippocampal neurons [The activity of extrasynaptic receptors mediates an alternative form of synaptic inhibition. The impact of this form of tonic inhibition is critically related to its impact on membrane conductance and membrane potential with time constants considerably lower than the one of receptors located in the terminals . The tonpocampus  and in lpocampus , albeit  neurons , in a re neurons . B-Rs with LTD induced by presynaptic before postsynaptic spikes. The fact that the sign of plasticity can be reversed during development makes this form of plasticity a suitable candidate for disinhibition during the auditory critical period [Unlike excitatory synapses, learning rules for inhibitory plasticity have not yet been extensively investigated . Howeverl period . Differel period . This mel period . At the l period . Neverthl period , as welll period , could pThe precise identification of the functional role of inhibitory plasticity is still an open issue. Nonetheless, the recurrent leitmotif regarding GABAergic plasticity is the maintenance of a constant E/I balance in a circuit that can compensate for changes in the excitatory driving force triggered by plasticity at glutamatergic synapses. This homeostatic regulation can be obtained by reducing both feed-forward inhibition and excitation . AlternaFrom a purely computational perspective, the power of the brain is traditionally linked to the complex connectivity of neuronal networks, whereas single neurons are considered as linear integrators and thresholding devices. It is indeed clear that a wide series of non-linear mechanisms converting synaptic input into output firing is employed by single neurons to process information. These mechanisms include synaptic noise, inhibitory conductance, and notably synaptic plasticity . As a geIn order to analyze the impact of IP onto neuronal network computation, researchers tend to employ circuits models with a limited number of parameters to control. The simplest network is composed of basic computational units, for instance, integrate and fire neurons, endowed with excitatory and inhibitory synapses randomly connected with a sparse architecture . In suchSynaptic plasticity is also thought to be the cellular and molecular counterpart of learning and memory. In particular, memories that can be recalled by contextual cues or commands must involve plasticity mechanisms to be exploited . In HopfThe analysis of inhibitory plasticity presented so far is limited to the molecular characterization of the mechanisms underlying IPs together with the effects at the circuit level. However, a univocal determination of the roles for the IPs in circuit functions is still lacking, and evidences have been collected regarding the consequences of inhibitory plasticity at the integrative level. It is well known in fact that alterations of inhibitory circuits can contribute to the induction of neurological disorders. In particular, the regulation of the E/I balance, which is inherently bound to IP impacts the induction of excitatory LTP and LTD and can indeed shift the threshold required to switch between the two plastic conditions. It has recently been observed, in fact, that the inhibitory LTP, by modulating E/I balance, can effectively restore the hippocampal excitatory LTD preventing memory impairment related to Aβ protein accumulation . SimilarBesides the obvious interest in the biomedical and clinical fields, the analysis of brain functions and particularly of synaptic connections also arouse great interest in computer science and, more generally, in the field of neuromorphic electronic and artificial intelligence. The goal in fact to build large artificial neural networks with vast amounts of computing elements has rendered the task of creating low consuming artificial synapses a high priority. The visionary idea of electrical elements behaving like synapses called memristors is now becoming true . These p