Cell cycle kinases represent an important component of the cell machinery that controls signal transduction involved in cell proliferation, growth, and differentiation. Nek2 is a mitotic Ser/Thr kinase that localizes predominantly to centrosomes and kinetochores and orchestrates centrosome disjunction and faithful chromosomal segregation. Its activity is tightly regulated during the cell cycle with the help of other kinases and phosphatases and via proteasomal degradation. Increased levels of Nek2 kinase can promote centrosome amplification (CA), mitotic defects, chromosome instability (CIN), tumor growth, and cancer metastasis. While it remains a highly attractive target for the development of anti-cancer therapeutics, several new roles of the Nek2 enzyme have recently emerged: these include drug resistance, bone, ciliopathies, immune and kidney diseases, and parasitic diseases such as malaria. Therefore, Nek2 is at the interface of multiple cellular processes and can influence numerous cellular signaling networks. Herein, we provide a critical overview of Nek2 kinase biology and discuss the signaling roles it plays in both normal and diseased human physiology. While the majority of research efforts over the last two decades have focused on the roles of Nek2 kinase in tumor development and cancer metastasis, the signaling mechanisms involving the key players associated with several other notable human diseases are highlighted here. We summarize the efforts made so far to develop Nek2 inhibitory small molecules, illustrate their action modalities, and provide our opinion on the future of Nek2-targeted therapeutics. It is anticipated that the functional inhibition of Nek2 kinase will be a key strategy going forward in drug development, with applications across multiple human diseases. Aspergillus nidulans and shown to be essential for mitotic entry of the cell [The human kinome represents the second most important class of therapeutic target after G-protein-coupled receptors (GPCRs). It is estimated that about 30% of all drug development projects in pharmaceutical companies are directed at kinases and their inhibition . Not surthe cell . Human Nthe cell ,6,7. Intthe cell . It shouthe cell . Nek2 prthe cell ,11,12,13the cell ,15,16. Ithe cell . Other nthe cell ,15,16. Sthe cell . High expression levels (2-5-fold above baseline) of the Nek2 kinase in a variety of highly aggressive solid tumors, including triple negative breast cancer, makes this kinase a high value target for developing anti-cancer drugs ,22,23,24Mitosis is a critically important cellular process that must be regulated with exquisite precision. Nek2 is involved in the disassembly of the tether (inter-centriolar linker) at the onset of mitosis to facilitate the centrosome separation that follows the establishment of the mitotic spindle . AlthouDuring interphase of the cell cycle, Nek2 exists as a complex with protein phosphatase 1 (PP1) and mammalian STE20-like protein kinase 2 (MST2), an important component of the Hippo pathway; it is maintained in an inactive state through dephosphorylation by PP1 ,32. PoloExperimental evidence suggests that Nek2 localizes to the kinetochore and plays an important role in spindle assembly checkpoint (SAC) signaling for faithful chromosome segregation by mediating interactions with Hec1 and MAD1 proteins  41,42,4,442,43. Primary cilium, a microtubule-based projection from the cell surface, plays critical communication roles in quiescent and differentiated cells in response to external stimuli . Cell cyPolycystic kidney diseases (PKDs) are genetic disorders that primarily affect kidney function in both adults and children; they may also affect related organs such as the liver and pancreas. Kidneys in PKD contain multiple fluid-filled cysts that results from uncontrolled cell proliferation due to aberrant cilium signaling . Using Seminal work by Zhou et al. showed that Nek2 expression was highly correlated with drug resistance, resulting in poor prognosis of multiple cancer types . This stNoting that higher expression of the Nek2 kinase was directly correlated with bone lytic phenotypes in multiple myeloma patients, Hao et al. investigated its role in bone remodeling . OverexpFaithful DNA replication during cell division and its maintenance throughout the life span of an organism are critically important events. Chemical damage to DNA can, however, occur during the normal life span via several mechanisms, such as a reaction with radiation-induced free radicals, exposure to environmental toxins, and reactive oxygen species (ROS) ,78. DamaCell cycle kinases are considered attractive targets for the development of anti-cancer agents ,102,103.Another evolutionary pathway often dysregulated in tumors where the Nek2 kinase is involved is Wnt signaling pathway. A key player in this pathway is β-catenin, a multifunctional protein involved in key cellular functions, such as centrosome disjunction, bipolar spindle formation, and cell-cell adhesion ,129. It Drosophila melanogaster model where dNek2 overexpression with a GFP tag was achieved in a tissue-specific manner: eye, thorax, and wing  or oncogenic RET (MENdRet2) resulted in enhanced survival and aggressive metastasis, and this cooperativity relied on synergistic PI3K/Akt signaling. Using a quantitative assay on a distant seeding phenotype, we further showed that both genetic and pharmacological inhibition of the components  of the PI3K/Akt pathway reduced metastasis. Finally, using this Nek2 fly model, we discovered a novel non-toxic quinoline-based pharmacophore that inhibited Nek2 function both in vitro and in vivo  data sets and gaining an overall understanding of proteogenomics at a systems level. This database contains mass spectrometry-based proteomic and phosphoproteomic data sets conducted on human tumor samples from different origins. Taking advantage of this, Deb et al. recently mined and analyzed proteomic and phosphoproteomic data sets of tumor samples derived from six different cancers types: ovarian cancer, breast cancer, clear cell renal cell carcinoma (CCRCC), uterine corpus endometrial carcinoma (UCEC), colon cancer, and lung adenocarcinoma (LUAD) . Using aPlasmodium falciparum is primarily responsible for morbidity and mortality in human malaria and is considered a valuable target for the development of effective therapeutics. Unfortunately, several resistance mechanisms to the existing drugs have emerged, making it a real threat to human life, especially in developing countries [Plasmodium via both mosquito vector and human host, making them interesting new targets for drug development [Plasmodium’s life cycle [Xestospongia sp. that showed good antiplasmodial activity against an FCB1 P. falciparum strain (IC50: 3 μM); it was also active in vivo at 5 mg/kg in P. berghei NK65 infected mice [50: 1 μM) [P. falciparum. In 2019 alone, the World Health Organization (WHO) estimated 229 million cases of malaria worldwide with an estimated number of deaths of 409,000 . Childreountries ,163,164.elopment ,167,168.elopment ,170. Whife cycle ,172,173.fe cycle . They alted mice . A simil0: 1 μM) . Further(a)++ binding site, and allows interaction with the hinge region via H-bonding [Type I: Chemotypes that occupy the ATP-binding site of an active kinase; this does not mandate specific conformation of key structural elements, such as αC helix or the DFG/Mg-bonding ,179.(b)Type I 1/2: Chemotypes that inhibit the ATP-binding site of an inactive target kinase with the DFG motif’s Asp ‘in’ and αC helix ‘out’ conformation ,180.(c)Type II: Chemotypes that occupy the ATP-binding pocket of an inactive kinase with DFG ‘out’ conformation ,179.(d)Type III: Allosteric modulators that bind within the cleft, formed by the small and large lobes, adjacent to ATP-binding pocket ,179,181.(e)Type IV: Allosteric modulators that bind next to ATP-binding pocket; however, unlike type III inhibitors, they bind outside of the cleft and the phosphoacceptor region ,181.(f)Type V: Chemotypes that occupy two distinct sites at once. This type of inhibitor has been further divided into two subcategories. These are (i) Bisubstrate analog inhibitors, which span over the ATP and substrate-binding site and (ii) Bivalent inhibitors, which span over the ATP-binding site along with any other site on the protein except at the substrate binding site ,182.(g)Type VI: Chemotypes with a built-in electrophilic warhead that trap the accessible nucleophilic protein residue to form a covalent adduct with the target kinase .For almost three decades now, protein kinases have garnered much-coveted attention as potential drug targets for various human diseases. This effort has culminated in more than 70 FDA-approved small molecule drugs so far. The era of kinase drug discovery was started by the discovery of staurosporine—a pan-kinase inhibitor—which, despite its large structure, outcompetes ATP and occupies the ATP-binding site ,177. Desin vitro and in vivo. Furthermore, another class of chemotype, a protein-protein interaction (PPI) inhibitor, which attenuates Nek2 protein activity by disrupting its interaction with Hec1 protein, has also emerged. In the following section, we discuss the emergence and advancement of the different classes of Nek2 inhibitors, categorize them to specific inhibitor types, and outline the possible impact of Nek2-selective inhibitory chemotype development on future drug discovery efforts.Nek2 has invigorated attention in recent years due to its involvement in several pathophysiological conditions. The biological implications of Nek2 inhibition have primarily been investigated using various biochemical techniques, such as the RNAi knockdown model. Although this approach excavated important findings on Nek2 kinase function, it did not mimic therapeutic inhibition of the target by the small molecules. This has inspired the discovery of several chemotypes in recent years for sensitizing Nek2 activity both The structural analysis of the first reported Nek2 inhibitor, SU11652, an ATP-competitive tyrosine kinase inhibitor with cross-reactivity toward several serine/threonine kinases, emerged as the lead guiding factor for Nek2 inhibitor development ,185,186.50 value of 8 µM [mK ~ 90 µM, catk ~ 17 min−1). Interestingly, SU11248—a structural analog of SU11652—exhibited a marginally weaker inhibition profile [SU11652 showed modest Nek2 inhibition with an IC of 8 µM  when det profile . This is profile . Althoug50: 2 nM), when evaluated against a panel of kinases, exhibited promising Nek2 inhibition. Although none of these compounds showed selectivity towards the Nek2 kinase, the findings turned out to be crucial for the development of Nek2-selective inhibitors. This will be further elaborated in the later sections.The thiophene-based motif garnered recognition as a potential Nek2 inhibitory scaffold during a drug discovery effort aiming to target the polo-like kinase 1 (Plk1) . The Plk3a and 3b exhibited antiproliferative activity toward osteosarcoma U2OS cells and cervical cancer HeLa cells. Viridin analogs also negatively regulated premature centrosome splitting in Nek2A-inducible U20S cells. The authors further treated the same cell line with the selective Cdk2 inhibitor, as Cdk2 is known for inducing premature centrosome splitting [High-throughput screening played a major role in Nek2 inhibitor discovery as Hayward et al. screened approximately 73,000 compounds and identified viridin/wortmannin-like compounds—known ATP site binding inhibitors of other kinase family members—as potential Nek2 inhibitors ,188,189.plitting . Interesplitting ,192,193.50: 0.87 ± 0.34 µM) but suffered from low cell-permeability. This was attributed to the presence of readily ionizable free carboxyl group along with relatively high topological polar surface area. The authors primarily altered the substitutions at 2- and 6-positions of the aminopyrazine ring to address the issues associated with the lead compound. Crystal structure analysis of the unphosphorylated Nek2-AP-1 complex showed that it inherits many attributes of common kinase-inhibitor interactions  and showed remarkable selectivity over Chk1—a kinase involved in cell-cycle regulation that does not share structural similarity with the Nek2 kinase. In addition, compound 4 exhibited > 80-fold selectivity over Plk1 (19.4 µM). However, it displayed compromised selectivity when evaluated against either form of the phosphorylated or unphosphorylated Nek1 (IC50: 0.17 µM) kinase. Finally, most of the developed aminopyrazine derivatives failed to exhibit improved cell-permeability. Overall, the study revealed a potential inhibitory scaffold for targeting the Nek2 kinase and provided valuable information on ligand-induced protein conformation that has been leveraged for future inhibitor discovery and will be discussed in upcoming sections.In a noteworthy study, Whelligan et al. discovered a new class of chemical phenotype, which—for the first time—inhibited both phosphorylated and unphosphorylated forms of the Nek2 kinase . The autractions . Howevertions 4,  exhibite(R) isomer of the racemic inhibitor, rac-1, was observed in the crystal structure which indicated that (R) configuration is favored over the (S) counterpart for targeting the Nek2 kinase. Taking clues from the above observation, the authors further performed computational studies and developed a series of compounds by retaining the benzimidazole core but replacing the thiophene with a phenyl ring. Although the most potent analog of this series, compound 5  kinase in A549 human lung cancer cell lines but lacked efficacy against the C22V Nek2 mutant. Interestingly, compound 6 did not influence mitosis events, which further showed that the inhibitor did not interfere with other regulatory proteins—such as Cdk1, Plk1, AurB, and MPS1. Overall, this study presented a small molecule irreversible Nek2 inhibitor for the first time that not only shed light on the cellular roles of the Nek2 function but also provided the premise for developing Nek2-selective inhibitors, targeting the non-conserved Cys22 residue.In a seminal study, Henise et al. developed propynamide-containing irreversible inhibitors of the Nek2 kinase . The tar7, which was comprised of: (a) 2-aminopyridine—a moiety known to interact with the hinge region by establishing two hydrogen bonds with Glu87 and Cys89 residues; (b) substituted benzamide, in which the phenyl ring formed a sandwiched interaction between the gatekeeper Met86 and Phe148 residues and the amide group forming hydrogen bonding interactions with the Asp159 of the DFG-motif; (c) allylic ether, which occupied a hydrophobic pocket formed by part of the glycine-rich loop; and (d) substituted thiophene that engaged in additional hydrophobic interactions with the Gly92 (R) isomer inhibited Nek2 kinase activity with a low nanomolar IC50 value (7) and exhibited remarkable selectivity over other mitosis regulatory kinases, such as Plk1, MPS1, AurA, and CDK2. In addition, 7 maintained its selectivity profile over a panel of 24 other kinases, except for a few non-cell cycle related ones. When tested in osteosarcoma U2OS cells, compound 7 inhibited C-NAP 1 phosphorylation, a physiological substrate of the Nek2 that is phosphorylated at S2179 position, in a dose-dependent manner and exhibited modest GI50 values against different cell lines. Although this inhibitor exhibited high permeability at physiological pH, as confirmed by parallel artificial membrane permeability assay, it suffered from high metabolic degradation. The developed Nek2 pharmacophore exhibited several positive traits of a good inhibitor, such as high potency and selectivity with improved cell-permeability, and it certainly added substantive values to the existing armory of Nek2-focused drug discovery efforts.Innocenti et al. duly realized the certain shortcomings of the existing 2-aminopyrazine- and benzimidazole-based Nek2 inhibitors and, thus, constructed a novel series of hybrid chemotypes that preserved and stimulated certain key structural features of the inhibitor classes discussed above  143,144144,146. he Gly92  and Ile1in vivo at the molecular level and help SAR optimization for enhanced anti-cancer activities. As described earlier, our developed whole animal Nek2 overexpression model in flies led to the deregulation of several cell migration markers—Rho1, Rac1, and Ecad—along with unique tissue phenotypes, amenable for drug screening in vivo. dNek2 overexpression, in conjunction with receptor tyrosine kinase and mitogen-activated protein kinase, promoted local invasion, distant cell seeding, and metastasis. The Nek2 kinase was established as a key mediator that cooperated with other oncogenic signaling pathways to promote aggressive colonization of tumorigenic cells into adult fly tissues. With the goal of identifying a novel type of non-toxic Nek2 pharmacophore, we undertook a bioinformatics-based computational approach. We analyzed the primary sequence and crystallographic structures of the Nek2 kinase and noted the presence of a unique helical motif (αT), just outside of the DFG motif, which was also present in receptor tyrosine kinases, EGFR and HER2 [in silico on an existing library of advanced-stage clinical inhibitory agents of EGFR/HER2 kinases and discovered that pelitinib (EKB-569) and neratinib (HKI-272)  , Entry 8An investigation by Zhou et al. indicated that Nek2 kinase overabundance resulted in poor prognosis, rapid cell proliferation, and drug resistance in multiple myeloma . Almost in vivo results barred any of the developed scaffolds from being considered viable clinical candidates. Fang et al. addressed this issue by discovering a new compound, MBM-5 [in vivo activity [50 value towards the Nek2 kinase. The computational study predicted that MBM-5 would occupy the ATP-binding site and establish a hydrogen bonding interaction with Cys89 and Asp159 residues, along with additional pi-stacking interaction with Phe148 residue. The cell-based assay revealed that MBM-5 effectively reduced Hec1 phosphorylation and, even more intriguingly, led to reduced levels of Hec1 protein. Notably, MBM-5 did not affect the Aurora A kinase activity. When tested on multiple cancer cell lines, MBM-5 was found to be more effective towards leukemia, gastric, and colorectal cancer cell lines as the EC50 values were in the low micromolar range. In line with other Nek2 inhibitors, MBM-5 caused mitotic abnormalities and induced apoptosis. Most importantly, MBM-5 exerted significant efficacy in suppressing cancer growth in MGC-803 gastric and HCT-116 xenografts mice. Although MBM-5 showed some promising traits of a desirable clinical candidate, it suffered from low half-life in vivo, barring its advancement to the next level. Despite this, MBM-5 represented a new scaffold for Nek2 selective inhibitor design that could potentially be exploited for developing an improved pharmacophore in the future.While significant progress was made towards developing Nek2 inhibitors thus far, a lack of desirable activity . The autIn an interesting study, Wang et al. noted that Nek2 acts as a functional protein binding partner of enhancer of zeste homolog 2 (EZH2) and post-translationally regulates EZH2 in glioma stem cells (GSCs) . Nek2 ph12a (12b), which exhibited 10-fold selectivity over the CDK2 kinase. This compound was also found to be effective in growth inhibition assays involving U2OS, MDA-MB-231, and HeLa cell lines. Unfortunately, 12 was found to be unstable under simulated physiological conditions; thus, it could not progress further as a clinical candidate.Purine-based pharmacophores were introduced by Coxon et al. as ATP-competitive reversible inhibitors of Nek2 and CDK2 kinases . After p12a A bound t12a A. Furthe13  inhibit tumor cell proliferation, (b) promote mitotic abnormalities, and (c) retard tumor growth. These findings are the hallmark of the inhibition of the Hec1/Nek2 axis. This study ushered an alternative approach to the targeting of Nek2 that was subsequently adopted for building improved Nek2 inhibitory phenotypes.Several key studies have revealed the important features of Nek2-associated protein-protein interactions as key regulatory events during mitosis ,36,42,4415  in Sprague Dawley (SD) rats and improved the tumor suppression ability in mice bearing human MDA-MB-231 xenografts. A relentless pursuit of developing a clinical candidate for destabilizing the Hec1/Nek2 axis has led to the discovery of TAI-95 (The ongoing optimization of the thiazole core has, further, led to the discovery of a TAI-1 derivative, compound 19,  which coin vitro and in vivo settings. They allow target identification and validation, proteome-wide analysis of off-targets, and subsequent analysis of signal transduction pathways [in vivo endpoints—the inhibition of Nek2 and related pathways with low whole animal toxicity [The extensive research reported so far strongly suggests that the inhibition of Nek2 activity in diseased states may lead to successful therapeutics. The advent of several classes of small molecule Nek2 inhibitors have significantly broadened our prospect of developing novel Nek2-targeting therapeutic modalities. Despite the focused efforts over the last two decades, only one PPI inhibitor chemotype, T-1101 Tosylate, has made it to clinical trials. Given the involvement of the Nek2 kinase in several diseases, it would be important to further enrich the Nek2 inhibitory pipeline for successful clinical trials . In thispathways . This sttoxicity ,216,217.


The stability of Antarctica and its contribution to sea-level rise are determined by the evolution of its ice shelves, which are vast expanses of floating ice that buttress the continent. Ice shelves have been undergoing major changes in recent decades, many of them collapsing. The presumption is that these events are caused by hydrofracturing and unusual wave forcing. We find that a main control on fracturing is the thickness of the ice mélange encased in and around preexisting rifts that penetrate the entire ice shelf thickness. If the ice mélange thins beyond a threshold value, the rifts reactivate and trigger iceberg calving. This process linking climate forcing and ice shelf retreat is missing from models and does not require hydrofracture. The sudden propagation of a major preexisting rift (full-thickness crack) in late 2016 on the Larsen C Ice Shelf, Antarctica led to the calving of tabular iceberg A68 in July 2017, one of the largest icebergs on record, posing a threat for the stability of the remaining ice shelf. As with other ice shelves, the physical processes that led to the activation of the A68 rift and controlled its propagation have not been elucidated. Here, we model the response of the ice shelf stress balance to ice shelf thinning and thinning of the ice mélange encased in and around preexisting rifts. We find that ice shelf thinning does not reactivate the rifts, but heals them. In contrast, thinning of the mélange controls the opening rate of the rift, with an above-linear dependence on thinning. The simulations indicate that thinning of the ice mélange by 10 to 20 m is sufficient to reactivate the rifts and trigger a major calving event, thereby establishing a link between climate forcing and ice shelf retreat that has not been included in ice sheet models. Rift activation could initiate ice shelf retreat decades prior to hydrofracture caused by water ponding at the ice shelf surface. The Larsen A and Larsen B ice shelves, in the Antarctic Peninsula, collapsed in spectacular fashion in 1995 and 2002, respectively, as a result of climate warming . The Lar4The Larsen C Ice Shelf, immediately south of Larsen A and B , is the The prevailing view for explaining the evolution of Larsen A and B and their collapse is the hydrofracture theory –13. In t14Calving events on ice shelves dominantly originate from ice front-parallel rifts that propagate in a direction transverse to the ice flow –25. WhenSeveral studies have attempted to quantitatively couple rift growth (followed by ice breakup) to its destabilizing effect on Antarctic ice shelves, although calving processes are not well understood and modeled see ref. . In thisIce shelves are composed of meteoric units fed by inland glaciers, glued together along suture zones. Suture zones in Larsen C form seaward of the Joerg and Churchill Peninsula and around Tonkin and Francis Islands, in places where the ice shelf rifts apart from stress singularities along the coastline . These f3439Prior work on Larsen B and C has shown that melting of accreted ice in rifted areas may alter the longitudinal stress orientation on the ice shelf, facilitating rifting through suture zones and potentially destabilizing the ice shelf , 29, 32.444955When combined together, processes such as hydrofracture and viscoelastic flexure of ice shelves can lead to runaway processes such as iceberg-capsize tsunamigenesis as described in ref. Iceberg A68 calved after the along-front propagation of a crack that had grown since 2005, stayed dormant for 10 y, opened up around 2014, stopped, and reopened up again in November 2016 , 30, to The first phase of rift propagation in year 2014 stopped at the suture zone downstream of Joerg Peninsula , 43 thatHere, we present a stress-balance analysis of the state of the ice shelf prior to the propagation that includes rifts. We evaluate the relationship between ice thinning and the opening rate of A68. We consider two major effects: 1) ice shelf thinning, which has been documented elsewhere , and 2) We model the evolution of ice shelf stress balance around actively propagating rifts as the ice shelf and/or ice mélange thickness changes. Irrespective of the propagation criterion, i.e., linear elastic fracture mechanics , 66, 67,Boundary conditions at the flanks of the rift depend on whether the rift is 1) actively opening, in which case water or mélange pressure applies as a normal boundary condition and we assume zero lateral shear, or 2) closing, in which case a nonpenetration normal boundary condition is applied [relying on application of contact mechanics  and penaOur model requires a full specification of the ice shelf geometry. The mesh domain A–C correThe ice shelf thickness is constrained by a high-quality TandemX (TDX) digital elevation model (DEM)  from 201Several forward runs are carried out where we vary the ice shelf thickness and the thickness of the mélange. Here, we define ice mélange as any part of the ice shelf where ice thickness is less than 250 m, which results in a regional map of accreted ice similar to what ref. A decrease in ice shelf thickness and mélange thickness leads to a decrease in the average opening rate for the GIR rift from 79 m/y (with 1 m thinning) to 36 m/y (with 14 m thinning) . This deA similar set of results is obtained for the case where we thin only the ice shelf , with an average opening rate decreasing from 79 to 22 m/y. Hence, the main cause of the deactivation of the rift in the prior simulation is ice shelf thinning.Finally, we examine the scenario where only the ice mélange is thinned. We find that the opening rate increases from 76 to 112 m/y, with strong variations of the rate along the rift. Opening rates for the eastward rift are largest in the middle of the rift at 250 m/y. Opening rates for the rift closest to GIR reach upward values of 400 m/y at the tip near the ice rise. We find that the opening rate of the eastern rift saturates at 150 m/y for an overall thinning greater than 18 m. Further thinning does not increase the opening rate, which remains at 150 m/y. Thinning of the ice mélange therefore exerts a direct control on rift propagation and increases the rate of opening as the mélange thins. Beyond a threshold thickness of the ice mélange, the system reacts as if the mélange no longer existed.Laser altimetry data collected by NASA Operation IceBridge in 2008 and 2016 along a flow line that crosses the rifted zone provide information on the evolution of the ice mélange in the intervening 8 y after compensation for the horizontal motion of the ice shelf . Rift 2aOur results demonstrate a strong sensitivity of the opening of the A68 rift to changes in ice mélange thickness close to GIR. Conversely, they suggest a lesser and even negative impact of ice shelf thinning on the opening rate. Ice shelf or ice mélange thicknesses are strong contributors to the buttressing capability of an ice shelf , 48 baseWe have a similar situation here with the calving of A68 around GIR. But our approach quantifies this physical process in more detail because we evaluate the impact of the rate of ice mélange thinning instead of examining the end states “thick mélange” versus “no mélange.” Our simulation explains why the rift that originated in the GIR area may have become unstable following a prolonged period of mélange thinning, either from above (warmer air temperature) or from below (warmer ocean temperature), or both, or from mechanical failure of the mélange , which rThe hydrofracture mechanism induced by water ponding at the surface –12 requiThe ice mélange is typically one order of magnitude thinner than the ice shelf proper  and hencStrong oceanic circulation in the GIR area may have melted accreted marine ice , 83. ThiAt present, we do not have sufficient information about the time evolution of the ice mélange within the rifts, especially over time scales of decades, and about the surface and ocean heat fluxes that control the growth of the mélange to identify which physical processes may have reduced its thickness. We recommend more studies of the ice mélange in the future to better understand its time evolution and its impact on ice shelf stability.We posit that the physical processes that control the stability of nascent rifts in the Peninsula are the same that operate on ice shelves farther south. An important aspect of the ice mélange is that it could start thinning independent of melt water ponding at the surface of ice shelves, for instance as the annual sea ice cover starts receding , possiblWe present a modeling study of Larsen C conducive to the calving of iceberg A68 that provides insights into the physical processes responsible for the calving. We find a strong relationship between the thickness of ice mélange near GIR and the opening rate of the rift responsible for the calving of iceberg A68. Conversely, we find that ice shelf thinning cannot explain the propagation of the rift. In fact, ice shelf thinning has the opposite effect of stabilizing the rifts. Given that ice mélange thickness depends on the ocean circulation underneath ice shelves and on radiation fluxes at the ice surface, our analysis may offer a link between climate forcing and ice shelf stability that has not been brought up to prominence and does not require extensive melt water ponding at the ice shelf surface. We posit that the ice mélange in nascent rifts near ice shelf fronts of the Antarctic Peninsula may thin decades prior to hydrofracture, thereby explaining why ice shelf fronts started to retreat well before the point of collapse through hydrofracture or reaching the compressive arch. Numerical models currently assume that ice shelves are at risk only if melt water ponding occurs. We find that a sufficient condition for their retreat could be the thinning of the ice mélange. Further investigation of the ice mélange is warranted to elucidate its role and evolution in more detail.


Pyramidal neurons (PNs) are covered by thousands of dendritic spines receiving excitatory synaptic inputs. The ultrastructure of dendritic spines shapes signal compartmentalization, but ultrastructural diversity is rarely taken into account in computational models of synaptic integration. Here, we developed a 3D correlative light–electron microscopy (3D-CLEM) approach allowing the analysis of specific populations of synapses in genetically defined neuronal types in intact brain circuits. We used it to reconstruct segments of basal dendrites of layer 2/3 PNs of adult mouse somatosensory cortex and quantify spine ultrastructural diversity. We found that 10% of spines were dually innervated and 38% of inhibitory synapses localized to spines. Using our morphometric data to constrain a model of synaptic signal compartmentalization, we assessed the impact of spinous versus dendritic shaft inhibition. Our results indicate that spinous inhibition is locally more efficient than shaft inhibition and that it can decouple voltage and calcium signaling, potentially impacting synaptic plasticity. Pyramidal neurons are covered by thousands of dendritic spines receiving excitatory synaptic inputs. A new 3D correlative light-electron microscopy (3D-CLEM) approach enables the cartography of synaptic ultrastructure with cell type-, dendrite type- and synapse type-specific resolution in brain tissues, revealing unique properties of spines that receive dual innervation. To expand our distribution of DiSs and shaft iPSDs  activation of the ePSD of the DiS and a shaft iPSD placed next to it (Δx = 0.7 μm)  or presence  of inhibition and computed the drop in depolarization amplitude inhV(Δt) = 1 − ΔVmax,E+I / ΔVmax,E. InhV = 0 indicates no inhibition of the EPSP, while inhV = 1 indicates complete inhibition of the EPSP. InhV was an asymmetrical function of Δt  ; and (2) 0.7 μm) . To quanon of Δt  36,128],128N = 3inh[Ca2+] (Δt) = 1 − [Ca2+]max,E+I / [Ca2+]max,E. Inh[Ca2+] peaked at Δt = 0 ms for both spinous and shaft inhibition. More precisely, spinous inhibition reduced calcium transient amplitude by 10% in average, reaching >36% in the top 10% simulations, while shaft inhibition reduced it by 8.6% in average and >28% in the top 10% simulations . We have previously shown that SRGAP2C regulates synapse density, spine neck length, and DiS proportion in oblique apical dendrites )In vivo 2-photon imaging experiments have shown that DiSs are among the most stable spines along the dendrites of L2/3 PNs , induced by dendritic (blue) or spinous (orange) IPSPs as a function of Δt. The inhibition of calcium signals was approximately 40% lower in neurons expressing SRGAP2C than in control neurons . CLEM, correlative light–electron microscopy; DiS, dually innervated spine; ePSD, excitatory postsynaptic density; GFP-GPHN, GFP-tagged gephyrin; iPSD, inhibitory PSD; IPSP, inhibitory postsynaptic potential; LM, light microscopy; L2/3, layer 2/3; PN, pyramidal neuron; SRGAP2C, Slit-Robo Rho GTPAse-activating protein 2C; SSC, somatosensory cortex; WT, wild-type; 3D-EM, three-dimensional electron microscopy.(A) Z-projection of the confocal stack showing a segment of basal dendrite of an L2/3 PN expressing cytosolic TdTomato (in magenta), GFP-GPHN (in green), and endrites ,127 sugg neurons . The dat(TIF)Click here for additional data file.S1 TableePSD, Rneck, and Ldend) are sorted by decreasing contribution to the variance of the simulation outputs, as estimated with a GLM. Numbers indicate to which proportion input variables accounted for the variance of considered output.Highest-ranking input parameters (A(XLSX)Click here for additional data file.S1 DataThe first sheet of the .xlsx file describes the samples used in our experiments. The second sheet reports scores for log-normality and intersample variability tests for each morphological variable. The 2 last sheets report spine and PSD anatomy in all segmented dendrites. Legends are included in the rightmost column.(XLSX)Click here for additional data file.


Diabetes mellitus (DM) eventually leads to chronic vascular complications, resulting in cardiovascular diseases. DM-associated endothelial dysfunction (ED) plays an important role in the development of chronic vascular complications. Low endothelial nitric oxide synthase (eNOS) activity, inflammation, and oxidative stress all contribute to ED. The G protein–coupled receptor Takeda G protein–coupled receptor 5 (TGR5) is a membrane receptor for bile acids that plays an important role in the regulation of glucose metabolism. Recent studies have shown that TGR5 is involved in the regulation of various mediators of ED, which suggests that TGR5 may represent a target for the treatment of DM-associated ED. In this review, we summarize the principal mechanisms of DM-associated ED, then propose TGR5 as a novel therapeutic target on the basis of its mechanistic involvement, and suggest potential directions for future research. Diabetes mellitus (DM) is a global public health problem that is associated with a high financial burden for healthcare systems. The progression of DM eventually involves the development of chronic vascular complications, which result in cardiovascular diseases. These cardiovascular complications are the main causes of death in diabetes patients around the world . AlthougThere are many potential complications of DM, which have differing mechanisms. DM is considered to be a vascular disease in addition to a metabolic disease  because In patients with DM, the risk of vascular disease has increased by two-to-four times in recent decades . MacrovaTakeda G protein–coupled receptor 5 (TGR5) is a G protein–coupled receptor that is expressed in many organs and tissues, but is also widely expressed in almost all types of endothelial cells (ECs). Recent studies have shown that TGR5 agonists are beneficial in DM and TGR5 has become a promising target for its treatment . TherefoThe endothelium is a layer of squamous epithelial cells that lines the inner surface of vascular systems. ECs form a barrier between blood vessels and tissues, and control the flux of substances in and out of tissues. They provide a metabolic interface between blood and tissue and are therefore essential for the maintenance of vascular homeostasis . The inaED forms the basis of the chronic microvascular and macrovascular complications of DM. Microvascular complications include DR, nephropathy, and neuropathy, and macrovascular complications affect coronary and peripheral arteries, causing cardiovascular and cerebrovascular diseases and stroke . In recel-arginine by the enzyme eNOS (Furchgott and Zawadzki first described the importance of the intact endothelium for acetylcholine-induced vasodilatation in 1980 . The medyme eNOS . eNOS isyme eNOS . Howeveryme eNOS . The mec2− instead of NO. This superoxide quickly combines with NO to form nitrogen-peroxynitrite (ONOO−), which is a highly active oxidant (4) to dihydrobiopterin (BH2) by ONOO− and H2O2 limits the availability of eNOS substrates and prevents the production of NO , an NO precursor, increases the NO concentration by stimulating the production of eNOS. They found that the glomerular filtration rate of diabetic rats increased when they were administered LA and provided evidence that this effect may be the result of increases in eNOS expression and urinary cGMP, which leads to renal microvascular dilation and the amelioration of ED. Liu  . ROS arepathways , throughn of ROS .via nuclear factor kappa-B (NF-κB)  . TherefoThe pathogenesis of ED in DM involves oxidative stress, chronic inflammation, and low NO production. TGR5 is a G protein–coupled receptor that is present in many organs and tissues and is widely expressed in almost all types of EC. Recent studies have shown that TGR5 mediates beneficial effects in DM and regulates various molecules that mediate DM-associated ED .et al. found that activation of TGR5 using oleanolic acid reduced the serum glucose and insulin concentrations of high-fat-diet-fed mice (TGR5 (Gpbar1) is a G protein–coupled bile acid receptor that has been widely studied in recent years. It is expressed in various tissues and cell types, such as the gall bladder, intestine, and placenta . The rolfed mice . In addifed mice . Such imThe role of the NO produced by eNOS activity in the maintenance of vascular health and function has been well described . The reivia the “bile-salt-TGR5-cAMP pathway,” which involves binding to the cAMP response element of the human eNOS promoter  is an intestinal hormone that has been shown to improve endothelial function , and theAs discussed, activation of the bile-salt-TGR5-cAMP and TGR5-GLP-1-PI3K-eNOS pathways induces eNOS expression, which should protect against the ED induced by DM. However, eNOS supplementation does not always protect the endothelium. via the NF-κB pathway represents targets for the treatment of such ED. Previous studies have shown that the activation of TGR5 by INT-777 reduces oxidative stress in human podocytes exposed to a high-glucose environment (et al. showed that the effect of INT-777 to reduce oxidative stress is achieved via a reduction in the production of ROS and apoptosis in cardiomyocytes exposed to a high-glucose environment (As described above, oxidative stress and inflammation are important mechanisms whereby DM leads to endothelial damage. The inhibition of ROS production or inflammatory signaling ironment . In addiironment . These sironment . Guo et ironment . MoreoveLow eNOS expression, inflammation, and oxidative stress are the principal mechanisms of ED in DM, and the means whereby conventional hypoglycemic drugs protect against ED mainly involve antagonism of these mechanisms . Many stin vitro evidence to suggest that TGR5 agonists may induce cancer cell proliferation (Although the use of TGR5 agonists for the treatment of ED in DM holds great promise, a series of questions remain to be resolved. First, TGR5 is expressed in many organs, which may imply that an agonist would be prone to side effects in other organs. For example, feration .Second, even in the same organ, the effects of TGR5 may differ, because of the existence of various distinct binding sites. For example, TGR5 is expressed in the primary cilia of cholangiocytes, where it is coupled to G-α and inhibits cell proliferation, but it is also expressed on the apical plasma membrane, where it is also coupled to G-α proteins but initiates cell proliferation .Third, TGR5 binds a wide range of ligands, including endogenous and synthetic bile acids and neurosteroids, and also synthetic agonists with a nonsteroidal core . AlthougFourth, differing concentrations of TGR5 agonists may have different outcomes. For example, compound 18, a new type of TGR5 agonist, has a significant effect on biliary secretion at a dose as low as 3 mg/kg, whereas doses of compound 18 above 30 mg/kg have a significant effect on GLP-1 secretion .Finally, even if an TGR5 agonist is used at the same dose, the results obtained in animal models and humans differ, because of the biological differences between species . TherefoNevertheless, in recent years, substantial efforts have been made to identify suitable TGR5 agonists, and there have been some promising results. For example, The high prevalence of and numerous risk factors for CVD make it the leading cause of death in the world . However


Staphylococcus pseudintermedius, Streptococcus canis or Escherichia coli was determined in presence of stimulated neutrophils. Additionally, free DNA and nucleosomes as NET marker were analyzed in supernatants and neutrophils were assessed for NET formation by immunofluorescence microscopy. Methylprednisolone concentrations of 62.5 and 625 µg/mL enhanced the neutrophil killing of Gram positive bacteria, whereas no significant influence was detected for the Gram negative Escherichia coli. Interestingly, higher amounts of free DNA were detected under methylprednisolone stimulation in a concentration dependency and in the presence of Streptococcus canis and Escherichia coli. The nucleosome release by neutrophils is induced by bacterial infection and differs depending on the concentration of methylprednisolone. Furthermore, immunofluorescence microscopy analysis identified methylprednisolone at a concentration of 62.5 µg/mL as a NET inducer. In summary, methylprednisolone enhances NET-formation and time-dependent and concentration-dependent the bactericidal effect of canine neutrophils on Gram positive bacteria.Methylprednisolone is a glucocorticoid and can negatively influence immune defense mechanisms. During bacterial infections in the dog, neutrophils infiltrate infected tissue and mediate antimicrobial effects with different mechanisms such as phagocytosis and neutrophil extracellular trap (NET) formation. Here, we investigated the influence of methylprednisolone on canine NET formation and neutrophil killing efficiency of Gram positive and Gram negative bacteria. Therefore, canine blood derived neutrophils were treated with different concentrations of methylprednisolone over time. The survival factor of  The glucocorticoid methylprednisolone is used in veterinary medicine as an anti-inflammatory and immunosuppressive agent depending on the applied concentration ,2,3. TheDirofilaria immitis, Trypanosoma cruzi and Toxoplasma gondii [Escherichia coli or Streptococcus species [Neutrophils are the first line of defense of the innate immune system of the host. They are recruited to the site of infection and can recognize invading microorganisms such as bacteria. Neutrophils can counteract invading bacteria by different mechanisms, including production of reactive oxygen species (ROS) and the release of antimicrobial peptides ,13,14,15a gondii ,28,29. Ia gondii . Further species .Escherichia coli [Several studies investigated the influence of drugs from natural as well as conventional medicine on neutrophil functions and bacterial killing. For example, Gum Arabic, which is a water-soluble polysaccharide with sugars used as a traditional medicine in Africa and India, has immunomodulatory effects on neutrophils by increasing the ROS production and phagocytic activity against hia coli . The brehia coli  and the hia coli . Therefohia coli .The aim of our study was to determine the effect of methylprednisolone, which is described as an anti-inflammatory and immunosuppressive drug, on canine neutrophils in cases of concurrent experimentally induced bacterial infections.Staphylococcus (St.) pseudintermedius, Streptococcus (Sc.) canis and Escherichia (E.) coli with and without methylprednisolone stimulation for a short period of incubation of one and three hours.To analyze the influence of methylprednisolone on bacterial infections in dogs, we conducted neutrophil killing assays with St. pseudintermedius is an opportunistic Gram positive pathogen and is part of the normal skin flora of most dogs. If the defense mechanisms of the host are reduced, skin lesions with the occurrence of a systemic inflammatory response syndrome are described [St. pseudintermedius was found to be associated with urinary tract infections and respiratory tract disease [Sc. canis is another commensal Gram positive bacterium found in the flora from skin and mucosa of dogs. However, an imbalance of the immune system can be associated with a toxic shock syndrome, necrotizing fasciitis, fibrinous bronchopneumonia and urinary tract infections triggered by Sc. canis [E. coli as a normal commensal inhabitant starting directly after birth. A disturbance of the microbiome results in an imbalance of the normal intestinal flora. E. coli can cause gastrointestinal and extra-intestinal diseases such as urogenital tract infection in dogs [escribed ,37,38. F disease ,40,41. Sc. canis ,45,46,47 in dogs ,50,51,52St. pseudintermedius showed after one and three hours of incubation a statistical lower survival factor (SF) at a methylprednisolone concentration of 62.5 µg/mL and after three hours incubation time additionally at 625 µg/mL compared to the SF of bacteria incubated with neutrophils alone  were adapted to described plasma concentrations after systemic administration in the dog ,54. St. ls alone A,B. The .5 µg/mL C,D. Intenisolone E,F. ThesSc. canis. Furthermore, after three hours incubation no significant bactericidal effect by all tested concentrations of methylprednisolone was observed. Therefore, the enhanced bactericidal effect of neutrophils by methylprednisolone cannot be explained by a direct bactericidal effect on the bacteria.In order to exclude a possible direct bactericidal effect of methylprednisolone on the investigated bacteria, the assay was repeated without canine neutrophils . In the E. coli bacteremia [E. coli sepsis in an experimental model after methylprednisolone administration could be detected [In general, an immunosuppressive treatment is described to affect the immune system by increasing susceptibility to infections . Latent cteremia . During detected .St. pseudintermedius and Sc. canis.Our results implicate that methylprednisolone enhances the bactericidal effect of canine neutrophils for infections with the Gram positive bacteria Since we could show a concentration-dependent enhanced killing of Gram positive bacteria by neutrophils, we investigated the possible responsible mechanism. Bacteria can be killed by neutrophils with different mechanisms including phagocytosis, degranulation and the generation of ROS as well as the formation of NETs ,17,18,63Sc. canis ) was evaluated in the supernatants of the performed killing assay B, as DNathe NETs ,67,68,69eumoniae . DNases eumoniae . An exceeumoniae  and is deumoniae ,19,71. OSt. pseudintermedius induced a notable higher release of free DNA from neutrophils only after one hour of incubation and only at a concentration of 625 µg/mL  were quantified in the supernatants of treated neutrophils . In accot manner C. The renisolone D–G. At t E. coli D–G. ThisSt. pseudintermedius, could be explained with the existence of nuclease activity produced by the bacterial species. St. pseudintermedius showed significant lower survival factors  plasma. For the plasma itself, a nuclease activity could not be ruled out completely and EDTA as a calcium and magnesium chelator could have an impact on NET release. Therefore, an assay without plasma was finally performed to confirm our assumed thesis that methylprednisolone triggers NET release.p value of 0.0603 was calculated. After Grubbs’ test an additional significance at a concentration of 12.5 µg/mL was noted  (accessed on 27 April 2021). All blood taking procedures were conducted with oral consent from the dog owners.The collection of blood from healthy dogs was registered at the Lower Saxonian State Office for Consumer Protection and Food Safety . It was conducted in line with the recommendations of the German Society for Laboratory Animal Science (Gesellschaft für Versuchstierkunde) and the German Veterinary Association for the Protection of Animals (Tierärztliche Vereinigung für Tierschutz e. V.)  to exclude white blood cell abnormalities , Golden Retriever, Malinois, Mixed breed dog (2), German wirehaired pointer and White shepherd dog) and two females (Mixed breed dogs). The maximum age for passing the inclusion criteria was defined at 8 years. From each dog, 10–13 mL of blood from the ties See .g without brake, 20 °C). After removing plasma, peripheral blood mononuclear cells and Pancoll, the polymorphonuclear cells were harvested and diluted in 1 × endotoxin-free PBS. After centrifugation , the pelleted granulocytes were lysed for three times until the pellet was white. The hypotonic lysis was started after adding 6 mL sterile ice cold 0.2% sodium chloride (NaCl) to the pellet and stopped after 30 s by adding 6 mL of sterile ice cold 1.6% NaCl. Each lysis step was followed by a centrifugation . The purified pellet was resuspended in 1 mL Roswell Park Memorial Institute (RPMI) 1640 Medium  and the number of polymorphonuclear cells was determined by counting the trypan blue  negative cells using a Neubauer chamber.Purification of canine neutrophils were performed by density gradient centrifugation in combination with a hypotonic lysis of the erythrocytes. The density gradient was composed of 2 mL Histopaque 1.119 g/mL  and 2 mL human Pancoll 1.077 g/mL . The EDTA blood was diluted 1:2 with 1 x endotoxin-free PBS  . The amount of 6.5 mL blood-PBS mixture was layered on top of the gradient in a 15 mL falcon tube and the gradient was centrifuged  while applying the Software MACSQuantify Software, . Ten thousand live-gated events were counted and the population of the granulocytes were detected by analysis of the cell size and granularity of the sample by forward scatter (FSC) and side scatter (SSC). On average, a purity of 90.5 ± 0.6% in the FACS analysis was achieved.The purity of the isolated canine granulocyte population was assessed by flow cytometry. Therefore, 1 mL propidium iodide  was diluted in 49 mL cell staining buffer . For analysis by fluorescence-activating cell sorting (FACS), 20 µL of the cell suspension was mixed with 180 µL of the propidium iodide solution. The measurement was performed by using a MACSQuantStreptococcus (Sc.) canis (IMT48617), Staphylococcus (St.) pseudintermedius (IMT49419) and Escherichia (E.) coli (IMT45929). All strains were isolated from the wounds of dogs during diagnostic procedures at the Centre for Infection Medicine, Institute of Microbiology and Epizootics, Freie Universität Berlin, Germany. Bacterial strains were grown on a blood agar plate  at 37 °C (20–24 h).In this study the following bacterial strains were used: St. pseudintermedius or E. coli was inoculated in 10 mL tryptic soy broth (TSB) without dextrose  and incubated overnight at 37 °C, 200 rpm. A 1:50 dilution from the overnight culture was conducted with pre-warmed TSB  and incubated at 37 °C 200 rpm until it reached the late exponential growth phase .Working cryostocks were produced as the following: One colony of Sc. canis were inoculated in 10 mL Todd Hewitt Broth (THB)  and incubated at 37 °C 5% CO2 for around 16 h in a melting ice-bath to delay the start of growth. A 1:50 dilution from the overnight culture was conducted in pre-warmed THB  and incubated at 37 °C, 5% CO2 until the late exponential growth phase (OD600nm 0.9 ± 0.05).Three colonies of Immediately, after the cultures reached the respective optical density, they were mixed with glycerol  and aliquots in 1.5 mL tubes were shock frosted in liquid nitrogen. The working cryostocks were stored at −80 °C until they were used and only thawed once. The colony forming units (CFU/mL) were determined by the plating of serial dilutions on agar plates for each batch.5 cells in a final volume of 200 µL/well) and infected with the three different bacterial strains from the working cryostocks (multiplicity of infection (MOI) = 1). Methylprednisolone  in three different concentrations 625 µg/mL, 62.5 µg/mL and 12.5 µg/mL were additionally added. In the untreated sample (0 mg/mL), RPMI was added. Samples were incubated with 10% of autologous canine plasma. The plates were centrifuged  and incubated afterwards for 1 and 3 h at 37 °C 5% CO2, respectively. After incubation, serial dilutions were conducted and plated on blood agar plates (St. pseudintermedius and Sc. canis) or on luria broth (LB) agar plates (E. coli). Plates were incubated for 20 h at 37 °C and the CFU/mL was determined for 0, 1 and 3 h. Finally, the survival factor was calculated as described previously [The neutrophil killing assay was conducted as described previously  with theeviously  for eachg at 20 °C) and the supernatant was collected and stored at −20 °C for later analysis of free DNA (see below).Furthermore, at the end of the neutrophil killing assays, the 48 well plates were centrifuged  or on luria broth (LB) agar plates (E. coli). Plates were incubated for 20 h at 37 °C and the CFU/mL was determined for 1 and 3 h.The above-described neutrophil killing assay was conducted without neutrophils. The plates were incubated for 1 and 3 h at 37 °C 5% CO5/well). The final volume was 200 µL in each well. The cells were stimulated with methylprednisolone  in three different concentrations, as described above. Negative control RPMI medium was added. A positive control methyl-β-cyclodextrin  was added. After centrifugation , the plates were incubated for 3 h at 37 °C and 5% CO2. Finally, cells were fixed with paraformaldehyde . The plates were wrapped with parafilm and stored at 4 °C until the immunofluorescence staining was performed.For the NET induction, assay cover slips  were placed in 48 well plates . The slides were coated in accordance with the manufacturer’s instructions with poly-L-lysine  and handled afterwards, as previously described . Harvest2. After centrifugation for 5 min, 400× g at 20 °C the supernatants were collected and stored at −20 °C for later analysis with a Pico Green assay (see below).For the analysis of free DNA, cells and stimuli were co-incubated in 1.5 mL tubes in the same amount and concentrations as described for the 48 well plate experiment. Half-closed tubes were incubated for 3 h at 37 °C and 5% CO®Gold antifade reagent .NETs were stained as previously described ,107. Bri2) and finally fixed with 4% paraformaldehyde. NETs were stained with a mouse monoclonal-antibody against DNA/histone 1 followed by an incubation with an ALEXA Flur488 Plus, as described above. Sc. canis was stained with a rabbit anti-Streptococcus suis antibody . E. coli was stained with a rabbit anti E. coli . As the secondary antibody, all bacteria were stained with a goat anti-rabbit Alexa 633-conjugated antibody  for 1 h. The staining was finally conducted as described above. Respective isotype controls were used as described above.For visualization of bacteria and NETs by immunofluorescence microscopy, 48 well plates and coverslips were prepared as described above in the NET induction assay. The samples were prepared with neutrophils, 10% autologous plasma and bacteria as described above in the neutrophil killing assay and co-incubated with methylprednisolone (625 µg/mL). Afterwards, centrifugation samples were incubated for 3 h . StStained coverslips were recorded using a confocal inverted-base fluorescence microscope (Leica TCS SP5) with a HCX PL APO 40 × 0.75–1.25 oil immersion objective. Settings were adjusted with control preparations using an isotype control antibody. For each sample 6 randomly selected images were acquired and used for quantification. The cells present in the pictures were counted manually using the ImageJ software . The total number of neutrophils and positive neutrophils (activated or NET-releasing) were counted. A neutrophil was counted as positive if an evident off-shoot of DNA was visible or if at least two of the following criteria were found: enlarged nucleus, decondensed nucleus or blurry rim. The percentage of NET-positive neutrophils was calculated. For each sample, a minimum of 200 cells was counted and an average from the six pictures was calculated.For detection of free DNA released from neutrophils without methylprednisolone stimulation, neutrophils were incubated as described in the neutrophil killing assays for 1 and 3 h. All samples were centrifuged and incubated and the supernatants were harvested after final centrifugation and stored at −20 °C for later analysis.TM PicoGreen®dsDNA kit; Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommendation as previously described [All collected samples from all assays were carefully defrosted and free DNA was quantified with a Pico Green Assay  was used to determine the Dnase activity in the supernatant of the neutrophil killing assay and the respective controls. The test was performed following the manufacturer’s instructions with 25 µL for each sample.The cell death detection ELISA PLUS kit  was used to quantify the number of histone-associated-DNA-fragments (mono- and oligo-nucleosomes) in the supernatant of the neutrophil killing assay and the respective controls. The assay was conducted following the manufacturer’s instructions.2. Subsequently 2′,7′-dichlorofluorescin-diacetate  with a final concentration of 10 µM was added to each sample and all samples were incubated at 37 °C and 5% CO2 for another 30 min. All samples were analyzed in duplicates and a respective background control without DCFH-DA was included. Intracellular ROS production was measured by flow cytometry , filter BL1 = 530/30) and a total of 10,000 events were recorded. The mean green fluorescence intensity of all cells (X-Mean of BL-1) was determined as relative measurement of ROS production. The gating strategy included only singlets of the neutrophil population . Values higher than 0.05 and lower than or equal to 0.1 are presented as Arabic numbers. All values were analysed for normality with the Shapiro–Wilk test. Very few individual values did not pass the normality test, however, the statistical calculation was conducted for all values in all experiements equal.Data were analyzed using Excel (Microsoft Office) and GraphPad Prism 8.4.3 and 9.1.0 . For detection of statistical significances between two groups, an one-tailed paired Student’s Staphylococcus pseudintermedius and Streptococcus canis.In conclusion, methylprednisolone induces NET formation and intracellular ROS production in vitro. A concentration-dependent and time-dependent enhanced bactericidal effect of canine neutrophils was identified under methylprednisolone stimulation for the Gram positive The clinical impact of these findings must be interpreted carefully. Recently, the use of low dose glucocorticoids (dexamethasone) gained attention as the mortality rate of hospitalized patients with COVID-19 who needed respiratory support was reduced . In the


Platysternon megacephalum. A total of 11 big‐headed turtles were captured, and all individuals were used in this mark–recapture study. The site fidelity results showed that the adult big‐headed turtles (n = 4) had a 71.43% recapture rate in the original site after their release at the same site, whereas the juveniles (n = 1) showed lower recapture rates (0%). Moreover, the homing behavior results showed that the adults (n = 5) had an 83.33% homing rate after displacement. Adult big‐headed turtles were able to return to their initial capture sites (home) from 150 to 2,400 m away and precisely to their home sites from either upstream or downstream of their capture sites or even from other streams. However, none of the juveniles (n = 4) returned home, despite only being displaced 25–150 m away. These results indicated that the adult big‐headed turtles showed high fidelity to their home site and strong homing ability. In contrast, the juvenile turtles may show an opposite trend but further research is needed.Site fidelity refers to the restriction of dispersal distance of an animal and its tendency to return to a stationary site. To our knowledge, the homing ability of freshwater turtles and their fidelity is reportedly very low in Asia. We examined mark–recapture data spanning a 4‐year period in Diaoluoshan National Nature Reserve, Hainan Province, China, to investigate the site fidelity and homing behavior of big‐headed turtles  Platysternon megacephalum). Our results indicated that the adult big‐headed turtles, but not juveniles, showed a high fidelity to their home site and strong homing ability.We examined mark–recapture data spanning a 4‐year period in Diaoluoshan National Nature Reserve, Hainan Province, China, to investigate the site fidelity and homing behavior of big‐headed turtles ( Population density of the big‐headed turtle was 1.65 individuals per km in our study area, and they have been less affected by hunting in recent years under the strict management of the nature reserve . These individuals were used in this mark–recapture study. The site fidelity experiment between 2016 and 2019 revealed that adult big‐headed turtles  showed a high recapture rate in the original site  after their release at the original site, whereas the juveniles showed lower recapture rates   showed a very high homing rate after displacement, that is, as high as 83.33%  returned home, despite only being displaced 25–150 m away (Table From 2015 to 2019 (except for 2017), a total of 11 big‐headed turtles were captured ; Investigation ; Writing‐original draft . Rongping Bu: Investigation ; Writing‐original draft . Liu Lin: Investigation . Jichao Wang: Writing‐review & editing ; Haitao Shi: Conceptualization ; Writing‐review & editing .All fieldwork was conducted in strict accordance with the guidelines of the Animal Research Ethics Committee of Hainan Provincial Education Centre for Ecology and Environment, Hainan Normal University (HNECEE‐2014–002), which conforms to the Law of the People's Republic of China.


Our measurements indicate the existence of a density-driven water circulation in the cavity beneath the ice shelf of Langhovde Glacier, similar to that proposed for warm-ocean cavities of larger Antarctic ice shelves.Basal melting of ice shelves is considered to be the principal driver of recent ice mass loss in Antarctica. Nevertheless, in-situ oceanic data covering the extensive areas of a subshelf cavity are sparse. Here we show comprehensive structures of temperature, salinity and current measured in January 2018 through four boreholes drilled at a ~3-km-long ice shelf of Langhovde Glacier in East Antarctica. The measurements were performed in 302–12 m-thick ocean cavity beneath 234–412 m-thick ice shelf. The data indicate that Modified Warm Deep Water is transported into the grounding zone beneath a stratified buoyant plume. Water at the ice-ocean interface was warmer than the in-situ freezing point by 0.65–0.95°C, leading to a mean basal melt rate estimate of 1.42 m a Basal melting of ice shelves is the principal driver of recent ice mass loss in Antarctica. The study reports comprehensive structures of temperature, salinity and current under an ice shelf in East Antarctica obtained by borehole measurements. The glaciers and ice streams form floating ice tongues and ice shelves, accounting for 74% of the coastal margins of the ice sheet2. Because approximately half of the ablation of the Antarctic Ice Sheet occurs due to basal melting of floating ice4, processes at the ice–ocean interface are very important for the ice sheet mass budget. Furthermore, thinning or loss of an ice shelf causes reduction in the buttressing force acting on inland ice. This accelerates ice discharge into the ocean, as observed in the Antarctic Peninsula region5. The most rapidly melting ice shelves are observed in the Bellingshausen–Amundsen seas sector of West Antarctica6. There, warm and dense circumpolar deep water (CDW) with a temperature of ~1°C (~3°C above the in situ freezing point) flows into subshelf cavities, supplying heat that enables rapid melting7. Observations and numerical modeling have indicated strong temporal and spatial variability in the basal-melt rates of ice shelves in the Bellingshausen and Amundsen seas9, where mass loss of the ice sheet has recently been increasing10. Recent observations at Totten Glacier demonstrated that rapid ice shelf base melting occurs in East Antarctica as well12. Heat for the basal melting in the Totten Glacier region is carried by modified CDW, which is slightly colder than CDW but still sufficiently warm for melting.The Antarctic Ice Sheet feeds ice into the ocean through fast-flowing outlet glaciers and ice streamsT, salinity S, and current speed U in the boundary layer beneath an ice shelf13 (see full equations in “Methods”)Tf is the freezing point of the seawater in the mixed layer beneath the ice shelf, but calculated using the pressure of the ice shelf base. Therefore, direct measurements of temperature, salinity, and water current underneath an ice shelf are crucial to estimate the melt rate based on physical processes at the ice–ocean boundary. In addition, in situ oceanic data from a subshelf cavity provides critical information for furthering our understanding of basal melting and water circulation. The equation for estimating melt rate assumes the transfer of ocean heat from a fully turbulent boundary layer to a hydraulically smooth ice shelf base13. However, turbulence in the boundary layer and the roughness of the ice-surface topography can only be investigated using direct measurements.Circulation beneath these rapidly melting ice shelves is driven by an out-flowing buoyant plume generated by basal melting. This density-driven circulation allows for the advection of warmer, saltier, and denser water to the grounding line. The basal-melt rate 14. In Mode 1, salty water at freezing points is generated by brine rejection during sea ice production and flows into the subshelf cavity. Since the freezing point drops as pressure increases, heat is available in the water to melt ice at the depth of the ice shelf base and a part of the upwelling cooled water might refreeze before it exits the cavity. In Mode 2, warm CDW flows onto the continental shelf and into the cavity. CDW causes rapid basal melting since it is warmer than the freezing point at the ice shelf base, typically by ~3°C. In Mode 3, Antarctic Surface Water (ASW) carries heat supplied by the atmosphere into the cavity, causing a significant amount of basal melting near the ice shelf front during the summer16. Although these modes are widely accepted in the community, in situ oceanic data with coverage of an entire cavity is required for validation of the proposed processes.Subshelf density-driven circulation is categorized into three major modes, which are associated with the magnitude of ocean heat transport into a subshelf cavity17. However, understanding of mixing processes in the vicinity of grounding lines is limited due to the difficulty of obtaining measurements. Theoretical studies suggest that tidal currents erode stratification and induce vertical mixing near the grounding line18. On the other hand, recent observations found a lack of tidal mixing in the grounding zone of Ross Ice Shelf, where vertical mixing was instead driven by relatively inefficient diffusive convection19. To better understand the processes controlling basal melting, in situ observations of thermohaline structure, and circulation regimes will be required over extensive areas of a subshelf cavity.Studies have shown melting near the ice shelf grounding lines to be a key for the future evolution of the ice sheetIn this study, we made direct measurements of ocean properties beneath the ice shelf of Langhovde Glacier, East Antarctica in January 2018, using boreholes drilled with a hot-water system through ice 234–412-m thick. Four boreholes were located along a glacier flowline from the ice shelf front to the grounding line. Water temperature, salinity, and currents were measured in the subshelf ocean cavity, which had thicknesses between 302 and 12 m. Our observations confirmed the advection of warm water into the cavity, and revealed the circulation regime under the ice shelf of Langhovde Glacier. Based on these data, we quantify the rate of basal melting and the stability of the water column, providing an overview of the ice–ocean interaction of the ice shelf of a small outlet glacier in East Antarctica.−1 (ref. 20), modulated by tides21. Surface topography indicates that the glacier has a several kilometer long ice shelf with a grounding line protruding toward the ocean in its central part22 , which is a mixture of WDW and Winter Water (WW)15.Lützow-Holm Bay is located near the eastern boundary of the Weddell Gyre, where the Antarctic Circumpolar Current approaches the coast25 Fig. . The cloBorehole drilling revealed ice and ocean bed geometries of the Langhovde Glacier ice shelf at the drill sites Fig. . At the Ice thickness measured at boreholes BH1801, 02, and 04 agrees with those calculations based on the hydrostatic equilibrium assumption to within 3%  increased from the ice to the seafloor  showed local maxima at 18–48 m below the ice base  toward the grounding line was observed at a depth of 260–420 m. The flow below 420 m was relatively weak (10–30 mm s−1) and changed its direction between high and low tides  corresponds to a travel time of 3–0.7 days between the ice shelf front and the grounding zone.At BH1801, water flowed toward the open ocean within ~50 m of the ice base, whereas a relatively strong current 40 mm s0 mm s−1 13 using previously proposed parameters30  and water temperature (0.3 m a−1/0.1°C at SA = 34.45 g kg−1 and U = 20 mm s−1). The melt rate at BH1803 (2.46 m a−1) was 40% greater than the mean over the four locations  and a relatively strong current    at 370-m depth increased downglacier from 27.58 kg m−3 at BH1804 to 27.61 kg m−3 at BH1803  and low thermal forcing (<0.1°C)19. The stratification was attributed to the absence of tidal mixing, and diffusive convection was suggested as a key process for driving basal melting. Thermal forcing of subshelf water at Langhovde Glacier was an order of magnitude higher than that under Ross Ice Shelf. Presumably, more rapid melting and a steeper basal ice slope generates an upslope-flowing plume, resulting in shear instabilities in the water column in the grounding zone of Langhovde Glacier. We conclude that the mixing conditions near the grounding line are controlled by the strength of basal melting, basal ice topography, and the magnitude of the tidal current, all of which will differ for different ice shelves.Near the grounding line, a complex water circulation was observed Fig. . Within 40. Observations that cover the entire field of a cavity are sparse7, thus our data serve as an important validation of theories and models used for estimation of basal melting. Our results indicate warm water inflow into the cavity and relatively high melt rates at the base of the Langhovde Glacier ice shelf. Similar processes are expected at other Antarctic outlet glaciers in similar settings. Despite the abundance of small ice shelves distributed around Antarctica, their size means that they are not resolved in numerical models used for predicting future sea-level change40. Further investigation and long-term monitoring of such ice shelves is crucial for an understanding of the response of the Antarctic Ice Sheet to a changing climate.The thermohaline structure and circulation presented in this study are consistent with those proposed by observation and theoretical analysis of Antarctic ice shelves−1 with a hot-water drilling system, which had been previously used on Langhovde Glacier23. The system consists of three high-pressure, hot-water machines (Kärcher HDS1000BE), a 1/2-inch diameter hose, and a winch system41. Meltwater was supplied to the system from a supraglacial stream or pond. Ice thicknesses and ocean bed elevation were determined to an accuracy of ±1 m, using water temperature, salinity, and pressure profiles described in the next section. At each borehole site, ice-surface elevation was measured using a dual-frequency GNSS (Leica Geosystems System 1200) with the static method using data from a reference station  located on the western flank of the glacier. In order to observe lower surface ice topography, an upward-looking video camera  enclosed in a pressure vessel was lowered into the borehole at BH1802. Roughness of the ice surface was measured by visual inspection of the imagery.In December 2017 and January 2018, we drilled four boreholes along a flowline of the ice shelf of Langhovde Glacier. Boreholes were drilled 0.55, 1, 1.6, and 2.5 km from the ice shelf front and referred to as BH1801, BH1802, BH1803, and BH1804 . The sample rate was 1 Hz. During the subshelf measurements the profiler, suspended on a Kevlar rope, was pulled upwards at a rate of 1 m s−1. To achieve a higher vertical resolution near the ice–ocean boundary, the profiler was moved more slowly, at a rate of 0.1 m s−1. Water depth was derived from pressure and water density computed from salinity and temperature. The magnitude and direction of the water current were measured with a current meter  combined with the CTD profiler  profiler  using the standard equations43. To investigate mixing of meltwater and MWDW, we used a Θ-SA diagram. As MWDW mixes with meltwater, temperature and salinity show variations on the diagram along the Gade line or meltwater-mixing line44, which connects water properties of MWDW and meltwater. The meltwater-mixing line was obtained using equations defined ratio of SA to Θ changes when ice melts into seawater45, using the Gibbs SeaWater Oceanographic Toolbox of TEOS-1043. MWDW is a mixture of WDW and WW, thus MWDW is identified on the Θ-SA diagram along a line connecting properties of WDW and WW. To find the lower boundary of the ice–ocean boundary layer, we analyzed the Brunt-Väisälä frequency (N)ρ is water density, g is the gravitational acceleration, and z is the depth below the ocean surface. Negative or zero N2 values indicate that the water column is unstable, and the stability increases as N2 takes on a larger value. In order to minimize the influence of small-scale instabilities, water density was smoothed using a Gaussian filter with a half-width of 50-m depth. A half-width of 20 m was used at BH1804 because the water column was thin. The local maximum in the calculated N2 was used to define the lower limit of the boundary layer at BH1801–03. At BH1804, we assumed the entire water column to be a boundary layer.Water temperature and salinity were converted into conservative temperature (Θ) and absolute salinity b was estimated to be 0.015°C m−1 from borehole temperature data obtained in a previous campaign23. The freezing point at the ice–ocean interface T0 was given by absolute salinity at the interface S0 and pressure P as defined by the “gsw_t_freezing” function43.Current, temperature, and salinity measurements were used to calculate the basal-melt rate, γT and γS, for a fully developed turbulent flow over a hydraulically smooth boundary are given byK is the ice-surface drag coefficient, Pr and Sc are the Prandtl and Schmidt numbers of seawater, and Re is the Reynolds number defined by Re = UD/v, with v being the kinematic viscosity13. Measured T, SA, P, and U were averaged over the ice–ocean boundary layer D  with thSupplementary infoUpward-looking movie showing the lower surface of the ice shelf at BH1802.Description of additional supplementary files


Mauritia flexuosa palms for fruit harvesting. Such degradation could significantly damage peat deposits by altering C fluxes through fine root productivity, mortality, and decomposition rates which contribute to and regulate peat accumulation. Along a same peat formation, we studied an undegraded site (Intact), a moderately degraded site (mDeg) and a heavily degraded site (hDeg) over 11 months. Fine root C stocks and fluxes were monthly sampled by sequential coring. Concomitantly, fine root decomposition was investigated using litter bags. In the experimental design, fine root stocks and dynamics were assessed separately according to vegetation type  and M. flexuosa age class. Furthermore, results obtained from individual palms and trees were site-scaled by using forest composition and structure.Amazon palm swamp peatlands are major carbon (C) sinks and reservoirs. In Peru, this ecosystem is widely threatened owing to the recurrent practice of cutting M. flexuosa adults was higher at the mDeg site than at the Intact and hDeg sites, while in trees it was lowest at the hDeg site. Site-scale fine root biomass (Mg C ha−1) was higher at the mDeg site (0.58 ± 0.05) than at the Intact (0.48 ± 0.05) and hDeg sites (0.32 ± 0.03). Site-scale annual fine root mortality rate was not significantly different between sites  while productivity (same unit) was lower at the hDeg site (1.5 ± 0.8) than at the Intact site (3.7 ± 1.2), the mDeg site being intermediate (2.3 ± 0.9). Decomposition was slow with 63.5−74.4% of mass remaining after 300 days and it was similar among sites and vegetation types.At the scale of individuals, fine root C biomass in The significant lower fine root C stock and annual productivity rate at the hDeg site than at the Intact site suggests a potential for strong degradation to disrupt peat accretion. These results stress the need for a sustainable management of these forests to maintain their C sink function.The online version contains supplementary material available at 10.1186/s13021-021-00197-0. Mauritia flexuosa, a dioecious species that can establish nearly monodominant stands  which was calculated as [t−1 is the necromass at time t − 1 and k is the decay constant generated from the fine root decomposition experiment (see section on fine root decomposition).Fine root production and mortality rates were estimated from the sequential coring data applying the widely used compartmental flow model according to the decision matrix from Fairley and Alexander , adaptedlated as :\documenM. flexuosa seedlings, juveniles, adults) (see methods in the section on C and nitrogen (N) determinations). Fine root biomass and necromass were averaged across the 11 monitoring months per vegetation category and site. Plot-scale results were computed using the relative densities derived from the vegetation inventory  constructed from 1.5 mm fiberglass screen. Fine roots were collected from randomly selected adult individuals of M. flexuosa and trees using a 5 cm diameter Russian corer. The sampling depth and position from the trunk were the same as for fine root stock measurements and samples were carefully inspected to not mix roots from M. flexuosa and trees. At the beginning of the experiment 60 bags were placed at each site, 30 bags containing roots of M. flexuosa and 30 bags containing roots of trees. Each bag was filled with 2 g of washed, air-dried fine roots and tagged with an aluminum plate for later identification. The bags were randomly buried at a distance of 1 m from the trunk of a palm or a tree, depending on their content, 15 cm under the soil surface, for a total period of 10 months (March 2016−January 2017). Six bags were monthly collected per site, three with M. flexuosa roots and three with dicot tree roots, following the method by Hoyos-Santillan et al. [t / Y0 is the percentage ratio of remaining dry mass at time t (days) to initial dry mass Y0 and k is the decay rate constant (day−1). The initial dry mass (1.8 g) was computed from the air-dried mass that was placed in bags (2 g) by applying an oven-dry mass to air-dried mass ratio measured from ten samples (0.9).The experiment was performed by distinguishing trees from n et al. . Upon coM. flexuosa). No distinction was made between live and dead roots. The samples were dried at 60 °C to constant mass, milled and homogenized. Their C and N content was determined using the induction furnace method .Additional fine root cores for C and N analyses were collected from the soil top 25 cm using a 5 cm diameter Russian corer. Samples were taken at each site around three randomly selected individuals per vegetation category  and soil temperature were monthly monitored concurrently with the measurements of fine roots stocks. Monthly precipitation was obtained from the daily data of the Puerto Almendra weather station, located near the study sites . The WT relative to the soil surface was measured in perforated PVC tubes  installed permanently in the soil. Water depth during flooding was measured with a graduated pole. Soil temperature at 10 cm depth was measured using a portable digital thermometer. Sampling considered the hummock and hollow microtopography of the soil, with nine replicates per microtopographical position for a total of 18 replicates at the Intact site and 18 replicates per microtopographical position for a total of 36 replicates at each of the degraded sites. These environmental parameters were collected as part of a long-term experiment monitoring soil greenhouse gas emissions at the studied sites . We usedM. flexuosa versus trees at the end of the decomposition experiment and the Kruskal–Wallis test for comparison of fine root biomass and necromass stocks, fine root productivity and mortality rates and C and N values between sites within a vegetation category and between vegetation categories within a site. Site-scale results were considered significantly different between sites when their value/mean ± standard error did not overlap.Statistical analysis was performed using the software InfoStat , with a Uncertainty estimates are reported as standard errors. In all calculations, the Gaussian error propagation method was used for propagating uncertainties. This method is adequate for step-by-step calculations that are intended to compute ecological quantities that can be expressed as an analytical equation using addition, subtraction, multiplication and division, such as C stocks or fluxes . The metM. flexuosa adults and trees was slightly lower at the mDeg site than at the Intact site while the density of M. flexuosa seedlings and juveniles was, respectively, six and three times higher at the mDeg site than at the Intact site , by contrast no such a difference was observed for trees (Table M. flexuosa adults was higher at the mDeg site than at the other sites (P = 0.0115), while in trees fine root biomass was the lowest at the hDeg site (P = 0.0176). Fine root C necromass in trees differed among sites with a higher stock at the mDeg site than elsewhere (P = 0.003). Regarding dissimilarities between vegetation categories, fine root C biomass was higher in M. flexuosa adults than in seedlings and trees at both degraded sites (P = 0.0001 and 0.0002 at the mDeg and hDeg sites); a result which remained the same when merging all sites (P = 0.001). Finally, at the mDeg site, fine root C necromass was higher in trees than in M. flexuosa adults and seedlings (P = 0.0017), which was also valid when the sites were considered all together (P = 0.005).Fine root C biomass was about twice fine root C necromass for es Table . Fine roSite-scale fine root C biomass followed the order mDeg > Intact > hDeg, while site-scale fine root C necromass was higher at the mDeg site than at the hDeg, with the Intact site displaying an intermediate stock not significantly different from the stocks at the degraded sites Table .P > 0.317 in all cases). On the other hand, fine root productivity rate tended to increase linearly with raising soil temperature of the preceding month , while fine root mortality tended to decrease linearly with increased water table level of the previous month   Fig. .Fig. 3MoAnnual productivity and mortality rates of fine roots were similar among sites within a vegetation category or among vegetation categories within a site Table . Site-scM. flexuosa adults) amounted to 69.2 ± 3.1%. Remaining fine root percentage at day 300 was not different either between sites  or between vegetation categories within a site . Decay rates were on average 0.0014 ± 0.0003 d−1 and 0.0010 ± 0.0001 d−1 for fine roots of M. flexuosa adults and trees, respectively. Decay rates were not related to root chemistry  (P > 0.05).The remaining percentage of initial fine root mass (2 g) after 300 days (43 weeks) varied between 63.5 ± 13.6% and 74.4 ± 8.1%  . At the hDeg site, fine root N content was lower in M. flexuosa seedlings and juveniles than in trees (P = 0.0324). At the same site, the C:N ratio of fine roots was higher in M. flexuosa juveniles than in trees (P = 0.0415).Fine root C content was higher at the mDeg site than at the hDeg site in all vegetation categories (5) Table . Fine roM. flexuosa and trees, but also the age distribution of M. flexuosa palms. At the hDeg site, the densities of M. flexuosa seedlings, juveniles, and adults and the density of trees decreased by, respectively, 56%, 43%, 81% and 64% as compared to densities at the Intact site  as an undisturbed reference and from the site with a ratio of 10 (site 12) as a highly degraded stand). Similar as at the highly degraded site observed by Horn et al. [M. flexuosa female adults at the hDeg site  favors M. flexuosa recruitment dynamics, probably as the result of increased light availability in gaps created by logging. The existence of seed and seedling banks waiting for appropriate light conditions for their growth has been widely recognized for many forest species under different conditions [M. flexuosa establishment strategies rely on a combination of seed recalcitrance and seed dormancy, which maintain seed and seedling banks [At the mDeg site, the density of trees and n et al.  who founnditions –57. M. fng banks , 59. Thing banks .M. flexuosa and trees at the mDeg site than at the hDeg site  (Table M. flexuosa seedlings and juvenile and their lower C:N ratio than that of M. flexuosa juveniles (Table The higher C content in fine roots of s) Table , suggestes Table  suggest −1 yr−1) are comparable with the average fine root productivity reported by Chimner and Ewel [−1 yr−1) and relatively higher than values measured by Finér and Line [−1 yr−1) in northern peatland forests. Fine root mortality rates at site scale (1.5−3.4 Mg C ha−1 yr−1) were above the average for peat swamp forests of Southeast Asia computed by Hergoualc'h and Verchot [−1 year−1).Site-scale annual fine root productivity rates  and necromass (0.2 − 0.4 Mg C ha−1) (Table −1 in the soil top 30 cm). The fine root biomass to necromass proportion differed according to plant functional group  and trees (k = 0.0010 d−1)  measured in Raphia taedigera palm swamp peatlands of Panama [k = 0.0027 d−1) measured at our sites by van Lent [Fine root decomposition was slow and uniform among sites and vegetation type. Our 69.2% cross-treatment average of remaining percentage after 300 days is in accordance with the 72% remaining percentage after 252 days found in a tropical forested peatland of Micronesia . Averagek = 0.001 d−1 and f Panama . In agref Panama  and Hoyof Panama , fine rovan Lent . Therefovan Lent , 80. Thek = 0.001 d−1 and M. flexuosa palms and trees reduced site-scale fine root productivity by 37% and 42% at the mDeg and hDeg sites, respectively  may affect root C stocks and fluxes. Furthermore, given the local scope of the study additional work is needed to test the response of fine roots to degradation elsewhere in the Peruvian Amazon or in tropical peat swamp forests in other regions where logging activities are taking place. The role that fine roots play in peat C budgets remains largely understudied and deserves much more attention, especially in tropical climates.Additional file 1: Figure S1. Location and length (m) of the transects for monitoring M. flexuosa felling at the moderately degraded site (mDeg). Lateral visibility within each transect was 6 m for AB; 10 m for BC; 7 m for CD, CE and EF; and 5 m for FB. Source: Google Earth Pro. Image from February 2017. Figure S2. Monthly rate of M. flexuosa cutting at the moderately degraded (mDeg) site. Figure S3. Sequential coring to estimate fine root production and mortality rates. Table S3. Decision matrix for estimating fine root production and mortality according to Fairley and Alexander [Figure S4. Monthly mean water table level and soil temperature at the Intact, moderately (mDeg) and heavily (hDeg) degraded sites. Error bars are standard error. Figure S5. Relationship between M. flexuosa male: female ratio and M. flexuosa density in palm swamps stands of the Peruvian Amazon. The figure is based on the data by Horn et al. [lexander , adaptedlexander . Figure n et al. . Seedlin