KRAS) gene mitigate these beneficial effects by promoting a negative feedback-loop comprising extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen-activated kinase kinase 1/2 (MEK1/2)-dependent inactivation of PPARγ. To overcome this inhibitory mechanism, we searched for novel post-translational regulators of PPARγ. Phosphoinositide phosphatase Myotubularin-Related-Protein-7 (MTMR7) was identified as cytosolic interaction partner of PPARγ. Synthetic peptides were designed resembling the regulatory coiled-coil (CC) domain of MTMR7, and their activities studied in human cancer cell lines and C57BL6/J mice. MTMR7 formed a complex with PPARγ and increased its transcriptional activity by inhibiting ERK1/2-dependent phosphorylation of PPARγ. MTMR7-CC peptides mimicked PPARγ-activation in vitro and in vivo due to LXXLL motifs in the CC domain. Molecular dynamics simulations and docking predicted that peptides interact with the steroid receptor coactivator 1 (SRC1)-binding site of PPARγ. Thus, MTMR7 is a positive regulator of PPARγ, and its mimicry by synthetic peptides overcomes inhibitory mechanisms active in cancer cells possibly contributing to the failure of clinical studies targeting PPARγ.Peroxisome proliferator-activated receptor-gamma (PPARγ) is a transcription factor drugable by agonists approved for treatment of type 2 diabetes, but also inhibits carcinogenesis and cell proliferation in vivo. Activating mutations in the Kirsten rat sarcoma viral oncogene homologue ( It is the latter characteristic that renders the nuclear receptor a promising target in gastrointestinal tumours with frequent, activating mutations in the RAS-ERK1/2 signalling cascade, e.g. in colorectal cancer (CRC)7. In this setting, mutations in RAS genes are a major obstacle for effective treatment in advanced disease8, and new drugable targets which inhibit RAS-ERK1/2 signalling are needed9. However, serious adverse effects limit the long-term monotherapy with PPARγ-ligands in metabolic diseases10. Nonetheless, combination with chemo- or biological therapies may offer novel strategies against cancer12 Clinical trials investigating the use of PPARγ-agonists have yet failed to show sufficient efficacy14.The nuclear transcription factor PPARγ has been established as a target in type 2 diabetes for many years. In addition to its lipid lowering and insulin sensitizing properties, pharmacological activation of PPARγ shows benefits in malignant and inflammatory human diseases16 and others18 demonstrated that downstream effectors of RAS inhibit PPARγ, e.g. by ERK1/2-dependent phosphorylation as well as by nuclear export and cytosolic sequestration through MEK1. In addition to this regulatory mechanism, off-target side effects of the first generations of PPARγ-agonists even resulted in an increased proliferation rate of tumour and vascular cells, as they involve PPARγ-receptor independent (“non-genomic”) activation of RAS19 and phosphoinositide 3-kinase (PI3K)20 signalling, especially at higher dosages.One reason for this discrepancy between preclinical and clinical studies may rely on the complex regulation of PPARγ by the RAS-ERK1/2 signalling cascade, which has not been taken into account in any of the before mentioned trials: weRAS genes, which are primarily unresponsive to PPARγ activation.We therefore hypothesized that the resulting decrease in nuclear transcriptional activity of PPARγ, due to its cytosolic sequestration in the presence of an active RAS cascade promotes its targeting to so far unknown cytosolic effectors. Therefore, unravelling novel effectors or modulators of PPARγ could be a promising approach to overcome this obstacle, especially concerning tumours harbouring activating mutations of 22. Homo- and heterodimerization between a catalytically active member of the family with an enzymatically inactive one, e.g. MTMR6/7/8 with MTMR9, is mediated via the CC domain resulting in an increased enzymatic activity23. For murine MTMR7, a truncated 54 kDa isoform has been described lacking this domain24. The active enzyme then dephosphorylates phosphatidyl-inositol-3-monophosphate (PI(3)P) and -3,5-bisphosphate P2). MTMs are membrane-bound and localize to endosomes, with the exception of MTMR7, being present in a soluble form in the cytoplasm24 using free inositol-1,3-bisphosphate P2) as a substrate.In this context, we identified 76 kDa myotubularin-related protein 7 (MTMR7), a member of the myotubularin (MTM) family of lipid phosphatases, as a novel interaction partner of PPARγ. MTMs consist of N-terminal plextrin homology (PH), central protein tyrosine phosphatase (PTP), SET-interaction (SID) and C-terminal coiled-coil (CC) domains24, we detected MTMR7 in the gastrointestinal tract25. In contrast to other MTMs, characterized as “survival phosphatases”22, we demonstrated that MTMR7 reduces proliferation of CRC cells in vitro, even in the presence of activating mutations of KRAS and active insulin signalling, due to inhibition of both RAS-ERK1/2 and PI3K-AKT-mTOR signalling25.In addition to the previously reported expression of MTMR7 in brain, muscle, liver and kidneyIn the present study, we describe a novel regulatory mechanism of PPARγ which augments its transcriptional activity via its interaction with MTMR7. In addition, we offer new insights into the subcellular distribution of MTMR7 in response to external stimuli and identified the CC domain of MTMR7, by designing and modifying a peptide resembling this domain, as a potential novel pharmacological activator of PPARγ in vitro and in vivo.26. However, the function of cytosolic PPARγ is unknown. To identify novel binding partners which may act as regulators or effectors for cytosolic PPARγ, a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) protein interaction screen was performed in the aneuploid human CRC cell line SW480, which has mutated alleles of the KRASG12V gene27 and a high amount of extra-nuclear PPARγ. SW480 cells were disrupted by hypotonic lysis without detergents, a procedure which extracts cytosolic proteins. Coimmunoprecipitation (CoIP) was then performed with PPARγ antibody (Ab) or control IgG followed by detection of precipitated bands by silver staining. Peptides within a band of 54 kDa were coprecipitated by the PPARγ Ab and identified to correspond to human MTMR7 by MALDI-MS sequencing  MTMR7 (76 kDa) protein25. Subcellular fractionation (SCF) of normal cycling HCT116 cells was performed, and cytosolic lysates were immunoprecipitated with MTMR7 or PPARγ Ab-conjugated or unconjugated beads. Immunoblotting (IB) was performed with MTMR7 Ab  protein for 24 h. After SCF, CoIP from cytosolic lysates was performed using MTMR7 or PPARγ-Ab-conjugated beads. IB against MTMR7 detected an 80 kDa band, representing GFP-PPARγ after MTMR7 pulldown  proteinown Fig.  or GFP-Pown Fig.  expressi25. In this setting, predominant cytosolic localization could be detected . In addition, we evaluated formalin-fixed paraffin-embedded (FFPE) tissue specimens from CRC patients (n = 67 cases) with regard to MTMR7 expression and subcellular localization by means of immunohistochemistry (IHC). Of these patients, 41 (61%) showed a positive MTMR7 staining. In 19 (46%) of these MTMR7 positive cases, a nuclear staining was detected .For 25 samples, an additional staining for PPARγ was observed, 16 (64%) of which exhibited a positive nuclear staining for PPARγ Fig. . Nuclearp = 0.0173, R1 p = 0.0431), while there was a trend for a predominant cytoplasmic localization in starved cells (p = 0.11) . Thus, endogenous MTMR7 translocates from the cytosol to the nucleus in response to serum or PPARγ-activation.To further study stimulation-dependent nuclear translocation of MTMR7, immunofluorescence microscopy was performed in HCT116 cells expressing high endogenous MTMR7 FL protein. Cells were serum-deprived for 16 h, followed by incubation with PPARγ-agonist rosiglitazone (rosi) , epidermal growth factor (EGF) (50 ng/ml) or foetal calf serum (FCS) (20% v/v) for 1 h Fig. . Upon st25. We therefore studied subcellular localization in HEK293T cells which had low endogenous MTMR7 FL protein. Cells were starved for 16 h and then stimulated with serum or PPARγ-agonist, followed by SCF and IB against MTMR7 . All Abs used in the present study recognized the C-terminal region of the MTMR7 FL protein  KRAS. The PDOs were incubated for 48 h in medium containing 1 µM rosi or vehicle control  . Thus, PPARγ-agonist facilitates nuclear accumulation of MTMR7 in vitro and ex vivo in patient-derived cancer stem cells.To characterize the subcellular localization of MTMR7 in situ, we cultivated patient-derived organoids (PDOs) of four patients harbouring activating SO) Fig. : On basep < 0.0001) and ligand-dependent  reporter gene activity compared with EV control .To assess, whether MTMR7 alters the transcriptional activity of PPARγ, we performed reporter gene assays using a luciferase plasmid with a PPARγ-responsive enhancer element (PPRE) in front of a basal promoter. HCT116, SW480 and HEK293T cells were transfected with empty vector (EV) or MTMR7 (FL) expression plasmid followed by stimulation with rosi (0.1–10 μM) for 48 h Fig. . MTMR7 o28. MTMR7 mRNA expression ≤1 S.D. of the mean positively correlated with a reduced expression of a broad set of PPARγ-target genes in the CRC dataset provided by TCGA Nature 2012 .To explore whether these results correlate with increased PPARγ-target gene expression, in silico analysis using the cBioportal database of cancer genomics was conducted012 Fig. . This fi16.To elucidate a possible mechanism underlying the observed increased PPARγ-activity in presence of MTMR7, we performed SCF followed by IB for phosphorylated PPARγ, using an Ab specific for the bona fide ERK1/2-phosphorylation site at serine 82/84. To this end, HEK293T cells were transfected with EV or MTMR7 (FL) and treated with vehicle control (VC), 20% (v/v) FCS or 1 μM rosi (R1) for 24 h, respectively Fig. . After M30, we investigated the effect of MTMR7 overexpression on rosi-mediated ERK1/2 phosphorylation. To this end, HCT116 cells were transiently transfected with EV or MTMR7 (FL), serum-deprived for 16 h and subsequently incubated with 10 µM rosi, a concentration higher than its IC50 and shown to stimulate ERK1/230. This effect was abrogated by overexpression of MTMR7, leading to reduced amounts of phosphorylated ERK1/2 , particularly after 10 min of rosi stimulation  (n = 3 replicates)  may be mediated by the aberrant activation of MEK1/2-ERK1/2 signallinges) Fig. . A similes) Fig. .31. We therefore hypothesized that the CC domain of MTMR7 is the region of the protein responsible for the interaction with PPARγ. To test this idea, we designed a synthetic peptide to substitute for the MTMR7 FL protein. There is no crystal structure of the MTMR7’s CC domain or any other MTM available. However, Kim et al. identified and characterized the CC domain of MTMR232. Performing multiple alignment analysis of the amino acid (aa) sequences of MTMR7, MTMR2, MTMR6, MTMR8 and MTMR9, we identified a stretch of 30 amino acids at the C-terminus of MTMR7  domainMR7 Fig. a1.33 (including PPARγ) was designed . A similar effect was observed in HCT116 (n = 3) and SW480 (n = 5) replicates . Treatment with concentrations of rosi higher than its IC50 (>1 µM) reduced PPARγ activity, presumably due to a “non-genomic” activation of the MEK1/2-ERK1/2-pathway and subsequent phosphorylation and inactivation of PPARγ16. Of note, exposure of cells to MP and PEP reversed this effect: In the presence of both peptides, increased PPARγ activity was detectable even after treatment with 10 μM rosi . To address the question whether this in vitro efficacy could be translated into the in vivo situation, C57BL6/J mice (pCEA-SV40-Tag)34 were treated for 14 days with MP, PEP (both at 30 mg/kg*d) or vehicle control (VC) by intraperitoneal injection (four times per week), respectively. RT-qPCRs (Tab. P21(Cip1/Waf1) and Cd36 mRNAs were increased in animals treated with either of the peptides . This effect was accompanied by an increase in body weight  and spleen weight  , MP or PEP (both at 1 µM) was performed in presence or absence of rosi (1 and 10 μM). As for overexpression of MTMR7 FL protein, treatment with both peptides increased the transcriptional activity of PPARγ  Fig. . Taken t35 and Agadir36  were predicted to form α-helical secondary structures by the web servers PredictProtein37 predicted the residues surrounding the agonist-binding pocket of PPARγ to be the most likely interaction site on the protein38  predictors, Waggawagga40 and DeepCoil41, predicted only helix 10/11 as a possible site for coiled-coil interaction on PPARγ. Helix 10 mediates heterodimerization between PPARγ and RXRα and forms a coiled-coil with helix 10 of RXRα  of PPARγ Fig. . This ann38 Fig. , mainly XRα Fig. . The PINARγ Fig. .Fig. 6Pr47 docked the MP exclusively with the canonical LXXLL motif at the coactivator binding site. The PEP was also docked exclusively to this site either with its LXXXL or LXXL motifs. The second tool used was the web server ClusPro48. The ClusPro docking resulted in complexes with the peptides docked to varying sites on PPARγ, including helix 10/11 and the proximity of the coactivator interaction site.As the heterodimerization and the coactivator-binding sites were already experimentally shown to be binding sites for α-helical peptides and protein regions, they were thought to be more likely interaction sites than the agonist-binding site. Docking was performed using two different web servers that employ different approaches: The web server GalaxyPepDock39, the binding mode of MP seemed to be similar to SRC1, involving residues on helices 3, 4 and 12  simulations followed by calculations of binding free energies were performed on 23 complexes from the two web servers with MP and PEP bound to different sites on the nuclear receptor. Peptides were bound either to the heterodimerization site or the coactivator interaction site. The results of the free energy calculations indicated that the coactivator interaction site is a more favourable binding site than the heterodimerization site Tab. . Interac 12 Tab. , includi 12 Tab. . These t39. For both peptides, this leucine was calculated to interact with F306 inside this cleft, an interaction residue not shown for SRC1.PEP was not found to interact with residues on helix 12 in more than 80% of the 120 ns MD simulation and, therefore, is unlikely to make use of the charged clamp for positioning Fig. . The leuThe structures of the complete complexes of PPARγ with PEP or MP at the last frame of the MD simulations are shown in Supplementary Fig. In conclusion, the in silico prediction of the structures of peptide/PPARγ complexes indicates a coactivator-like binding mode that may explain the capacity of the peptides to stimulate the transcriptional activity of PPARγ.49. The here described MTMR7-PPARγ interaction complex offers a novel mode of PPARγ regulation by compartmentalization and a possible explanation for the pro- vs. anti-tumour effects of PPARγ-agonists52. Previously, we characterized MTMR7 as an inhibitor of ERK1/2 and AKT/mTOR signalling25, thus acting as a dual blockage of two interconnected and compensatory oncogenic pathways downstream of receptor tyrosine kinases (e.g. the EGF receptor) and RAS54. MTMR7 may thus enforce the classical nuclear function of PPARγ by inhibiting the RAS-ERK1/2 cascade, which otherwise evokes inactivation of PPARγ by ERK1/2-mediated phosphorylation and MEK1/2-dependent sequestration of PPARγ in the cytosol16, where it interacts with molecules that restrain PPARγ activity including caveolin-126 and heat shock proteins55.In the present study, we describe a novel role for MTMR7 in human CRC, identifying it as a binding partner and positive regulator of PPARγ. The transcriptional activity of PPARγ is regulated by ligand binding, post-translational modifications and subcellular localisation57. Through this mechanism, which has been shown to be mediated by EGF- or G-protein-coupled receptors (e.g. GPR40) in the plasma membrane, MTMR7 may prevent the adverse side effect of this class of PPARγ-ligands on cell proliferation and organ damage52. This finding is of particular significance in light of the plethora of negative clinical trials using PPARγ agonists in the therapy of gastrointestinal malignancies58: since the loss of MTMR7 is a common event in CRC, induced by stimuli including insulin or insulin-like growth factors25, the MTMR7 expression status might be a predictive parameter when targeting PPARγ in the setting of anti-proliferative therapies.In the current study, we provide evidence that MTMR7 also reduces the rapid “non-genomic activation” of ERK1/2 by the PPARγ-agonist rosi22, we collected data suggesting that MTMR7 shuttles between the cytosol and the nucleus in response to stimuli of the RAS signalling cascade or to PPARγ agonist (here exemplary for rosi). In mice24, full-length (FL) 76 kDa and truncated 54 kDa isoforms of MTMR7 are expressed. The latter lacks the C-terminal coiled-coil (CC) domain and may thus be incapable of oligomerization with other MTMs24. The peptides identified in SW480 cells by MALDI-MS also suggest the existence of a 54 kDa isoform of MTMR7 in human CRC cells, forming the complex with PPARγ. Western blot analyses using four different Abs against the C-terminal domain of MTMR7, as well as previously published exon-selective RT-PCRs from several human CRC cell lines25 confirmed the presence of truncated <54 kDa MTMR7  domain of the phosphatase, which was able to activate PPARγ in vitro and in vivo: Notably, it induced transcription of PPARγ target genes as well as body weight gain, a well-known adverse effect attributed to PPARγ agonists of the thiazolidinedione class (e.g. rosi)38. Complexes covering this interaction site were more stable and energetically favourable compared with any other candidate sites like the heterodimerization interface or the agonist-binding pocket. MP and PEP interacted with residues that were also reported for SRC1 by Nolte et al.39. As such, MP made use of the charged clamp between E471 and K301 and interacted with several residues on helix 12. Both peptides were also found to interact with F306, implicating that the LXXLL/XXXL motifs might reach further into the cleft and contribute to the stable binding of the peptides to the coactivator interaction site. Notably, stretches of the peptides unfolded from their initial helical conformation assigned, whereas other parts, and especially the ones bound to the coactivator-binding site, stayed helical throughout the MD simulations. The stable helix formation of this peptide region agrees with reports on the behaviour of the LXXLL motif in SRC1 that is thought to be unstructured in its apo-form but to form a short amphiphatic α-helix upon binding to the coactivator-binding site62.Exploitation of different web servers for binding site prediction and docking, in combination with MD simulations, pointed at the coactivator interaction site of PPARγ being the most likely binding site for the peptides, similar to those of the bona fide coactivator SRC1, inserting the leucine at position 5 of its LXXLL motif into a hydrophobic cleft between helices 3, 4, 5 and 1262. The fact that MP gave more favourable binding free energies than PEP supports the reported importance of all three leucines. Cell-free competition assays with both peptides and SRC1 on recombinant PPARγ-LBD protein would be necessary to experimentally confirm our in silico predictions.While the computational methods favour the coactivator interaction site and resulted in a seemingly plausible interaction prediction, one important short-coming of this model for the PPARγ/PEP complex is that PEP does not contain the canonical LXXLL motif that was deemed to be necessary for this kind of coactivator-like interaction in the pastPreliminary findings revealed that the peptides were unable to displace rosi from the PPARγ-LBD, thus unlikely to act as true agonists; instead, rosi increased MTMR7/PPARγ complex formation and MTMR7 protein expression in CRC cells (not shown). These observations may indicate that, alike MTMR9, ligand-activated PPARγ could stabilize the MTMR7 protein, and, vice versa, MTMR7 may lead to a quasi-allosteric activation of PPARγ via the SRC1 coactivator site. However, future in depth nuclear magnetic resonance (NMR) or co-crystallization studies will be required to experimentally prove the predicted in silico models.Taken together, our study identified a novel positive regulator of PPARγ based on (i) inhibition of ERK1/2 signalling and (ii) direct interaction with the CC domain of MTMR7. Additional studies will be necessary to characterize the pharmacological properties of the peptides to allow further development of this novel PPARγ activator for future clinical applications.15. Human full-length (FL) MTMR7 cDNA  was in pTarget (pT) vector  with or without GFP-tag25. Transient transfection and luciferase assays were performed as described63.Chemicals were from Merck/Sigma . Antibodies (Abs) used are listed in Table. LMAVKEETQQLEEELEALEERLEKIQKVQL) (Tab. https://web.expasy.org/randseq/: VLQEEILEMTEEKALLQALLKKEQERVQEE) generating a canonical LXXLL nuclear receptor coactivator motif. Both peptides were synthesized by automated, solid phase peptide synthesis on Rink amide resin and coupled at the N-terminus with myristidic acid . The resulting peptides were characterised by HPLC and MALDI-MS for purity and sequence confirmation and provided as lyophilized powders64.PEP corresponded to the original leucine-rich aa sequence of MTMR7  Tab. . MP was 26. PDOs from CRC tissues were cultivated as published in refs. 66. All cultures were routinely tested for contamination with Mycoplasma .Human embryonic kidney (HEK293T) and colon adenocarcinoma cell lines  were maintained as beforesupplement methods R2supplement figure legends R2supplement figure S1supplement figure S2supplement figure S3supplement figure S4supplement figure S5supplement tables R2


Occurrences in land use, human activities and climate change have both direct and indirect influences on the environment. Of interest for this study is mining; a common activity in developing countries such as Nigeria which is endowed with over 34 solid minerals. The gold mining sites in the Southwest region of the country is predominantly by Artisanal and Small-Scale Mining (ASM). Though the benefits are known, its induced consequences are enormous. To understand its extent of floristic diversity, identification of functional plants and plant species surviving on the mined sites ; this study compared the floristic composition of an abandoned mining site (Site 1), an active mining site (Site 2) and an undisturbed vegetation sites (Control) of similar vegetation zone.Acanthus montanus and Icacina trichantha found on the Control sites are native and significance but species such as Capsicum frutescens and Crassocephalum crepidioides on Site 2 are due to human inference while most species on Site 1 shows both original and altered floristic composition (e.g. Adenia venenata and Grewia flavescens).A total of 54, 28 and 37 species belonging to 31, 20 and 23 families were found on Site 1, Site 2 and the control site, respectively. It shows that the floristic composition of all the sites has been altered due to its past intense agricultural colonization and human activities, but severe on Site 1 and 2 due to mining. Lots of the identified species are functional species and stand as ecological indicators. Species such as Apart from the on-going farming activities, ASM activities such as pollution, deforestation and exposure of the forest soils to direct sunlight has greatly stressed and disturbed the floristic composition, species richness, life form patterns, of the mined sites as well as introduction of non-native plant species. It is therefore necessary to develop effective approaches and policies to curb these illegal ASM activities, empower the community , stabilize the economy and establish sustainable development strategies with adequate reclamation measures. Biodiversity loss is not just region specific; it is a global challenge and can be attributed to different occurrences in land use, human activities as well as climate changes . DependiIn Nigeria, there are several identified factors that are responsible for modifying the original composition  of the rainforest. This includes human activities such as agriculture, increase in rural settlements, mining, rural road networks evolution, government policies, etc. , 38, 39.Artisanal and Small-scale Mining (ASM) is the most predominant in the southwest region of Nigeria. It accounts for over 95% of the entire mining activities and has been in existence since 1902 which connotes the advent of the colonial mining , 25, 37.Milicia excelsa; listed as an IUCN near threatened species and ought to be worked with based on appropriate permission. Such regulation is necessary, it helps to conserve, regulate and monitor any ecological imbalance. Altering of ecological balance influences the economic, social, environmental and even cultural values of the community. In addition is the level of importance usually attached to some species in providing necessary goods and services; the importance of some of these species to Nigeria was highlighted by Nigeria Fifth National Biodiversity Report. National Reports [Being in the lush tropical rainforest forest estate, this area is known as a biodiversity sanctuary with the complex terrestrial ecosystem . Due to  Reports . These s Reports , 40. How Reports , 41.This study was therefore conducted in order to understand the impact of mining on the floristic association of the gold mined sites within the southwest region of Nigeria. It aims to know the current status of its floristic diversity, identification of functional plants and ecological indicators as well as characterized plant species surviving on the mined sites (despite its high level of disturbance from the mining activities and contamination). This may also serve as fundamental and applied reasons especially towards reclamation of the mined sites.2,9 = 28.953, p < 0.001). A pair-wise test further shows significant differences between Site 1 and Site 2 (p = 0.03), between Site 1 and the reference (p = 0.03) and between Site 2 and the reference (p = 0.03). Simple results for species contributions at 50% cut off for low contribution revealed average similarity of 73% for Site 1, 78% for Site 2 and 80% for the reference site. The most contributing species for Site 1 include Melanthera scandens, Melochia corchorifolia, Paullinia pinnata, Sida pilosa, Desmodium adscendens, all contributing about 23% out of the total 73%. Site 2 were characterised by Palisota ambigua, Justicia insularis, Drynaria laurentii, Pentodon pentandrus, Chromolaena odorata, Cissus quadrangularis and Sida pilosa all contributing about 53% of the average similarities within Site 2.The biological diversity of the 3 studied sites are presented in Table Theobroma cacao, Pteris togoensis, Crinum jagus, Drynaria laurentii, Justicia insularis all contributing cumulative 23% of the average similarities within the reference site. Site 1 and Site 2 shared about 70% average dissimilarity, while Site 1 and the reference site shared 64% average dissimilarity, and also 64% average dissimilarity exist between Site 2 and the reference site. The Non-metric Multi-Dimensional scaling (nDMS) analysis using bootstrap averages revealed a clear separation of the sites in terms of species composition , it grows in deciduous, semi-deciduous or evergreen, primary or secondary forest. It is a considered pioneer species, categorized as Lower Risk/near threatened by the IUCN Red List of Threatened species, with low characterized potential of competition with climbers and shrubs in young secondary forest [Ficus mucuso and Cola nitida [Alchornea laxiflora (an understory shrub) indicates disturbance within the last 100 years [Voacanga africana is known to be original occupant of the forest, and species such as Adenia cissampeloides, Sida acuta, Cissampelos owarensis, Aneilema aequinoctiale are common and wide spread in the forest regions. Mondia whitei is characterized to be rare in rainforest [For both Site 1 and the control site; 13 plant species were similar most of which are significant indicators. The species include a et al. , Albiziaoa farms . Similary forest . Also, ta nitida . Their a00 years , while Vinforest , 36.Justicia insularis, Drynaria laurentii and Pentodon pentandrus. All the 3 species are typical for this region with wide distribution. Justicia insularis is known as weed of plantation and it is usually characterized with competing potentials [Drynaria laurentii; found in the wet tropical environments with native range extending from equatorial Africa to tropical south and East Asia, and Pentodon pentandrus found in wet environments, and considered of least concern by IUCN Red List [Common to both Site 2 and the control site are only 3 plant species which include tentials . Also isRed List .Alchornea cordifolia, Cissus quadrangularis, Clerodendrum volubile, Ficus asperifolia, Pouzolzia guineensis and Spondias mombin. According to [Spondias mombin is a deciduous tree, though known as an ancient introduction from America; but also possibly a native of West Africa. It is widespread and common in farmlands, regrowth and villages especially in the forest regions, as well as in the savanna. In fact, it was classified as a savanna species by Salami [Pouzolzia guineensis is found mainly in the tropics and it is an evidence of succession, while Ficus asperifolia is usually found in river areas; its presence on the two mined site is probably because of the availability of water used in washing the mined gold unlike on the control site [Alchornea cordifolia is a forest disturbance indicator, largely due to anthropogenic activities, the plant extracts from leaf and back exudates has medicinal properties, hence used locally by villagers. Clerodendrum volubile is a climber found in forest or thicket; it is also medicinal and ornamental in nature.A total of 6 plant species characterized both Sites 1 and 2 where mining activities has been done. They include rding to , Spondiay Salami  and arearol site . AlchornAcanthus montanus, Adenia lobata, Blepharis maderaspatensis, Brachiaria deflexa, Combretum hispidum, Crinum jagus, Elaeis guineensis, Hewittia sublobata, Icacina trichantha, Pentodon pentandrus, Phyllanthus amarus and Sterculia tragacantha. Acanthus montanus is majorly found in high forest, with wide distribution within Central and West Africa, it is classified under plant with less concern according to the IUCN, but is easily threatened by land and water pollution Ghogue [Icacina trichantha, a small tropical family, in forest and forest regrowth vegetation, found mainly within few countries in West Africa; Nigeria, Ivory Coast, and Benin [Sterculia tragacantha is a deciduous tree, remnant of original forest with distributed undergrowth while Elaeis guineensis , is a cultivated crop (indicating human interference), but its survival on contaminated soils is very low. Also, Adenia lobata occurs in the secondary rainforest, while Combretum hispidum; a scandent shrub is found majorly in the west part of Nigeria. Brachiaria deflexa originated from Africa, and has reached tropics of the new and old world like Middle East, India and south East Asian countries. Crinum jagus is a native range of tropical Africa found in the secondary/primary forest, swamp forest, and riverine vegetation while Phyllanthus amarus, and Hewittia sublobata are widespread in the tropics,On the control site, 11 plant species were observed which include n Ghogue . This sund Benin . SterculCapsicum frutescens, Crassocephalum crepidioides, Laggera pterodonta, Momordica foetida, Pallisota ambigua, Rauvolfia vomitoria, Spilanthes filicaulis and Trema orientalis. Capsicum frutescens is widely dispersed throughout the tropics, cultivated but sometimes naturalized and it is an evidence of human activities [Crassocephalum crepidioides, which also indicates very prominent human disturbance. But Trema orientalis is an indicator of forest regrowth while Laggera pterodonta, Momordica foetida are species widespread in tropical Africa and Pallisota ambigua in lowland rainforest. Rauvolfia vomitoria is also locally called “Asofeyeje” in Yoruba, a shrub common in secondary forest and used medicinally by the locals as medicinal, making of dye, and planted as a shade tree for cocoa.8 different plant species were observed on Site 2 compared to the other two sites. These include tivities . SimilarAdenia venenata, Aneilema beniniense, Aspilia africana, Chasmanthera dependens, Clerodendrum splendens, Colocasia esculentum, Desmodium adscendens, Dissotis rotundifolia, Gliricidia sepium, Grewia flavescens, Hippocratea indica, Holarrhena floribunda, Laportea aestuans, Lonchocarpus cyanescens, Manihot esculenta, Melochia corchorifolia, Morinda lucida, Momordica charantia, Mucuna pruriens, Musa paradisiaca, Newbouldia laevis, Senna occidentalis, Talinum triangulare, Triumfetta cordifolia and Vigna unguiculata. Typical plantation crops common in this region which might not have been identified on other sites include Manihot esculenta and Musa paradisiaca. Talinum triangulare (Water leave) is a cosmopolitan/naturalized weed, sometimes cultivated, edible and useful as ethnomedicine [Mucuna pruriens, Vigna unguiculata (Wild), Hippocratea indica. Lonchocarpus cyanescens (the Yoruba Indigo), Newbouldia laevis, and Clerodendrum splendens, as well as Holarrhena floribunda, Morinda lucida, Clerodendrum splendens, Aneilema beniniense in the rainforest, with Colocasia esculentum which is native to tropical Asia and South-West Pacific, listed as least concern according to IUCN with no major threats [Dissotis rotundifolia are often in damp places as well as Triumfetta cordifolia. Melochia corchorifolia is common in wet places and widespread in the tropics of the old world, Cissampelos owariensis is a twiner mainly in secondary growth in forest regions, while Adenia venenata is majorly found in Nigeria; suggesting that this area still maintains some original vegetation significance. But there are some identified savanna plant species which indicate the high intense of disturbance this site has been subjected to. These include Grewia flavescens, and Chasmanthera dependens, and similar to that are Momordica charantia, but Aspilia africana is common and widespread in the warmer part of the world.On Site 1, 25 different species were identified compared to other two sites. These include  threats . HoweverAcanthus montanus and Icacina trichantha), species found on Site 2  are due to human inference while the species on Site 1 shows both original and disturbed floristic composition (e.g. Adenia venenata and Grewia flavescens). In addition is the presence of Ficus asperifolia usually associated with gold found on the two mined sites but not on the control site. Though all the studied sites have been stressed; resulting in less number of species characterized for these sites compared to previous studies. However, the mined sites had not only been stressed but had also been greatly disturbed. This can be traced to the on-going farming activities and ASM activities such as pollution, deforestation and exposure of the forest soils to direct sunlight. The stress decreases the biodiversity of the species while the disturbance increases its productivity. The washing and processing of the mined gold might have also influenced the substrate conditions of the mine sites which act as an ‘environmental sieve’ especially for the groundwater. It is therefore necessary to develop effective approaches and policies that will curb illegal ASM activities within the south-west region of Nigeria. In doing this, it is also of importance to investigate its supply chains; this is because several nationalities are sometimes noticed illegally on the sites. The community  need to be empowered to discourage this illegal source of income. Coupled with that is conservation and management measures that can enhance the sustainable development and stabilize the economy of this area, thereby discouraging the continuation of ASM mining and encourage adequate reclamation.It could be deduced from the study that the floristic composition of all the 3 sites has been altered with significant differences from one site to the other. This can be attributed to the intense agricultural colonization and human activities on the sites in time past. However, the ASM activities have as well significantly influenced the floristic composition of the mined sites, its species richness, life form patterns, with occurrence of non-native plant species on the mined sites. Many of the differently identified species found on the control sites are native and significance (e.g. The study areas consisted of an abandoned mining site (Site 1) an active mining site (Site 2) and an undisturbed vegetation site for control. Site 1 is situated at Okutu-Omo , Site 2 at Itagunmodi village  and the Control site (or reference site) at Igila  in Atakunmosa west Local Government Area (LGA) of Osun state, within the southwest region of Nigeria  was used for analyzing the data. The significance of difference among the study sites were analyzed using PERMANOVA  and pairwise comparison between the sites. Vegetation data were log transformed (LogX + 1)) to improve homogeneity of variances. Species richness, number of individuals, Pielous evenness, Shannon and Simpson indices were estimated from the log transformed data with the “DIVERSE” function in PRIMER 7  was crea) to impr


The anti-CD20 monoclonal antibodies rituximab and obinutuzumab differ in their mechanisms of action, with obinutuzumab evoking greater direct B cell death. To characterize the signaling processes responsible for improved B cell killing by obinutuzumab, we undertook a phosphoproteomics approach and demonstrate that rituximab and obinutuzumab differentially activate pathways downstream of the B cell receptor. Although both antibodies induce strong ERK and MYC activation sufficient to promote cell-cycle arrest and B cell death, obinutuzumab exceeds rituximab in supporting apoptosis induction by means of aberrant SYK phosphorylation. In contrast, rituximab elicits stronger anti-apoptotic signals by activating AKT, by impairing pro-apoptotic BAD, and by releasing membrane-bound NOTCH1 to up-regulate pro-survival target genes. As a consequence, rituximab appears to reinforce BCL2-mediated apoptosis resistance. The unexpected complexity and differences by which rituximab and obinutuzumab interfere with signaling pathways essential for lymphoma pathogenesis and treatment provide important impetus to optimize and personalize the application of different anti-CD20 treatments.  •Rituximab and obinutuzumab induce strong ERK and MYC activation•Obinutuzumab supports apoptosis induction by aberrant SYK phosphorylation•Rituximab elicits stronger anti-apoptotic signals by activating AKT•Rituximab elicits stronger pro-survival signals by enhancing NOTCH1 signaling Immunology; systems biology: proteomics; cancer We thereby uncovered an activation of pathways downstream of the BCR by rituximab as well as obinutuzumab treatment, identified differences between the two monoclonal antibodies, and discovered links between anti-CD20 treatment and NOTCH1 arising from an activation of the BCR signaling cascade.CCL4 and CCL3 expression as established surrogates for BCR activation 2 fragments (p < 0.001), but not trastuzumab (CCL4 and CCL3 expression levels (p < 0.01), diminished the increase in CCL3 expression after rituximab treatment , and completely abrogated CCL4 up-regulation by rituximab  representing an already de-activated form of the short-lived transcription factor , we found a strong increase in AKT Ser473 phosphorylation at 1 h after rituximab and rituximab F(ab’)2 treatment with fading signals at the 24 h time point (246. Only low levels of phospho-AKT Ser473 were detected after obinutuzumab treatment and no increase in AKT Ser473 phosphorylation was observed after isotype control trastuzumab treatment (Activation of the lipid kinase phosphatidylinositol 3-kinase (PI3K) was inferred by changes in the phosphorylation level of PI3K binding sites on CD19 and BCAP and by activity changes of downstream PI3K effectors . Increasreatment . Howeverreatment , likely tibodies . To valime point . This rereatment A.Figure 99, as an important AKT target site with key role in inhibition of apoptosis, was found phosphorylated only after rituximab treatment . In addition, rituximab more than obinutuzumab increased levels of Ser118 phosphorylation on BAD phosphorylation allows an adjustment of NOTCH1 signaling strength. ADAM10 was shown to cleave NOTCH1 after ligand binding, whereas ADAM17 has been associated with ligand-independent NOTCH1 activation 731 500 45849.This study did not generate new unique reagents.PXD023572 and 10.6019/PXD023572.The mass spectrometry proteomics data generated during this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier All methods can be found in the accompanying


Litopenaeus vannamei raised under ad libitum  and restricted  feeding regimes.Competition is a common social interaction among shrimp and depending on its intensity, it can affect heritable variation and response to selection. Little is known about the variance of indirect genetic effects (IGE) under competitive and non-competitive conditions in shrimp. In this study, we used extended mixed linear models to estimate genetic parameters for the direct genetic effect (DGE) and IGE on body weight in Estimates of heritabilities for body weight obtained with a traditional animal model (i.e. without accounting for IGE) were 0.11 ± 0.09 under AF and 0.25 ± 0.11 under RF. With extended animal models that accounted for IGE, the corresponding estimates for body weight were 0.07 ± 0.08 and 0.34 ± 0.11. Thus, heritabilities were higher under the RF regime than under the AF regime, regardless of whether IGE was accounted for or not. The log-likelihood ratio test revealed significant IGE under the RF regime. Although estimates of indirect genetic variance were low (0.0023 ± 0.0013 for AF and 0.0028 ± 0.0012 for RF), they contributed substantially to the total heritable variance: 66.8% for AF and 692.2% for RF. The total heritable variance was smaller under the RF regime (0.7 ± 1.3) than under the AF regime (5.8 ± 2.6) because of the high contribution of the negative covariance between DGE and IGE (− 7.03). Estimates of the correlation between DGE and IGE were 0.32 ± 0.47 under AF and − 0.93 ± 0.15 under RF, those of DGE and IGE for body weight between both regimes were 0.94 ± 0.07 and 0.67 ± 0.20, respectively, and those of IGE for body weight with DGE for survival were − 0.12 ± 0.22 under AF and − 0.58 ± 0.20 under RF.L. vannamei. Significant reranking and variation in IGE of individuals were observed between the two feeding regimes. Strong competitive interactions reduced the total heritable variation for body weight when food was restricted. These results indicate that the extent of competition among L. vannamei depends on the feeding regime applied and that this competition affects the genetic basis of body weight.These results indicate that strong competitive interactions occurred under the RF regime in  Salmo gairdneri), dominant individuals access more food and grow faster than subordinates. In teleost fish, up to 90% of the mortality is due to cannibalism [(Oreochromis niloticus) and Arctic charr  [Cannibalistic and aggressive behaviors are often observed in crustaceans , 2 and fnibalism . Inter-iinus L.) , 7. Highinus L.) , 9. Sociinus L.) .Gadus morhua) [For a family-based selective breeding program in aquaculture, candidates are usually tagged and tested in one or several communal rearing environments. Although observed social interactions may suggest the presence of IGE, these genetic effects cannot be estimated with a traditional animal model because of confounding with the direct genetic effect (DGE) and the lack of an effective experimental design. To estimate IGE, individuals need to be split into a large number of groups, which makes the data structure based on a traditional communal rearing environment unsuitable to separate DGE from IGE. However, ignoring IGE in selective breeding can lead to increased competition and a negative selection response , 11. Exp morhua)  were obt morhua) , 14. Sig morhua) –21.More than 60 selection experiments have been carried out for increased growth in aquaculture species. Estimates of genetic gain for body weight obtained in these experiments ranged from 2.3 to 42% per generation, with an overall average of 12.7% . EmpiricLitopenaeus vannamei, is an important farmed shrimp worldwide, with an annual production of 4.46 million metric tons, accounting for 80% of the total penaeid shrimp production [L. vannamei [L. vannamei, the variance of IGE on adult body weight was found to be large at low stocking density and under an ad libitum feed regime [The Pacific white shrimp, oduction . A familvannamei , 30. In d regime . There wd regime . SelectiL. vannamei. Moreover, the type and intensity of social interactions among individuals under two feeding regimes were evaluated by estimating correlations between DGE and IGE for body weight. To investigate the consequences of selection for rapid growth on aggressive and competitive behaviors, genotype-by-environment interaction for IGE was evaluated by estimating the correlation of IGE for body weight between two feeding regimes.Thus, the main objective of this study was to quantify DGE and IGE for body weight under ad libitum (AF) and restricted (RF) feeding regimes in 3 of a selection line that was established in 2012. Eight improved batches from different companies in the United States and Singapore were introduced as founder broodstocks. Each shrimp was tagged on one ocular peduncle with a numbered ring. The shrimp were checked for different viruses and bacterial strains and those that were pathogen-free were reserved as broodstocks. The healthy broodstocks with mature gonads were chosen after 1 month of temporary rearing. The base population (generation G0) of eight strains was established by an incomplete diallel cross design. Each generation, full-sib and half-sib families were produced using a nested mating design, in which two dams (sires) were mated to the same sire (dam) by artificial insemination. In total, 207 full-sib families  from 187 sires and 174 dams were produced in 2012. These families were tagged with a visible implant elastomer (VIE) and communally reared in two 100-m2 tanks for the grow-out test.The social interaction experiment started in 2015 at Hebei Xinhai aquaculture Technology Ltd  in Huanghua City, Hebei Province, China. The experimental shrimp belonged to generation G0 were ranked using a selection index that included the standardized individual estimated breeding values (EBV) for body weight and survival, with weights of 70 and 30%, respectively. The mating schemes for the selection and control populations in generation G1 were as follows: the selection population was generated by mating selected males and females with a high selection index, while maintaining a relatively large effective population size and controlling inbreeding . The control population was composed of 10 to 20 full-sib families produced by single-pair mating of 20 to 30 breeding candidates of generation G0. One hundred and five families  were produced in generation G1 from 100 sires and 91 dams. The same scheme was used to generate generations G2 and G3. Eighty-seven families  from 65 males and 68 females of generation G2 were selected for the analysis of IGE in generation G3 (Table Candidates in generation G3) in a rectangle concrete tank (100 m2), with restrictions to control coancestry coefficients  and to maintain a low coefficient of variation of body weight among the three families in a cage. Each cage had 42 shrimp from three families, which resulted in an average shrimp density of about 171 individuals/m3 (depth of the water of 0.5 m) at the beginning of the 71-day experiment. This stocking density is not high for most intensive culture models of L. vannamei [3 at the end of the experiment. The water exchange rate for each tank was about 20 to 30% every 3 days. Mortality rates under the AF and RF regimes were 21.40 and 21.43%, respectively. In total, 6720 shrimp were assigned to 160 cages within 1 day, and 5281 shrimp were harvested over 1 days after the 71-day growth-test  was defined as the total heritable effect of an individual on body weight in the studied population, and was calculated as :3\documeThe phenotypic variance . Because of convergence issues, IGE for the AF regime and the common environmental effects for full sibs  than under the RF regime (14.9 g), whereas the standard deviation and coefficient of variation of body weight were lower under the AF regime (3.7 g and 21.8%) than under the RF regime . The RF regime may have increased competition among individuals that shared the same environment and food. However, there was no difference in survival between the AF and RF regimes., P < 0.001). The small variances for IGE of body weight, 0.0023 ± 0.0013 and 0.0028 ± 0.0012 under the AF and RF regimes, respectively, contributed substantially to the total heritable variance. The contributions of the variance of IGE over the total heritable variance  indicates that strong competitive interactions occurred when the amount of food provided was limited. In contrast, the positive correlation between the DGE and IGE under the AF regime (0.32 ± 0.47) implies that there was no evidence of competitive interactions when food was available in excess.The negative and significant estimate of the correlation between DGE and IGE under the RF regime . This indicates that a re-ranking for IGE of the families occurred between the two competitive levels. The high negative correlation estimate of DGE under the AF regime with IGE for the RF regime (− 0.81 ± 0.12) implies that retained individuals characterized by a rapid growth under the AF regime would show strong competition when tested under the RF regime.Estimates of genetic correlations to quantify genotype-by-environment interactions for DGE and IGE on body weight between the two feeding regimes are in Table The back-transformed heritabilities of survival under the AF and RF regimes were 0.08 ± 0.02 and 0.07 ± 0.02, respectively Table . The estThis study reports the first large-scale test for IGE under different competitive environments in aquaculture. A strong re-ranking of the genetic competitive abilities of families was detected between the two feeding regimes. Individuals that were genetically highly competitive showed better survival under the RF regime. The total heritable variance of body weight was greatly reduced because of the strong competitive interactions that occurred among individuals under the RF regime. The results indicate that an increase or a decrease in competition during artificial selection for growth depends on resource availability. Moreover, the results indicate a cryptic genetic variation for body weight when feed is limited.Differences in body weight between the AF and RF feeding regimes were relatively small although the RF regime represented 50% less feed intake compared to the AF regime Table . HoweverL. vannamei (0.17 ± 0.04 to 0.45 ± 0.09) [In this study, estimates of heritability for body weight under the AF regime (0.07 ± 0.08 and 0.11 ± 0.09) were lower than previously reported estimates in  ± 0.09) –41, whic ± 0.09) . Thus, tEstimates of correlations between IGE of body weight and DGE of survival may be biased because the P > 0.05). The estimate of the correlation between DGE and IGE for body weight under the AF regime was also small (0.32 ± 0.47). Moreover, the correlation of DGE between both regimes (0.94 ± 0.07) and the correlation between DGE and IGE under the RF regime (− 0.93 ± 0.15) were all high. Therefore, the bias in The estimate of the correlation of DGE on body weight under the AF regime with IGE on body weight under the RF regime  [3), relatively small standard errors were obtained for estimates of the IGE-DGE covariance (− 0.087 ± 0.028 to 0.013 ± 0.017). Nevertheless, a larger standard error for the estimate of the correlation between DGE and IGE under the AF regime (0.32 ± 0.47) than under the RF regime (− 0.93 ± 0.15) implies that competitive interactions were less frequent under the AF regime.Standard errors of the genetic parameter estimates for IGE were high and influenced by the number of test groups. For a dataset of 493 test groups in Nile tilapia obtained . A largeobtained , 17. Theobtained  when theuals/m3) , the staEucalyptus globulus), Silva et al. [Neovison vison, a positive genetic correlation (0.90 ± 0.15) and a large covariance between DGE and IGE  were obtained for total bite mark score [E. globulus, the covariance between DGE and IGE increased the heritable variance by 71% for Mycosphaerella leaf disease [Very strong competitive interactions were observed under the RF regime, based on the estimate of the genetic correlation between DGE and IGE of − 0.93 ± 0.15. The IGE of an individual for body weight rapidly spreads to all its mates, and its full impact is reflected in the TBV. The much smaller variance of TBV of body weight under the RF regime than under the AF regime 0.68 vs 5.79) implies that the total heritable variance was greatly reduced by the strong competitive interactions under the limited feeding condition. Although the difference between the estimates of the variance of IGE for the RF and AF regimes was small (0.0028 ± 0.0012 vs 0.0023 ± 0.0013), the difference between the estimates of the covariance between DGE and IGE for the RF and AF regimes was large (− 0.087 ± 0.028 vs 0.013 ± 0.017). The large negative covariance decreased the total heritable variance under the RF regime by 91% , a small additive genetic variance (0.085) for body size was detected under high salinity, but it increased significantly (3.289) when an oceanic population was reared under low salinity [In contrast to the small direct genetic variance (0.75 to 1.15) under the AF regime, a larger direct genetic variance (2.26 to 3.17) was observed under the RF regime. However, the phenotypic mean and variance for body weight were larger under the AF regime (mean BW = 16.8 g, ariation . In our salinity . CGV wassalinity , for plasalinity , and forsalinity  when abrL. vannamei when reared under conditions with limited food. The competitive abilities of families differed significantly between the ad libitum and restricted feeding regimes. Strong competitive interactions reduced the total heritable variance for body weight when the amount of food was limited. Our results support the assumption that an increase or a decrease in aggression and competition during selection depends on whether the amount of available food is limited or excessive.Our findings reveal strong competitive interactions among


Drosophila, the Warts (Wts) kinase, a component of the Hippo signalling pathway, plays an essential role in regulating transcription and growth by phosphorylating its substrate Yorkie (Yki). The phosphorylation of Yki critically influences its localisation and activity as a transcriptional coactivator. In this study, we identified the homeodomain-interacting protein kinase (Hipk) as another kinase that phosphorylates Yki and mapped several sites of Yki phosphorylated by Hipk, using in vitro analysis: Ser168, Ser169/Ser172 and Ser255. These sites might provide auxiliary input for Yki regulation in vivo, as transgenic flies with mutations in these show prominent phenotypes; Hipk, therefore, represents an additional upstream regulator of Yki that works in concert with Wts.Developmental growth and patterning are regulated by an interconnected signalling network of several pathways. In  Drosophila, four tumour-suppressor genes—warts (wts), salvador (sav), hippo (hpo) and mob as a tumour suppressor (mats)—were discovered to regulate organ growth. Their mutations in cell clones lead to massive overgrowth phenotypes, and they all belong to the same signalling pathway, the Hippo pathway, which is evolutionarily conserved up to mammals . The coiewed in ,3. Any Yiewed in ,4,5.Drosophila as well as in mammals, the phosphorylation of Ser168(Yki)/Ser127(YAP) by Wts/LATS1 critically impacts the outcome of the Hpo signalling pathway, as it inactivates the potential oncogene by excluding it from the nucleus through an interaction with the protein 14-3-3 ATP. A schematic overview of all the Yki fragments tested is shown in For better handling, we initially divided the protein sequence of Yki into smaller sections; each was amplified from the protein-coding region of We performed a mutational analysis of the three identified regions to identify the specific amino acid residues (serine or threonine) phosphorylated by Hipk; we introduced single amino acid changes from serine or threonine to alanine. As Hipk has been described as a proline-guided serine/threonine kinase with phosphorylation target sites that mostly appear in clusters of two or three adjacent residues around a proline according to the consensus S/X-R/X-S/T-P-S/X ,31,32, wAlthough Ser169 and Ser172 have been excluded as Wts-phosphorylation sites by some researchers, they have been examined for their influence on Ser168 phosphorylation . When boAnother Hipk-phosphorylation site was also suggested to exist within Yki-N2B; this fragment was positively tested in the above-mentioned assay. We decided to focus on Ser255 as a potential Hipk target based on our consensus. The mutation of Ser255 to alanine Yki-N2C; C completOur studies described thus far of the Yki-C9 fragment, multiply-mutated Yki-N2FG fragment and Yki-N2C fragment showed that, at least in vitro, there are no Hipk phosphorylation sites at the N-terminal end of Yki (aa 1–96) and no further Hipk phosphorylation sites within the C-terminal region of Yorkie (aa 117–418). Drosophila embryos  M{vas-int.Dm}ZH-2A w[*] PBac{y[+]-attP-3B}VK00033 (BL24871). Further effector lines used: P{UAS-yki.GFP}4-12-1 ; P{UAS-yki.S168A.GFP.HA}10-7-1 ; yellow white (yw67c23) (BL6599). The flies were reared on standard medium and raised at 25 or 18 °C as indicated.Fly strain for phiC31 integration: ® mounting medium. The wings were imaged using an Olympus SZX 12 microscope with a U-CMAD3 camera system, and area measurement was performed using the cellP imaging software . The shown images were processed with Adobe Photoshop and Adobe Illustrator.The fly heads were dissected from the bodies of adult female flies and mounted on coverslips. The wings were dissected from the adult female flies, rinsed in 70% EtOH and mounted on a coverslip in AquatexDrosophila melanogaster, and we mapped several Hipk-phosphorylation sites . In vivo functional relevance was demonstrated by expression studies with respective mutant forms of Yki. The effect of a Ser255 mutation in Yki clearly implies that Hipk can also regulate Yki in vivo via this site. Hipk is the second kinase, alongside Wts, known to phosphorylate the transcriptional coactivator Yki, implying that the regulation of Yki, in addition to the Hpo core pathway, is more complex than expected. Our results may be of general interest, as the Hpo signalling pathway is highly conserved in Drosophila as well as vertebrates and plays a pivotal role in maintaining homeostasis in cell proliferation and organ growth during both normal development and oncogenesis.In this study, we showed that the Hippo (Hpo)/Warts (Wts) signalling effector Yorkie (Yki) is an in vitro substrate of homeodomain-interacting protein kinase (Hipk) in


Cytotoxic T lymphocytes kill cancer or virally infected cells by exocytosis of lytic granules. This leads to perforin-mediated granzyme entry into the target cell, consequently killing the target cell. Granzymes and perforin are activated by cysteine cathepsins whose activity is regulated by the protein inhibitor cystatin F. Since cystatin F can be secreted by a range of cancer and immune cells in tumour microenvironments, we here investigated whether extracellular cystatin F can be taken up by and affect the function of cytotoxic T lymphocytes. We demonstrated cystatin F uptake into cytotoxic T lymphocytes, down-regulation of target peptidases, and reduced target cell killing. Overall, our results indicate that cystatin F is an important mediator that can impair the killing efficiency of cytotoxic T lymphocytes and thus suggest that it is a possible target for cancer immunotherapy.Cystatin F is a protein inhibitor of cysteine cathepsins, peptidases involved in the activation of the effector molecules of the perforin/granzyme pathway. Cystatin F was previously shown to regulate natural killer cell cytotoxicity. Here, we show that extracellular cystatin F has a role in regulating the killing efficiency of cytotoxic T lymphocytes (CTLs). Extracellular cystatin F was internalised into TALL-104 cells, a cytotoxic T cell line, and decreased their cathepsin C and H activity. Correspondingly, granzyme A and B activity was also decreased and, most importantly, the killing efficiency of TALL-104 cells as well as primary human CTLs was reduced. The N-terminally truncated form of cystatin F, which can directly inhibit cathepsin C (unlike the full-length form), was more effective than the full-length inhibitor. Furthermore, cystatin F decreased cathepsin L activity, which, however, did not affect perforin processing. Cystatin F derived from K-562 target cells could also decrease the cytotoxicity of TALL-104 cells. These results clearly show that, by inhibiting cysteine cathepsin proteolytic activity, extracellular cystatin F can decrease the cytotoxicity of CTLs and thus compromise their function. Cytotoxic T lymphocytes CD8+ (CTLs) and natural killer (NK) cells are crucial players in the immune response against virally infected cells and tumour cells . The maiIn cytotoxic lymphocytes, cathepsin C is the major progranzyme convertase, activating progranzymes by removing a dipeptide at their N-terminal end . In the In immune cells, cystatin F, an endogenous protein inhibitor, was described to play an important role in regulating cysteine cathepsin activity . CystatiIn cytotoxic cells, cystatin F is localised in cytotoxic granules in which it co-localises with its target cathepsins C, H, and L ,15,16 inIn this study, we demonstrate that extracellular cystatin F decreases CTL cytotoxicity, likely by affecting the activation of granzymes, effector molecules of the perforin/granzyme pathway. We first demonstrate internalisation of cystatin F into TALL-104 cells and interaction of internalised cystatin F with cathepsin C. We show that extracellular cystatin F attenuates the activities of cathepsins C, H, and L and granzymes A and B, while it does not affect perforin processing. Finally, we demonstrate that cystatin F secreted from target cells can also affect the cytotoxicity of CTLs.We first assessed recombinant cystatin F uptake into TALL-104 cells, a model cell line of cytotoxic T lymphocytes . WesternIn cytotoxic lymphocytes, cystatin F regulates the activities of cathepsins C, H, and L, which are implicated in granzyme and perforin activation ,13. The Next, we aimed to determine whether lower cathepsin C and H activity translates to decreased granzyme activity. Granzyme A and B activity was determined in whole-cell lysates of TALL-104 cells treated with full-length or N-terminally truncated cystatin F. Extracellularly added full-length cystatin F significantly inhibited the activity of granzymes A and B d,e in TANext, we tested whether treatment of TALL-104 cells with full-length or N-terminally truncated cystatin F affects their cytotoxic potential by calcein-AM release assay. The ability of TALL-104 cells to kill target K-562 cells was significantly reduced after treatment with either form of cystatin F f,g.Lastly, to further extend our findings in a more physiological setting we tested the effect of full-length cystatin F on granzyme activity and cytotoxicity of primary human SEA/SEB-stimulated CTLs. Indeed, activities of granzymes A and B were significantly decreased a,b. ImpoSince cystatin F is a potent inhibitor of cathepsin L, which is involved in the maturation of cathepsin C , we nextGiven the effect of cystatin F on TALL-104 cytotoxicity, we assessed whether the presence of cystatin F in the target cells themselves inhibits the cytotoxic effect of lymphocytes, thus conferring protection to target cells. Target K-562 cells that do not express endogenous cystatin F were incubated with recombinant cystatin F. Western blot analysis revealed that full-length as well as N-terminally truncated cystatin F were both internalised into target K-562 cells within 4 h . When cyCystatin F is a protein inhibitor of cysteine cathepsins that is found intracellularly in the lysosomal/endosomal pathway as well as extracellularly ,13. The Extracellular cystatin F was demonstrated to be internalised by several cell types, including human HeLa and HEK-293 cell lines ,14 and mTo confirm the effect of internalised cystatin F on target cathepsins, we analysed cathepsin C, H, and L activity in TALL-104 cells and found that they were significantly reduced. We also compared the effect between full-length and N-terminally truncated cystatin F on their activities. N-terminally truncated cystatin F is a monomeric form and can immediately interact with cathepsins after internalisation. Conversely, full-length cystatin F forms inactive dimers that must first be processed at the N-terminus and monomerise to become active inhibitors . Both foFinally, we investigated whether cystatin F in target cells could confer protection against CTL killing. Indeed, cystatin F-pretreated target K-562 cells were killed less efficiently by TALL-104 cells. In the timeframe of the cytotoxicity assay, cystatin F-loaded target K-562 cells released a large portion of internalised cystatin F. Therefore, we propose that target cells may protect themselves from CTL killing by secreting cystatin F, which is subsequently internalised into cytotoxic cells, in which it inhibits the activation of granzymes that are crucial for efficient target cell killing. A protective role of cystatin F in target cells might be especially important in tumour microenvironments in which cystatin F could be exploited by tumour cells to evade the anti-tumour immune response. Indeed, up-regulated cystatin F expression in tumour tissue was demonstrated in patients with colorectal cancer, and was even correlated with liver metastasis and worse prognoses . A similFor western blotting and immunofluorescence microscopy, the rabbit anti-cystatin F antibody from Sigma-Aldrich  and Davids Biotechnologie GmbH  was used, respectively. Additionally, the following antibodies were used: rabbit anti-β-actin (Sigma-Aldrich), rabbit and mouse anti-glyceraldehyde 3-phosphate dehydrogenase , rabbit anti-his-tag , rabbit anti-N-terminal part of cystatin F , mouse anti-cathepsin C, mouse anti-granzyme A, and mouse anti-perforin . The mouse anti-cathepsin H antibody 1D10  and sheeFull-length and N-terminally truncated human recombinant cystatin F were produced in the human freestyle HEK293F cell line and purified as described in .8/mL for 1 h and then re-suspended in RPMI (Lonza) with 10% foetal bovine serum (Gibco) and 100 IU/mL interleukin-2 (Bachem) at a cell density of 2 × 106/mL. After 5 days SEA/SEB-specific CTLs were isolated by positive selection according to manufacturer’s instructions . The purity of CTLs was determined by flow cytometry using fluorescence labelled antibodies against CD3, CD4, CD8, CD56, CD16, CD19 and TCRα/β  and was >95%.TALL-104 cells  were cultured in Iscove’s Modified Dulbecco’s Medium  with 20% foetal bovine serum , 100 IU/mL interleukin-2 , 2.5 µg/mL recombinant human albumin (Sigma-Aldrich), and 0.5 µg/mL D-mannitol (Sigma-Aldrich). HeLa cells  were cultured in Dulbecco’s Modified Eagle Medium  with 10% foetal bovine serum (Gibco), 100 U/mL penicillin , and 100 U/mL streptomycin (Lonza). K-562 cells  and Raji cells  were cultured in RPMI-1640 (Lonza) with 10% foetal bovine serum, 100 U/mL penicillin (Lonza), and 100 U/mL streptomycin (Lonza). Primary human CTLs were isolated from peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers at the Blood Transfusion Centre of Slovenia, Republic of Slovenia, according to institutional guidelines. PBMCs were stimulated with 5 µg/mL staphylococcal enterotoxin A  and 5 µg/mL staphylococcal enterotoxin B  at a cell density of 10Cells, untreated or incubated for 4 h with 100 nM full-length cystatin F, were washed once in phosphate-buffered saline (PBS), left to adhere to slides for 30 min at 37 °C, and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature (RT). After rinsing three times with PBS for 5 min each, specimens were blocked for 1 h in 5% goat serum and 0.3% Triton X-100 in PBS. Primary antibodies were diluted in 1% bovine serum albumin and 0.3% Triton X-100 in PBS to a final concentration of 10 μg/mL for anti-cystatin F, 2 μg/mL for anti-his-tag and 4 μg/mL for anti-perforin and anti-granzyme A antibodies and incubated overnight at 4 °C. Next, slides were rinsed three times in PBS for 5 min each and incubated with goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 555, diluted 1:1000 in the same buffer as primary antibodies, for 2 h at RT. After the final washing step, the slides were cover-slipped using Prolong Gold Antifade Mountant . Samples without one or both primary antibodies were used as controls. Images were acquired with a Carl Zeiss LSM 710 confocal microscope  with ZEN 2011 imaging software (Carl Zeiss).g for 20 min at 4 °C. Supernatants were transferred to fresh tubes, and protein concentrations were determined using the DC-Protein Assay Kit (Bio-Rad). The lysis buffer for western blot analysis comprised 50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA with added protease inhibitors (Roche). The lysis buffer for cathepsin activities comprised 0.1 M citrate buffer pH 6.2 with 1% Triton X-100. The lysis buffer for granzyme activities comprised 25 mM HEPES, 250 mM NaCl, 2.5 mM EDTA, and 0.1% Nonidet p-40, pH 7.4.Cells were either untreated or incubated with 100 nM full-length or N-terminally truncated cystatin F for 4 h and 24 h or with 20 μM E64d for 17 h. Cells were then washed in PBS, lysed in lysis buffer, incubated for 30 min on ice, and centrifuged at 16,000× Samples containing 30–50 µg of cell lysate total protein were resolved in SDS-PAGE using 12% polyacrylamide gel and transferred onto nitrocellulose membranes using the trans-blot turbo transfer system (Bio-Rad) and trans-blot turbo RTA transfer kits with nitrocellulose membrane (Bio-Rad). For some experiments, samples were resolved using 12% TGX stain-free polyacrylamide gels (Bio-Rad); these were activated before the transfer with UV light for 1 min using the ChemiDoc MP System (Bio-Rad). After the transfer, the membranes were imaged for stain-free labelling of total proteins with the ChemiDoc MP System. Next, membranes were blocked in 5% non-fat dry milk in PBS (for cystatin F), 5% non-fat dry milk in tris-buffered saline with 0.1% Tween-20 , and 1.5% non-fat dry milk/0.5% bovine serum albumin in tris-buffered saline with 0.1% Tween-20 (for perforin). The primary antibodies were diluted in blocking solution at 1:500 for cystatin F and N-terminal part of cystatin F, 1:200 for cathepsin C and perforin, and 1:600 for his-tag and cathepsins H and L and incubated overnight at 4 °C. After washing, the membranes were incubated with HRP- or fluorescently conjugated secondary antibodies diluted 1:5000 in blocking solution. Membranes with HRP-conjugated antibodies were incubated with Lumi-Light western blotting substrate (Roche). Images were acquired using a ChemiDoc MP System (Bio-Rad), and quantification analysis was performed in Image Lab, version 5.1 software (Bio-Rad).6) were either left untreated or were incubated for 4 h with 100 nM recombinant full-length cystatin F with his-tag. Afterwards, the cells were washed with PBS and lysed in lysis buffer  with protease inhibitors. After incubation on ice for 30 min, the cell lysates were centrifuged for 20 min at 16,000× g, and the supernatant was transferred to a new tube. The anti-his-tag antibody was added to the cell lysate at a final concentration of 2.7 µg/mL, and the samples were incubated at 4 °C overnight with constant shaking. Dynabeads protein A (Thermo Fisher Scientific) were washed once with lysis buffer, added to the cell lysate, and incubated at RT for 20 min with constant shaking. The beads were then washed three times in lysis buffer, resuspended in SDS loading buffer containing 40 mM dithiothreitol, and boiled for 5 min. The co-immunprecipitated proteins were analysed by western blot.TALL-104 cells (50 × 10Enzyme activities were determined using the following substrates: 70 µM H-Gly-Phe-7-amino-4-methylcoumarin (AMC) (Bachem) for cathepsin C, 20 µM H-Arg-AMC (Bachem) for cathepsin H, 50 µM Z-Phe-Arg-AMC for cathepsin L (Bachem), 50 µM acetyl-Ile-Glu-Pro-Asp-AMC for granzyme B (Bachem), and 200 µM Z-Gly-Pro-Arg-AMC for granzyme A (Bachem). The following assay buffers were used: 25 mM MES, 100 mM NaCl, 5 mM cysteine, pH 6 (for cathepsin C); 100 mM MES, 2 mM EDTA, 5 mM cysteine, pH 6.5 (for cathepsins H and L); 50 mM Tris-HCl, 100 mM NaCl, pH 7.4 (for granzyme B); and 20 mM Tris, 150 mM NaCl, pH 8.1 (for granzyme A). Whole-cell lysates were first activated in assay buffer for 15 min at RT for cathepsins or for 30 min at 37 °C for granzymes. The substrate was then added, and formation of fluorescent products was measured continuously on a microplate reader Infinite M1000 . To determine cathepsin L activity, 5 µM of irreversible inhibitor of cathepsin B, CA-074 (Bachem), was added before the addition of substrate.HeLa cells were grown in 6-well plates and transfected with the pcDNA3 plasmid containing wild-type cystatin F , using P6 cells) were determined using the inverse of the number of effector cells needed to lyse 30% of target cells × 100 [To determine the effect of cystatin F on cytotoxicity of effector cells (TALL-104 cells and primary human SEA/SEB-stimulated CTLs), effector cells were first incubated for 4 h with 100 nM full-length or N-terminally truncated cystatin F. K-562 were used as target cells for TALL-104 cells, while SEA/SEB-pulsed Raji cells were used as targets for SEA/SEB-stimulated CTLs. Raji cells were pulsed with 1 µg/mL SEA and 1 µg/mL SEB for 1 h at 37 °C. Alternatively, to test the effect of cystatin F present in target cells on cytotoxicity, target K-562 cells were incubated for 4 h with 100 nM full-length or N-terminally truncated cystatin F. Next, cystatin F was thoroughly washed away, and the calcein-AM release assay  was perfls × 100 .t-test when two groups were compared or with one-way ANOVA followed by Šidák’s multiple comparisons test when more than two groups were compared. Differences were considered significant when p ≤ 0.05.GraphPad Prism 6  was used for data analysis. Statistical significance was assessed with a In this study, we have demonstrated that extracellular cystatin F upon internalisation decreases the cytotoxicity of cytotoxic T cells by attenuating the activities of cathepsins C and H and consequently the activities of granzymes A and B. Furthermore, we showed that cystatin F inactivates cathepsin C by direct inhibition and not by affecting processing from its inactive precursor. Similarly, cystatin F does not affect perforin processing by cathepsin L. Importantly, cystatin F secreted from target cells can also decrease T cell cytotoxicity, indicating that cystatin F can provide a protective mechanism for target cells with which they can avoid cytotoxic attack.


Activity optimization of a pyrazolyl-pyrimidone against the iron transporter SLC11A2 (hDMT1) and mechanistic studies revealed that this class of inhibitors act by metal chelation. 50 = 1.1 μM) pyrazolyl-pyrimidone inhibitor of radiolabeled iron uptake in hDMT1 overexpressing HEK293 cells acting by a non-competitive mechanism, which however does not affect the electrophysiological properties of the transporter. Isothermal titration calorimetry, competition with calcein, induced precipitation of radioactive iron and cross inhibition of the unrelated iron transporter SLC39A8 (hZIP8) indicate that inhibition is mediated by metal chelation. Mapping the chemical space of thousands of pyrazolo-pyrimidones and similar 2,2′-diazabiaryls in ChEMBL suggests that their reported activities might partly reflect metal chelation. Such metal chelating groups are not listed in pan-assay interference compounds (PAINS) but should be checked when addressing SLCs.Solute carrier proteins (SLCs) control fluxes of ions and molecules across biological membranes and represent an emerging class of drug targets. SLC11A2 (hDMT1) mediates intestinal iron uptake and its inhibition might be used to treat iron overload diseases such as hereditary hemochromatosis. Here we report a micromolar (IC This transporter is expressed in the intestinal brush border membrane, where it acts as the key mediator of dietary iron uptake. hDMT1 is also linked to pathologies such as hereditary hemochromatosis, β-thalassemia, Parkinson's disease and Alzheimer's disease, highlighting that its pharmacological inhibition may be beneficial to treat human diseases.13Solute carrier proteins (SLCs) control fluxes of ions and other molecules across biological membranes and represent an emerging class of drug targets.1 and mesitylene 2,3  closer and understand its mechanism of action.Two families of small molecule hDMT1 inhibitors have been reported in the literature as the results of high-throughput screening campaigns, namely bis-cationic isothioureas such as dibenzofurans lene 2,3 .15 In ou1.6–25) of pyrazolyl-pyrimidone 5 by condensing aminoguanidines with 1,3-diketones and keto-esters to form pyrazoles 6–20 and pyrazolones 21–25 using known chemistry . With non-symmetrical diketones synthesis also mostly yielded a single product. X-ray crystal structures of 13 and 17 showed that the product from non-symmetrical diketones was the isomer with the larger pyrazole substituent pointing away from the aminopyridone. By analogy, the same arrangement was assigned to the other pyrazoles. The synthesis of pyrazolones 21–25 only gave a single product whose structures were confirmed by an X-ray crystal structure of 24 as hydrochloride salt  showed similar or improved inhibition compared to 5. On the other hand, pyrazoles 8–12, 14–15 and 20 as well as pyrazolones 21–25 did not show significant inhibition of iron uptake in our assay. Across all compounds tested, pyrazole 13 stood out as one of the most potent compounds with IC50 = 1.1 ± 0.01 μM, representing a 10-fold improvement over our initial inhibitor 5 while preserving the original ligand efficiency. Note that the bis-cyclohexane analog 18 was slightly more potent, however at the cost of higher hydrophobicity.Activity screening of the twenty synthesized analogs of 2.13 in the radioactive 55Fe2+ uptake assay showed that this compound was a non-competitive inhibitor with similar inhibition constants Ki = 2.0 μM ([Fe2+] → 0) and Kii = 1.7 μM (saturating iron), reproducing the behavior of the previously reported less potent pyrazolyl-pyrimidone 5. By comparison, isothiourea 1 showed a competitive inhibition with inhibition constant Ki = 1.4 μM ([Fe2+] → 0), Kii = 15.6 μM (saturating iron) determined by Dixon plot analysis,4 , but that pyrazolyl-pyrimidone 13 had no measurable effect on the recorded currents  with Cd2+, which we used as a redox stable analog of Fe2+ less prone to hydroxide formation at pH = 7.4 and which is also transported by DMT1. At a pH of 7.4, the ITC data indicated an apparent dissociation constant of KD = 6.4 μM with n = 0.54, consistent with the formation of a 2:1 ligand–metal complex .A first indication that pyrazolyl-pyrimidone  complex . At pH 5pitation . The les2+ chelation at pH 5.5 also indicated a significant level of iron chelation by 13, although the effect was weaker than with the positive control bipyridine or the literature inhibitor 3, and pyrazolyl-pyrimidone 5 did not show any iron chelation in this assay , a transporter of Zn2+ and Fe2+ unrelated to SLC11 family, might also be inhibited by our hDMT1 inhibitors. Both SLCs can be tested by inhibition of radioactive iron uptake into HEK293 cells expressing the transporter, at pH 5.5 for hDMT1 and at pH 7.4 for hZIP8.Complex formation with Fe3, our optimized pyrazolyl-pyrimidone 13, and to a lesser extent pyrazolyl-pyrimidone 5, all inhibited both transporters, although the inhibition by 3 was not consistently observed at higher concentrations, probably due to adsorption of the complex to the cell material and assay plate, resulting in apparent uptake. On the other hand, bis-isothiourea 1, which inhibits hDMT1 by competing in the iron binding site, only inhibited hDMT1 and had no measurable activity on hZIP8 –C:N substructures, similar to the well-known chelation by bipyridine forming a tight 3:1 complex with Fe2+. Acid–base titration of 3, 5 and 13 shows in each case two ionizable groups  can be attributed to the pyrazole hydroxyl group for 3 and to the pyrimidone NH group for 5 and 13, resulting in a transition from the neutral form below pH 7 to a mono-anionic form above pH 7 . However, this inhibitor did not modulate hDMT1-induced currents in Xenopus oocytes and exerted cross-inhibition of the unrelated iron transporter hZIP8.To date the pharmacology of divalent metal transporters is extremely limited. In view of the increasing importance of SLCs as drug targets, we investigated the inhibition of hDMT1 mediated iron transport by two previously reported inhibitors, namely pyrazolyl-pyridine 2+ in the assay medium by these molecules to form an insoluble precipitate. Although metal chelation is often exploited as a mechanism of drug action,Our combined experiments suggest that this non-selective inhibition reflects chelation of FeThere are no conflicts to declare.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.


El Niño Southern Oscillation (ENSO) drives interannual variability in West Antarctic climate through altering atmospheric circulation in the Amundsen Sea region (ASR). The El Niño–ASR teleconnection is known to be strongest in austral winter and spring, but its variation with El Niño amplitude is underexplored. This study uses experiments from the HadGEM3‐A climate model to investigate the El Niño–ASR teleconnection for a range of imposed SST perturbations spanning weak (0.75 K) to strong (3 K) amplitudes. In austral winter, the El Niño–ASR teleconnection behaves linearly for El Niño amplitudes up to 2.25 K, but is found to weaken for stronger forcing (3 K). The anomalous Rossby wave source in the subtropical South Pacific increases monotonically with El Niño amplitude. However, a Rossby wave reflection surface originally located in the western South Pacific sector extends progressively eastward with increasing El Niño amplitude, reducing wave propagation into the ASR. The wave reflection surface is associated with curvature in the upper tropospheric zonal winds which intensifies as the subtropical jet strengthens under El Niño forcing. In contrast, the El Niño–ASR teleconnection in austral summer, which more closely resembles the Southern Annular Mode, is found to increase linearly for El Niño amplitudes up to 3 K. The results explicitly demonstrate that a linear approximation of the El Niño teleconnection to the ASR is reasonable based on the range of El Niño amplitudes observed in recent history. The tropical Pacific SST anomalies imposed in the four El Niño experiments (shading), which are held constant year round. Contours show the annual mean outgoing long‐wave radiation anomalies in the experiments. Contour interval is 20 W·m−2. Dotted contours show negative OLR anomalies. We investigate the dynamical mechanisms identified in previous studies  and 85 vertical levels up to an altitude of ∼84 km   4.0) with N96 horizontal resolution , according to the functionα is a scaling factor and the other nomenclature is standard. In the four perturbation experiments, α is set to 0.58, 1.15, 1.73 and 2.30, which corresponds to Niño 3.4 index anomalies of 0.75, 1.5, 2.25 and 3.0 K, respectively =αfunctionδT=αThe imposed SST anomalies are held constant throughout the 54 years of each experiment. This is unrealistic because observed El Niños evolve over the seasonal cycle, generally being strongest in austral summer and weakest in austral winter. The 3.0 K anomaly is around twice the amplitude of the strongest Niño 3.4 anomaly observed in JJA over the period 1870–2018, but is comparable to the strongest event observed in DJF  can be defined asζ is the absolute vorticity, D is the divergence of the horizontal wind, vx is the zonal component of the divergent wind and vy is the meridional component of the divergent wind. The first term (ζD) represents the rate of change of vorticity due to vortex stretching and the second (vx∇ζx) and third (vy∇ζy) terms represent the rate of change of vorticity due to vorticity advection by the zonal and meridional components of the divergent wind, respectively. We use the RWS to identify where El Niño forcing alters the wave source regions that are important for teleconnections to high latitudes.Following Sardeshmukh and Hoskins , the Ros2.2.2et al.,ω is the frequency, u is the zonal wind, β* is the meridional gradient of absolute vorticity, K is the total wavenumber (K=l2+k2), k is the zonal wavenumber and l is the meridional wavenumber. Note that the meridional gradient of absolute vorticity (β*) can be expressed as β∗=β−uy, where β is the meridional gradient of planetary vorticity and uyy is the second derivative of the zonal wind with respect to latitude.Rossby wave ray tracing provides insights to how the background atmospheric state impacts the propagation of a linear Rossby wave . The group velocity of the waves isK=l2+k2 and Equation tan−1(dy/dx)) can be computed for any given k. Following Li et al. . In other cases, the ray will propagate in the direction given by Equation The rays are initialized within a 40° et al.  is 3.0 ± 0.7 hPa, 6.1 ± 0.8 hPa, 8.6 ± 0.7 hPa and 7.0 ± 0.7 hPa for the 0.75 K, 1.5 K, 2.25 K, 3.0 K El Niño experiments, respectively. The amplitude of the peak SLP anomaly in the ASR therefore increases by ∼3 hPa per 0.75 K for El Niño amplitudes up to 2.25 K. However, in the 3.0 K El Niño experiment, the peak SLP anomaly decreases by ∼1.6 hPa compared to the 2.25 K case despite the larger SST forcing. Observation‐based data have a much sparser sampling of El Niño magnitudes, which makes it difficult to extract a robust signal of how the teleconnection varies with forcing amplitude. Nevertheless, across all years since 1870, a seasonal variation in the relationship between the ASR‐averaged SLP anomaly and the Niño 3.4 anomaly is evident, with the magnitude being ∼60% larger in JJA than in DJF  SLP. The SLP patterns corresponding to EOFs 1–3 are shown in Figure Given the pronounced differences in the pattern of SLP response to El Niño between austral winter and summer seasons, Figure In JJA, the strongest projection across all El Niño amplitudes is onto EOF2 , but the response becomes sub‐linear for stronger forcing (>2.25 K in JJA). This is manifested as changes in the projection of the extratropical SLP response onto the PSA (EOF2) and SPW (EOF3) patterns in particular. In contrast, the amplitude of the ASR SLP anomalies in austral summer (DJF) increases approximately linearly over the range of El Niño amplitudes considered , changes to β* will be determined by uyy. The background upper tropospheric zonal wind is therefore important for the poleward propagation of Rossby waves emanating from the Tropics , regions where uyy ≥ 2.7 m−1 s−1 will be associated with Rossby wave reflection, while rays will propagate through regions where uyy< 2.7 m−1 s−1. Note that this threshold is approximate as it ignores the contribution from changes in β to β* and thus does not account for the small latitudinal variations in the location of the wave reflection surface. Nevertheless, the propagation and reflection of the ray traces plotted in Figure Figure uyy value between 30°S and 45°S as a function of longitude in the control and four El Niño experiments. The horizontal dotted lines show the most eastward location where uyy drops below the estimated threshold to permit wave propagation; this is at ∼215°E, 230°E, 240°E, 250°E and 265°E for the control and four El Niño experiments, respectively. Hence the eastward extent of the region in the South Pacific where Rossby wave reflection occurs increases with El Niño amplitude. Of course, a wave reflection zone ending at a certain point does not mean that all Rossby waves will be blocked at that longitude. This is because, as seen in the ray traces in Figure Figure uyy< 2.7 m−1 s−1, and the longitude of the peak 200 hPa RWS anomaly (blue line) for the different El Niño experiments. The former shows a larger relative change than the latter as El Niño amplitude increases. The anomalous RWS is predominantly a consequence of the shifting upper‐level divergence  varies as a function of El Niño amplitude. We analyse idealized experiments performed with the HadGEM3‐A climate model which impose an East Pacific El Niño SST anomaly corresponding to a Niño 3.4 anomaly of 0.75, 1.5, 2.25 and 3 K. In austral summer (DJF), the teleconnection to the ASR, as measured by the local SLP anomaly, is found to be approximately linear for all El Niño amplitudes explored and projects onto a negative phase of the SAM. In contrast, in austral winter (JJA) the teleconnection behaves approximately linearly for El Niño amplitudes up to 2.25 K, but for a stronger El Niño forcing of 3 K the teleconnection in the ASR weakens despite the larger SST forcing. This is associated with a switch from a negative to positive projection of the SLP response onto the South Pacific wave pattern (EOF3) and a relatively smaller increase in the projection onto PSA (EOF2). To investigate the causes of this behaviour, the changes in Rossby wave source and Rossby wave propagation under the different El Niño conditions were analysed. This revealed that the region of strongest RWS in the South Pacific moves progressively eastward with increasing El Niño amplitude, causing an eastward shift in the Rossby wave train and a change in the sign of the SLP anomalies in the Ross Sea. However, the wave reflection surface associated with curvature in the upper tropospheric zonal winds also moves eastward but at a faster rate. This means that for stronger El Niños the relative propensity for wave propagation to high latitudes is diminished and the teleconnection actually weakens despite the El Niño forcing being larger. We note that the amplitude at which this effect occurs in our experiments is larger than observed El Niños in austral winter and thus, for the purposes of present‐day observation‐based studies, a linear approximation for the El Niño teleconnection in both austral winter and summer is likely to be reasonable.Figure S1: Box plots of seasonal Nino 3.4 index from HadISST1 for 1870–2018. Data have been detrended.Figure S2: Scatterplot of SLP anomalies in the Amundsen Sea region against Nino 3.4 index for (a) DJF and (b) JJA seasons during 1870–2018. The red line shows a linear regression with the gradient in the legend.Figure S3: (a) The sea level pressure [hPa] anomaly  for JJA as a function of El Nino amplitude. Blue line shows model simulations, green shows linear model. Error bars show ±2 standard errors.Figure S4: Residual sea level pressure [hPa] response in Southern hemisphere not explained by the projection onto EOFs 1–3 for different El Nino amplitudes (columns) in (a–d) DJF and (e–h) JJA.−1) and absolute 200 hPa wind vectors in the four El Nino experiments.Figure S5: Anomalous 200 hPa divergence  k = 2 and (bottom) k = 4. Blue shading shows regions of wave reflection and red shading shows regions of wave evanescence. The ASR is marked by the grey box.Supplementary Information.Click here for additional data file.


Rab GTPases are master regulators of membrane trafficking in eukaryotic cells. Phosphorylation of Rab GTPases was characterized in the 1990s and there have been intermittent reports of its relevance to Rab functions. Phosphorylation as a regulatory mechanism has gained prominence through the identification of Rabs as physiological substrates of leucine‐rich repeat kinase 2 (LRRK2). LRRK2 is a Ser/Thr kinase that is associated with inherited and sporadic forms of Parkinson disease. In recent years, numerous kinases and their associated signaling pathways have been identified that lead to phosphorylation of Rabs. These emerging studies suggest that serine/threonine and tyrosine phosphorylation of Rabs may be a widespread and under‐appreciated mechanism for controlling their membrane trafficking functions. Here we survey current knowledge of Rab phosphorylation and discuss models for how this post‐translational mechanism exerts control of membrane trafficking. Phosphorylation of Rab GTPases by serine/threonine and tyrosine kinases is emerging as a critical regulatory mechanism in membrane trafficking. This post‐translational modification can tune the strength of binding between Rabs and their interacting proteins. Phosphorylation can also influence the lifetime of active Rabs on subcellular compartments. We survey the current literature and discuss the various cellular processes connected with Rab phosphorylation. Over the last 30 years, there have been intermittent reports of phosphorylation without detailed mechanistic implications. However, in the last few years, phosphorylation of Rabs has gained prominence as an additional layer of regulation that controls membrane trafficking. Phosphorylation of Rabs can alter the GTP/GDP cycle of activity by modulating the activity of GAPs, GEFs, effectors, accessory proteins, as well as the intrinsic rate of GTP hydrolysis and GTP/GDP affinities. The G protein fold comprises a 6‐stranded mixed β‐sheet flanked by 5 α‐helices is conserved in Rabs. Despite a broadly conserved fold, sequence motifs termed Rab family (RabF) and Rab subfamily (RabSF) are associated with distinct Rab functions.1.1There are few documented cases of tyrosine phosphorylation of Rab GTPases Table . When ov1.2cdc2.There are widespread reports of Ser/Thr phosphorylation of Rabs dating as far back as 1991.1.2.1A conserved Ser/Thr motif in switch 2 of Rab1 and Rab3b is a target of LRRK2 (see below), although no function has yet been ascribed to this modification. Similarly, reversible phosphorylation of Rab4 at S204 in the hypervariable C‐terminal domain (HVD) has been demonstrated.1.2.2LRRK2 is a Ser/Thr kinase that is associated with inherited and sporadic forms of Parkinson disease.1.2.3Autosomal recessive mutations in the mitochondrial kinase PINK1 are associated with Parkinson disease.1.2.4Sec4p is the yeast homolog of human Rab8/13 and regulates exocytosis. Using a genomic kinase screen, the activity of polo‐like kinase Cdc5p was correlated with phosphorylation of S8 in Sec4p.1.3The sites of phosphorylation from Table The cluster of phospho‐sites immediately following the α5 helix are intriguing Figure . Rab6 in2Phosphorylation of Rab GTPases at Ser/Thr and tyrosine residues adds a dimension of regulation to their roles in membrane trafficking. In the context of infection and immunity, irreversible post‐translational modification of Rabs by intracellular pathogens has been characterized as a mechanism to subvert the host trafficking machinery. For example, the Legionnaires' disease protein DrrA targets Rab1a for AMPylation at Tyr77 in switch 2, resulting in a constitutively active form that is insensitive to cellular GAPs.The authors declare that they have no conflicts of interest.https://publons.com/publon/10.1111/tra.12765.The peer review history for this article is available at