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In spite of the low agronomic performances of Ivorian cashew orchards, Cote d'Ivoire recently became the first worldwide producer and exporter of raw cashew nuts. To maintain this ringleader position, several factors should be understood including the ecological factors supporting this success. Pollination service due to bees was recognized to impact cashew production. Therefore, determining cashew trees that are more attractive to bees can contribute to promote pollination activities. To detect these cashew trees possessing the flowers preferred by bees, two types of investigations were undertaken: (1) a socioeconomic survey in 3167 cashew hectares belonging to 381 producers and (2) an experimental detection of the bees’ floral preference. As results, 58 of the 381 interviewed producers had identified the bees’ floral preference resulting in a detection of 681 cashew trees. The flowers of the preferred cashew trees were visited 5 times more and they attracted 3 times more bee foragers as compared to non-preferred cashew trees. These findings may be included in research programs focus on: (i) improvement of agronomic performances and (ii) effective management of bees’ community.

Mutation-induced activation of WNT-β-catenin signalling is a frequent driver event in human cancer. Sustained WNT-β-catenin pathway activation endows cancer cells with sustained self-renewing growth properties and is associated with therapy resistance. In healthy adult stem cells, WNT pathway activity is carefully controlled by core pathway tumour suppressors as well as negative feedback regulators. Gene inactivation experiments in mouse models unequivocally demonstrated the relevance of WNT tumour suppressor loss-of-function mutations for cancer growth. However, in human cancer, a far more complex picture has emerged in which missense or truncating mutations mediate stable expression of mutant proteins, with distinct functional and phenotypic ramifications. Herein, we review recent advances and challenges in our understanding of how different mutational subsets of WNT tumour suppressor genes link to distinct cancer types, clinical outcomes and treatment strategies.

Cystic fibrosis (CF) is a progressive multi organ autosomal recessive disease with devastating impact on the lungs caused by derangements of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Morbidity and mortality in CF are largely caused by lung complications due to the triad of impaired mucociliary clearance, microbial infections and chronic inflammation. P. aeruginosa is a main respiratory pathogen in individuals with CF infecting most patients in later stages. Despite its recognized clinical impact, the molecular mechanisms that underlie P. aeruginosa pathogenesis and the host response to P. aeruginosa infection remain incompletely understood. The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARγ) an important modulator of inflammation and host defense, has shown to be reduced in CF airways. In this study we sought to investigate the upstream mechanisms repressing PPARγ expression and its impact on airway epithelial host defense. Endoplasmic Reticulum-stress (ER-stress) triggered unfolded protein response (UPR) activated by misfolded CFTR and P. aeruginosa infection contributed to attenuated expression of PPARγ. Specifically, the PERK signaling pathway lead to the enhanced expression of the CCAAT-enhancer-binding-protein homologous protein (CHOP). CHOP induction led to the repression of PPARγ expression. Mechanistically, we show that CHOP induction mediated PPARγ attenuation, impacted the innate immune function of normal, ΔF508 primary airway epithelial as well as bronchial epithelial cells by reducing expression of anti-microbial peptide (AMP) and paraoxanse-2 (PON-2), as well as enhancing IL-8 expression. Furthermore, mitochondrial reactive oxygen species production (mt-ROS) and ER stress positive feedforward loop also dysregulated mitochondrial bioenergetics. Additionally, our findings implicate that PPARγ agonist pioglitazone (PIO) has beneficial effect on the host at the multicellular level ranging from host defense to mitochondrial re-energization.

Intestinal organoids grown from adult stem cells have emerged as prototype 3D organotypic models for studying tissue renewal and homeostasis. Owing to their strict dependence on Wnt signaling, intestinal organoids offer an unprecedented opportunity to examine Wnt pathway regulation in normal physiology and cancer. We review how alterations in growth factor dependency and organoid morphology can be exploited to identify Wnt signaling mechanisms, characterize mutated pathway components, and predict responses of patient-derived tumors to targeted therapy. We discuss current deficits in the understanding of genotype-phenotype relationships that are to be considered when interpreting mutation-induced changes in organoid morphology.

Summary The Wnt/β-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by β-catenin’s phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein β-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human β-catenin destruction complex from purified components, recapitulating complex assembly, β-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote β-catenin capture, phosphorylation, and ubiquitylation. APC facilitates β-catenin’s flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCFβ-TrCP E3 ligase complex in a β-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes β-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/β-catenin signaling., Graphical abstract, Highlights • β-Catenin destruction complex function recapitulated with purified proteins • AXIN1 polymers and APC promote β-catenin capture, phosphorylation, and ubiquitylation • Oncogenic APC truncation mutants are hypomorphs promoting β-catenin recruitment • APC directly binds the ubiquitylation machinery, Ranes et al. reconstitute the β-catenin destruction complex from purified proteins, thereby recapitulating its assembly and biochemical activities and enabling the mechanistic interrogation of its scaffolding proteins AXIN1 and APC. AXIN1 polymerization and APC facilitate the recruitment, phosphorylation, and ubiquitylation of β-catenin. Oncogenic truncation of APC partially preserves APC function.

The intestinal epithelium harbors a remarkable adaptability to undergo injury-induced repair. A key part of the regenerative response is the transient reprogramming of epithelial cells into a fetal-like state, which drives uniform proliferation, tissue remodeling, and subsequent restoration of the homeostatic state. In this review, we discuss how Wnt and YAP signaling pathways control the intestinal repair response and the transitioning of cell states, in comparison with the process of intestinal development. Furthermore, we highlight how organoid-based applications have contributed to the characterization of the mechanistic principles and key players that guide these developmental and regenerative events.

Human Lymphatic filariasis (LF) caused by three types of filarial worms; Wuchereria bancrofti, Brugia malayi and Brugiatimori is one of the neglected tropical diseases and spread by bites of infected Anopheles, Culex, Aedes, Ochleratus andMansoni mosquitoes. The study evaluated diagnostic methods using blood samples from 420 consented participants in threeLocal Government Areas of Cross River State. Blood samples were examined following one hour of administration ofDiethylcarbamazine citrate for LF microfilaria microscopy (Knott’s concentration) and for LF antigen usingimmunochromatographic (ICT) method (Alere filariasis test strip). Of the 420 samples examined, 1.7% was found to bepositive using microscopy while 4.8% were positive using ICT. There was no significant difference in the diagnosis oflymphatic filariasis using microscopy and ICT among participants in the local government areas (χ2= 21.84, p>0.05). Of the214 males and 206 females examined, 4.2% males and 5.3% females tested positive using ICT while 1.4% males and 1.9%females were found positive using microscopy. The difference in the infection between gender was statistically significant(χ2=0.298, p0.05). The study showed that LF still remains a public health problem in Cross RiverState. Mass drug administration should be scaled up in the state so as to reduce and finally eradicate the disease. Keywords: Diagnosis, Lymphatic filariasis, Immunochromatography, Microscopy

Transition through cell cycle phases requires temporal and spatial regulation of gene expression to ensure accurate chromosome duplication and segregation. This regulation involves dynamic reprogramming of gene expression at multiple transcriptional and posttranscriptional levels. In transcriptionally silent oocytes, the CPEB-family of RNA-binding proteins coordinates temporal and spatial translation regulation of stored maternal mRNAs to drive meiotic progression. CPEB1 mediates mRNA localization to the meiotic spindle, which is required to ensure proper chromosome segregation. Temporal translational regulation also takes place in mitosis, where a large repertoire of transcripts is activated or repressed in specific cell cycle phases. However, whether control of localized translation at the spindle is required for mitosis is unclear, as mitotic and acentriolar-meiotic spindles are functionally and structurally different. Furthermore, the large differences in scale-ratio between cell volume and spindle size in oocytes compared to somatic mitotic cells may generate distinct requirements for gene expression compartmentalization in meiosis and mitosis. Here we show that mitotic spindles contain CPE-localized mRNAs and translating ribosomes. Moreover, CPEB1 and CPEB4 localize in the spindles and they may function sequentially in promoting mitotic stage transitions and correct chromosome segregation. Thus, CPEB1 and CPEB4 bind to specific spindle-associated transcripts controlling the expression and/or localization of their encoded factors that, respectively, drive metaphase and anaphase/cytokinesis.

R-spondins (RSPOs) amplify WNT signaling during development and regenerative responses. We previously demonstrated that RSPOs 2 and 3 potentiate WNT/β-catenin signaling in cells lacking leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4, 5 and 6 (Lebensohn and Rohatgi, 2018). We now show that heparan sulfate proteoglycans (HSPGs) act as alternative co-receptors for RSPO3 using a combination of ligand mutagenesis and ligand engineering. Mutations in RSPO3 residues predicted to contact HSPGs impair its signaling capacity. Conversely, the HSPG-binding domains of RSPO3 can be entirely replaced with an antibody that recognizes heparan sulfate (HS) chains attached to multiple HSPGs without diminishing WNT-potentiating activity in cultured cells and intestinal organoids. A genome-wide screen for mediators of RSPO3 signaling in cells lacking LGRs 4, 5 and 6 failed to reveal other receptors. We conclude that HSPGs are RSPO co-receptors that potentiate WNT signaling in the presence and absence of LGRs.

Pseudomonas aeruginosa is a major health challenge that causes recalcitrant multidrug-resistant infections, especially in immunocompromised and hospitalized patients. P. aeruginosa is an important cause of nosocomial and ventilator-associated pneumonia characterized by high prevalence and fatality rates. P. aeruginosa also causes chronic lung infections in individuals with cystic fibrosis. Multidrug- and totally drug-resistant strains of P. aeruginosa are increasing threats that contribute to high mortality in these patients. The pathogenesis of many P. aeruginosa infections depends on its ability to form biofilms, structured bacterial communities that can coat mucosal surfaces or invasive devices. These biofilms make conditions more favorable for bacterial persistence, as embedded bacteria are inherently more difficult to eradicate than planktonic bacteria. The molecular mechanisms that underlie P. aeruginosa biofilm pathogenesis and the host response to P. aeruginosa biofilms remain to be fully defined. However, it is known that biofilms offer protection from the host immune response and are also extremely recalcitrant to antimicrobial therapy. Therefore, development of novel therapeutic strategies specifically aimed at biofilms is urgently needed. Here, we review the host response, key clinical implications of P. aeruginosa biofilms, and novel therapeutic approaches to treat biofilms relevant to lung infections. Greater understanding of P. aeruginosa biofilms will elucidate novel avenues to improve outcomes for P. aeruginosa pulmonary infections.

Pseudomonas aeruginosa is an important opportunistic pathogen causing a variety of acute infections including nosocomial pneumonias, sepsis, urinary tract infections, keratitis, wound and skin infections. P. aeruginosa continues to be a leading cause of infections in immunocompromised host including patients with cystic fibrosis and is among the most virulent of the opportunistic pathogens as listed by the Centers of Disease Control (CDC). P. aeruginosa has also developed mechanisms to colonize surfaces by coordinately expressing genes in a density dependent manner regulated by the production of small diffusible molecules called auto inducers or quorum sensing (QS) molecules. Activation of the QS cascade promotes formation of biofilms which provide an encapsulated communal structure that coats mucosal surfaces and invasive devices. These biofilms make conditions more favorable for bacterial persistence as embedded bacteria are inherently more difficult to eradicate by both antibiotic regimens as well as by innate immune systems as compared with those in the planktonic state. The objective of this report is to provide an overview; (i) propagation of P. aeruginosa biofilms; (ii) components of the biofilm matrix and their transcriptional regulation; (iii) key signaling pathways regulating C-di-GMP dependent biofilm dispersal; (iv) characterization of experimental models of biofilms.

We report solid-state Nuclear Magnetic Resonance (ssNMR) studies on amyloid-like protein complexes formed by DIX domains that mediate key protein interactions in the Wnt signalling pathway. Our results provide insight into the 3D fold of the self-associated Axin-DIX domain and identify a potential lipid cofactor.

The β-catenin destruction complex is a dynamic cytosolic multiprotein assembly that provides a key node in Wnt signalling regulation. The core components of the destruction complex comprise the scaffold proteins axin and adenomatous polyposis coli and the Ser/Thr kinases casein kinase 1 and glycogen synthase kinase 3. In unstimulated cells, the destruction complex efficiently drives degradation of the transcriptional coactivator β-catenin, thereby preventing the activation of the Wnt/β-catenin pathway. Mutational inactivation of the destruction complex is a major pathway in the pathogenesis of cancer. Here, we review recent insights in the regulation of the β-catenin destruction complex, including newly identified interaction interfaces, regulatory elements and post-translationally controlled mechanisms. In addition, we discuss how mutations in core destruction complex components deregulate Wnt signalling via distinct mechanisms and how these findings open up potential therapeutic approaches to restore destruction complex activity in cancer cells. Linked Articles This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc

The intestinal stem cell (ISC) marker LGR5 is a receptor for R‐spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self‐renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis, regeneration and cancer, little is known about the post‐translational regulation of LGR5. Here, we show that the HECT‐domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation, increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high‐grade adenomas. Mechanistically, we find that both NEDD4 and NEDD4L negatively regulate Wnt/β‐catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post‐translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers, which subsequently enhances tumour predisposition and progression., NEDD4 and NEDD4L E3 ligases control LGR5 receptor turnover in intestinal homeostasis and colorectal cancer.

Wnt/β‐catenin signaling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt‐hypersensitive tumors that are susceptible to anti‐Wnt‐based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that induce β‐catenin‐mediated transcription, despite exhibiting retained Wnt receptor downregulation. These mutations interfere with a ubiquitin‐independent suppressor role of the RNF43 cytosolic tail that involves Casein kinase 1 (CK1) binding and phosphorylation. Mechanistically, truncated RNF43 variants trap CK1 at the plasma membrane, thereby preventing β‐catenin turnover and propelling ligand‐independent target gene transcription. Gene editing of human colon stem cells shows that RNF43 truncations cooperate with p53 loss to drive a niche‐independent program for self‐renewal and proliferation. Moreover, these RNF43 variants confer decreased sensitivity to anti‐Wnt‐based therapy. Our data demonstrate the relevance of studying patient‐derived mutations for understanding disease mechanisms and improved applications of precision medicine., C‐terminal shortening of RNF43 promotes Wnt/β‐catenin by decreasing destruction complex activity without effects on its ubiquitin ligase activity.

The culturing of mini-organs (organoids) in three-dimensions (3D) presents a simple and powerful tool to investigate the principles underlying human organ development and tissue self-organization in both healthy and diseased states. Applications of single molecule analysis are highly informative for a comprehensive understanding of the complexity underlying tissue and organ physiology. To fully exploit the potential of single molecule technologies, the adjustment of protocols and tools to 3D tissue culture is required. Single molecule RNA fluorescence in situ hybridization (smFISH) is a robust technique for visualizing and quantifying individual transcripts. In addition, smFISH can be employed to study splice variants, fusion transcripts as well as transcripts of multiple genes at the same time. Here, we develop a 3-day protocol and validation method to perform smFISH in 3D in whole human organoids. We provide a number of applications to exemplify the diverse possibilities for the simultaneous detection of distinct mRNA transcripts, evaluation of their spatial distribution and the identification of divergent cell lineages in 3D in organoids., Summary: We present a sensitive single-molecule (sm)FISH application for the identification of individual mRNA transcripts and their spatial distribution in 3D in whole human colon organoids.

The pathogenicity of P. aeruginosa is dependent on quorum sensing (QS), an inter-bacterial communication system that can also modulate host biology. The innate immune function of the lung mucosal barrier is dependent on proper mitochondrial function. The purpose of this study was to define the mechanism by which bacterial factors modulate host lung epithelial cell mitochondrial function and to investigate novel therapies that ameliorate this effect. 3-oxo-C12-HSL disrupts mitochondrial morphology, attenuates mitochondrial bioenergetics, and induces mitochondrial DNA oxidative injury. Mechanistically, we show that 3-oxo-C12-HSL attenuates expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, antioxidant defense, and cellular respiration, and its downstream effectors in both BEAS-2B and primary lung epithelial cells. Overexpression of PGC-1α attenuates the inhibition in cellular respiration caused by 3-oxo-C12-HSL. Pharmacologic activation of PGC-1α restores barrier integrity in cells treated with 3-oxo-C12-HSL. These data demonstrate that the P. aeruginosa QS molecule, 3-oxo-C12-HSL, alters mitochondrial pathways critical for lung mucosal immunity. Genetic and pharmacologic strategies that activate the PGC-1α pathway enhance host epithelial cell mitochondrial function and improve the epithelial innate response to P. aeruginosa. Therapies that rescue PGC-1α function may provide a complementary approach in the treatment of P. aeruginosa infection.

Mammalian Wnt proteins are believed to act as short-range signals(1-4), yet have not been previously visualized in vivo. Selfrenewal, proliferation and differentiation are coordinated along a putative Wnt gradient in the intestinal crypt(5). Wnt3 is produced specifically by Paneth cells(6,7). Here we have generated an epitopetagged, functional Wnt3 knock-in allele. Wnt3 covers basolateral membranes of neighbouring stem cells. In intestinal organoids, Wnt3-transfer involves direct contact between Paneth cells and stem cells. Plasma membrane localization requires surface expression of Frizzled receptors, which in turn is regulated by the transmembrane E3 ligases Rnf43/Znrf3 and their antagonists Lgr4-5/R-spondin. By manipulating Wnt3 secretion and by arresting stem-cell proliferation, we demonstrate that Wnt3 mainly travels away from its source in a cell-bound manner through cell division, and not through diffusion. We conclude that stem-cell membranes constitute a reservoir for Wnt proteins, while Frizzled receptor turnover and ` plasma membrane dilution' through cell division shape the epithelial Wnt3 gradient.

Ants constitute an important part of arboreal arthropod biomass in rainforests. Nevertheless, there are only a few methods which permit a rapid assessment of these insects in the canopy layer. This study aims at evaluating the efficiency of a new variant type of pitfall trap i.e. “the funnel trap”, to sample arboreal ants in a secondary and gallery forest in Lamto reserve (Côte d’Ivoire). This method was compared to standard arboreal pitfall trap and beating. In total, the 3 methods yielded 7072 ant workers belonging to 43 species, 14 genera and 5 subfamilies. Tree beating recorded the highest ant’s numerical abundance (3670 workers), with 27 species, 12 genera and 3 subfamilies followed by the “funnel trap” that yielded 2800 ant workers, with 23 species belonging to 12 genera and 5 subfamilies. Finally, arboreal pitfall traps caught the lowest individual with 602 ant workers from 20 species belonging to 9 genera and 3 subfamilies. The composition of species which are caught by arboreal pitfall trap and “funnel trap” was similar at 53 percent. Tree beating showed a distinct species composition compared to arboreal pitfall trap and “funnel trap”. The “funnel trap” could be a fast and efficient way to quickly assess ant-biodiversity in forest canopies and agroecosystems as it looks like a non-destructive sampling method.

Differential WNT and Notch signaling regulates differentiation of Lgr5+ crypt-based columnar cells (CBCs) into intestinal cell lineages. Recently we showed that mitochondrial activity supports CBCs, while adjacent Paneth cells (PCs) show reduced mitochondrial activity. This implies that CBC differentiation into PCs involves a metabolic transition toward downregulation of mitochondrial dependency. Here we show that Forkhead box O (FoxO) transcription factors and Notch signaling interact in determining CBC fate. In agreement with the organoid data, Foxo1/3/4 deletion in mouse intestine induces secretory cell differentiation. Importantly, we show that FOXO and Notch signaling converge on regulation of mitochondrial fission, which in turn provokes stem cell differentiation into goblet cells and PCs. Finally, scRNA-seq-based reconstruction of CBC differentiation trajectories supports the role of FOXO, Notch, and mitochondria in secretory differentiation. Together, this points at a new signaling-metabolic axis in CBC differentiation and highlights the importance of mitochondria in determining stem cell fate.

An important issue in biodiversity conservation is the early detection of introduced and invasive organisms and identification of regions that may either be points of introduction or be particularly prone to invasion. Among insects, ants are easily transported worldwide by humans, and several species, such as Solenopsis geminata or Pheidole megacephala, can be highly invasive. To establish an initial baseline for urban ant monitoring in Côte d’Ivoire (Western Africa), fifteen cities - equally distributed over the three main ecoregions of the country - were surveyed using tuna baits as sampling method. A total of 103 ant species from 22 genera and 5 subfamilies were collected. We identified 10 potentially invasive species, amongst them two new species for the country: Solenopsis geminata and Solenopsis globularia. Another highly invasive species, Paratrechina longicornis, was the most abundant species collected throughout the country. Potentially invasive ant species co-occurred with non-invasive species, and contributed importantly to the structure of ant assemblages of all sampled urban habitats. The present study provides evidence that introduced and invasive ant species are established in cities of Côte d’Ivoire, often in high numbers and are occurring over large areas of invasion.

Blepharocheilodontic syndrome (BCDS) consists of lagophthalmia, ectropion of the lower eyelids, distichiasis, euryblepharon, cleft lip/palate and dental anomalies and has autosomal dominant inheritance with variable expression. We identified heterozygous variants in two genes of the cadherin-catenin complex, CDH1, encoding E-cadherin, and CTNND1, encoding p120 catenin delta1 in 15 of 17 BCDS index patients, as was recently described in a different publication. CDH1 plays an essential role in epithelial cell adherence; CTNND1 binds to CDH1 and controls the stability of the complex. Functional experiments in zebrafish and human cells showed that the CDH1 variants impair the cell adhesion function of the cadherin-catenin complex in a dominant-negative manner. Variants in CDH1 have been linked to familial hereditary diffuse gastric cancer and invasive lobular breast cancer; however, no cases of gastric or breast cancer have been reported in our BCDS cases. Functional experiments reported here indicated the BCDS variants comprise a distinct class of CDH1 variants. Altogether, we identified the genetic cause of BCDS enabling DNA diagnostics and counseling, in addition we describe a novel class of dominant negative CDH1 variants.

Deletion or mutation of the gene encoding the deubiquitinating enzyme CYLD is a common genomic aberration in multiple myeloma (MM). However, the functional consequence of CYLD loss and the mechanism underlying its putative role as a tumor suppressor gene in the pathogenesis of MM has not been established. Here, we show that CYLD expression is highly variable in myeloma cell lines and primary MMs and that low CYLD expression is associated with disease progression from monoclonal gammopathy of undetermined significance to MM, and with poor overall and progression free-survival of MM patients. Functional assays revealed that CYLD represses MM cell proliferation and survival. Furthermore, CYLD acts as a negative regulator of NF-kappa B and Wnt/beta-catenin signaling and loss of CYLD sensitizes MM cells to NF-kappa B-stimuli and Wnt ligands. Interestingly, in primary MMs, low CYLD expression strongly correlated with a proliferative and Wnt signaling-gene expression signature, but not with an NF kappa B target gene signature. Altogether, our findings identify CYLD as a negative regulator of NF-kappa B and Wnt/beta-catenin signaling in MM and indicate that loss of CYLD enhances MM aggressiveness through Wnt pathway activation. Thus, targeting the Wnt pathway could be a promising therapeutic strategy in MM with loss of CYLD activity

Urban ecosystems house artificial infrastructures such as ports, transport stations and commercial centres which are often introduction foci for exotic species. The connectivity of these structures to other centres specialised in commodity flows located in urban cores, enhances the accidental transport and spreading of exotic species which can become invasive. Despite this fact, targeted investigations to detect establishment of invasive species in tropical urban landscapes are lacking. A survey conducted on ant communities in 15 cities throughout Cote d'Ivoire (West Africa) allowed us to find the highly invasive tropical fire ant (TFA) Solenopsis geminata for the first time in the country. DNA analysis of mitochondrial cytochrome c oxidase I (COI) of two specimens of S. geminata found that they had a close genetic relationship with specimens from Venezuela. From the 15 towns surveyed throughout Cote d'Ivoire, this ant was discovered only in industrial areas of Abidjan near the port zone suggesting an early stage of invasion.

The mechanism by which Wnt receptors transduce signals to activate downstream beta-catenin-mediated target gene transcription remains incompletely understood but involves Frizzled (Fz) receptor-mediated plasma membrane recruitment and activation of the cytoplasmic effector Dishevelled (Dvl). Here, we identify the deubiquitinating enzyme CYLD, the familial cylindromatosis tumor suppressor gene, as a negative regulator of proximal events in Wnt/beta-catenin signaling. Depletion of CYLD from cultured cells markedly enhances Wnt-induced accumulation of beta-catenin and target gene activation. Moreover, we demonstrate hyperactive Wnt signaling in human cylindroma skin tumors that arise from mutations in CYLD. At the molecular level, CYLD interacts with and regulates K63-linked ubiquitination of Dvl. Enhanced ubiquitination of the polymerization-prone DIX domain in CYLD-deficient cells positively links to the signaling activity of Dvl. Together, our results argue that loss of CYLD instigates tumor growth in human cylindromatosis through a mechanism in which hyperubiquitination of polymerized Dvl drives enhancement of Wnt responses.

Rad18 functions at the cross-roads of three different DNA damage response (DDR) pathways involved in protecting stressed replication forks: homologous recombination repair, DNA inter-strand cross-link repair and DNA damage tolerance. Although Rad18 serves to facilitate replication of damaged genomes by promoting translesion synthesis (TLS), this comes at a cost of potentially error-prone lesion bypass. In contrast, loss of Rad18-dependent TLS potentiates the collapse of stalled forks and leads to incomplete genome replication. Given the pivotal nature with which Rad18 governs the fine balance between replication fidelity and genome stability, Rad18 levels and activity have a major impact on genomic integrity. Here, we identify the de-ubiquitylating enzyme USP7 as a critical regulator of Rad18 protein levels. Loss of USP7 destabilizes Rad18 and compromises UV-induced PCNA mono-ubiquitylation and Pol η recruitment to stalled replication forks. USP7-depleted cells also fail to elongate nascent daughter strand DNA following UV irradiation and show reduced DNA damage tolerance. We demonstrate that USP7 associates with Rad18 directly via a consensus USP7-binding motif and can disassemble Rad18-dependent poly-ubiquitin chains both in vitro and in vivo. Taken together, these observations identify USP7 as a novel component of the cellular DDR involved in preserving the genome stability.

Degradation of cytosolic beta-catenin by the APC/Axin1 destruction complex represents the key regulated step of the Wnt pathway. It is incompletely understood how the Axin1 complex exerts its Wnt-regulated function. Here, we examine the mechanism of Wnt signaling under endogenous levels of the Axin1 complex. Our results demonstrate that beta-catenin is not only phosphorylated inside the Axin1 complex, but also ubiquinated and degraded via the proteasome, all within an intact Axin1 complex. In disagreement with current views, we find neither a disassembly of the complex nor an inhibition of phosphorylation of Axin1-bound beta-catenin upon Wnt signaling. Similar observations are made in primary intestinal epithelium and in colorectal cancer cell lines carrying activating Wnt pathway mutations. Wnt signaling suppresses beta-catenin ubiquitination normally occurring within the complex, leading to complex saturation by accumulated phospho-beta-catenin. Subsequently, newly synthesized beta-catenin can accumulate in a free cytosolic form and engage nuclear TCF transcription factors.

Wnt signaling mediates key developmental and homeostatic processes including stem cell maintenance, growth and cell fate specification, cell polarity and migration. Inappropriate activation of Wnt signaling is linked to a range of human disorders, most notably cancer and neurodegenerative diseases. In the Wnt/β-catenin cascade, signaling events converge on the regulation of ubiquitin-mediated degradation of the crucial transcriptional regulator β-catenin. The emerging mechanisms by which ubiquitin modification of proteins controls cellular pathways comprise both proteolytic and nonproteolytic functions. In nonproteolytic functions, ubiquitin acts as a signaling device in the control of protein activity, subcellular localization and complex formation. Here, we review and discuss recent developments that implicate ubiquitin-mediated mechanisms at multiple steps of Wnt pathway activation.

Summary Organoid technology provides the possibility of culturing patient-derived colon tissue and colorectal cancers (CRCs) while maintaining all functional and phenotypic characteristics. Labeling stem cells, especially in normal and benign tumor organoids of human colon, is challenging and therefore limits maximal exploitation of organoid libraries for human stem cell research. Here, we developed STAR (stem cell Ascl2 reporter), a minimal enhancer/promoter element that reports transcriptional activity of ASCL2, a master regulator of LGR5+ intestinal stem cells. Using lentiviral infection, STAR drives specific expression in stem cells of normal organoids and in multiple engineered and patient-derived CRC organoids of different genetic makeup. STAR reveals that differentiation hierarchies and the potential for cell fate plasticity are present at all stages of human CRC development. Organoid technology, in combination with the user-friendly nature of STAR, will facilitate basic research into human adult stem cell biology., Graphical Abstract, Highlights • ASCL2-responsive minigene (STAR) is specific for intestinal stem cells • STAR is compatible with user-friendly techniques like lentiviral infections • STAR enables stem cell labeling in normal and in cancer organoids of human colon • Cellular plasticity is present at all stages of colorectal cancer development, Oost et al. present an ASCL2-responsive minigene (STAR) that enables stem cell labeling in patient-derived colorectal cancer organoids, as well as in normal and benign colorectal tumor samples. The user-friendly nature of STAR applications in combination with organoid technology will facilitate basic research into human adult stem cell biology.

Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells in the bone marrow (BM). Most MMs display aberrant Wnt/β-catenin signaling, which drives proliferation; however, they lack oncogenic Wnt-pathway mutations, suggesting activation by autocrine Wnt ligands and/or paracrine Wnts from the BM microenvironment. Expression of the heparan sulfate proteoglycan (HSPG) syndecan-1 is a hallmark of MM. Syndecan-1 is a critical player in the complex reciprocal interaction between MM cells and their BM niche, mediating growth factor/cytokine binding and signaling by its heparan sulfate (HS) chains. Here, by means of CRISPR/Cas9-mediated knockout and doxycycline-inducible shRNA-mediated knockdown of EXT1, a critical enzyme for HS polymerization, we demonstrate that the HS-chains decorating syndecan-1 mediate aberrant Wnt-pathway activation in MM. HS-deficient MM cells exhibited a strongly decreased autocrine Wnt/β-catenin pathway activity and a reduced Wnt-pathway-dependent proliferation. In addition, we demonstrate that Wnts bind to the HS side-chains of syndecan-1 and that this binding contributes to paracrine Wnt-pathway activation through the Wnt-receptor Frizzled. Furthermore, in a HS-dependent fashion, syndecan-1 also binds osteoblast-produced R-spondin, which represses Frizzled degradation by activation of LGR4, an R-spondin receptor aberrantly expressed on MM cells. Co-stimulation with R-spondin, and its binding to HS chains decorating syndecan-1, are indispensable for optimal stimulation of Wnt-signaling in MM. Taken together, our results identify syndecan-1 as a crucial component of the Wnt-signalosome in MM cells, binding Wnts and R-spondins to promote aberrant Wnt/β-catenin signaling and cell growth, and suggest HS and its biosynthetic enzymes as potential targets for the treatment of MM.

SummaryThe human Lkb1 kinase, encoded by the ortholog of the invertebrate Par4 polarity gene, is mutated in Peutz-Jeghers cancer syndrome. Lkb1 activity requires complex formation with the pseudokinase Strad and the adaptor protein Mo25. The complex can induce complete polarization in a single isolated intestinal epithelial cell. We describe an interaction between Mo25α and a human serine/threonine kinase termed Mst4. A homologous interaction occurs in the yeast Schizosaccharomyces pombe in the control of polar tip growth. Human Mst4 translocates from the Golgi to the subapical membrane compartment upon activation of Lkb1. Inhibition of Mst4 activity inhibits Lkb1-induced brush border formation, whereas other aspects of polarity such as the formation of lateral junctions remain unaffected. As an essential event in brush border formation, Mst4 phosphorylates the regulatory T567 residue of Ezrin. These data define a brush border induction pathway downstream of the Lkb1/Strad/Mo25 polarization complex, yet separate from other polarity events.

Two lipids (palmitate and palmitoleic acid) are appended onto Wnt proteins. It has been suggested that palmitate is required for signalling, whereas palmitoleic acid is necessary for progression through the secretory pathway. By mutating the relevant amino acids, we have investigated how these adducts contribute to the secretion and signalling activity of Wingless, the main Drosophila member of the Wnt family. Analysis of Wingless with a Cysteine 93 to Alanine mutation ([C93A]Wingless) shows that palmitoylation is essential for signalling activity in vivo (as well as in cultured cells). Moreover, without palmitate, Wingless fails to reach the surface of imaginal disc cells and, as electron microscopy (EM) analysis suggests, appears to accumulate in the endoplasmic reticulum (ER). Artificial targeting of palmitate-deficient Wingless to the plasma membrane does not rescue signalling activity. Therefore, palmitate at C93 has a dual role: in secretion and signalling. From our analysis of [S239A]Wingless, which lacks a conserved residue shown to be acylated in Wnt3a, we infer that palmitoleic acid is not, as previously suggested, absolutely required for secretion. Nevertheless, this mutant has poor signalling activity, suggesting that palmitoleic acid contributes significantly to signalling. We suggest that the overall level of lipidation affects signalling activity.

We studied 44 cases of Hodgkin's disease for the presence of Epstein-Barr virus (EBV) DNA, its localization and the expression of the EBV receptor on the tumour cells. EBV DNA was found in 52% (16/31) of the Hodgkin's lymphomas using the polymerase chain reaction. With a very sensitive non-radioactive DNA in situ hybridization technique in combination with immunohistochemistry for CD 30 or CD 15 antigens, EBV DNA was localized to Reed-Sternberg cells and its mononuclear variants. The relationship between the presence of EBV DNA and the expression of the EBV-receptor CR2 (CD 21) on Reed-Sternberg cells was studied using the same techniques and two different monoclonal anti-CD 21 antibodies. CR2 could be detected on a substantial number of the Reed-Sternberg cells in EBV DNA positive Hodgkin's lymphomas (9/12; 75%), whereas in EBV negative cases positivity with anti-CD 21 was rare (1/13; 8%). The results indicate that CR2 expression on Reed-Sternberg cells and the presence of EBV DNA sequences are frequently associated in Hodgkin's lymphomas.

Neuroblastoma (NB) is the most common solid malignancy in childhood and its prognosis is still generally poor. In contrast to many other cancers, mutations of the p53 tumor suppressor are rare. Instead, significant cytosolic sequestration of wtp53 is one of several mechanisms that attenuate p53 function in this cancer. Here, we report that aberrant p53 hyperubiquitylation contributes to p53 cytoplasmic sequestration in NB. NB lines constitutively harbor an elevated portion of wtp53 as stable ubiquitylated species confined to the cytoplasm. p53 hyperubiquitylation is not due to dysregulation by Hdm2 or proteasomal dysfunction. Instead, the defect lies in p53 regulation by HAUSP, a major p53-deubiquitylating enzyme. In contrast to non-NB cancer cells with nuclear p53 and normal ubiquitylation, p53 from NB cells shows impaired HAUSP interaction. Conversely, interference with p53 hyperubiquitylation in NB cells by Nutlin 3a or by a C-terminal p53 peptide (aa 305-393) results in p53 relocalization from the cytoplasm to the nucleus, and in case of Nutlin, in reactivation of p53's transcriptional and apoptotic functions. Moreover, nutlin and camptothecin act synergistically in inducing NB cell apoptosis. Hence, this study strengthens the rationale for targeting p53 deubiquitylation by drugs like Nutlin as a promising new strategy in NB therapy.

FOXO (Forkhead box O) transcription factors are important regulators of cellular metabolism, cell-cycle progression and cell death. FOXO activity is regulated by multiple post-translational modifications, including phosphorylation, acetylation and polyubiquitination. Here, we show that FOXO becomes monoubiquitinated in response to increased cellular oxidative stress, resulting in its re-localization to the nucleus and an increase in its transcriptional activity. Deubiquitination of FOXO requires the deubiquitinating enzyme USP7/HAUSP (herpesvirus-associated ubiquitin-specific protease), which interacts with and deubiquitinates FOXO in response to oxidative stress. Oxidative stress-induced ubiquitination and deubiquitination by USP7 do not influence FOXO protein half-life. However, USP7 does negatively regulate FOXO transcriptional activity towards endogenous promoters. Our results demonstrate a novel mechanism of FOXO regulation and indicate that USP7 has an important role in regulating FOXO-mediated stress responses.

Desmosterol is an immediate precursor of cholesterol in the Bloch pathway of sterol synthesis and an abundant membrane lipid in specific cell types. The significance of the difference between the two sterols, an additional double bond at position C24 in the tail of desmosterol, is not known. Here, we provide evidence that the biophysical and functional characteristics of the two sterols differ and that this is because the double bond at C24 significantly weakens the sterol ordering potential. In model membranes, desmosterol was significantly weaker than cholesterol in promoting the formation or stability of ordered domains, and in mammalian cell membranes, desmosterol associated less avidly than cholesterol with detergent-resistant membranes. Atomic scale molecular dynamics simulations showed that the double bond gives rise to additional stress in the tail, creating a rigid structure between C24 and C27 and favoring tilting of desmosterol distinct from cholesterol. Functional effects of desmosterol in cell membranes were assessed upon acutely exchanging ∼70% of cholesterol to desmosterol. This led to impaired raft-dependent signaling via the insulin receptor, whereas non-raft-dependent protein secretion was not affected. We suggest that the choice of cholesterol synthesis route may provide a physiological mechanism to modulate raft-dependent functions in cells.

A novel microscope system is presented for observation of corneal cells in a living mouse. It enables tracking of individual cells in all layers of the cornea at various times, thus allowing the generation of time-lapse recordings. The system consists of three major components: an upright fluorescence microscope for visualization of corneal cells, a mouse-holding unit for immobilization of the animal and the eye, and a set of gimbals which permit observation of a wide area of corneal surface without refocusing. The same cells could be observed at different limes with the help of fiducial marks in the cornea, allowing their changes in position to be determined under natural and experimental conditions. This technique should prove useful in investigation of the cell movement in normal and diseased corneas, including the study of wound healing after an injury or surgery.

Programmed cell death (apoptosis) is crucial for thymocyte development. We analyzed the role of the ubiquitin (Ub)–proteasome pathway in dexamethasone-triggered and TCR-mediated apoptosis in fetal thymic organ culture (FTOC). Proteasome activity was increased in apoptotic thymocytes, as visualized by active-site labeling of proteasomal β subunits. The activity of deubiquitinating enzymes in murine apoptotic thymocytes was likewise examined by active-site labeling. We show that the deubiquitinating enzyme USP7 (HAUSP) is proteolytically processed upon dexamethasone-, γ-irradiation-, and antigen-induced cell death. Such processing of HAUSP does not occur in caspase 3 −/− thymocytes, or upon pretreatment of wild type thymocytes with the general caspase inhibitor ZVAD-fmk. Thus, our results suggest that thymocyte apoptosis leads to modification of deubiquitinating enzymes by caspase activity and may provide an additional link between the ubiquitin–proteasome pathway and the caspase cascade during programmed cell death.

Crosslinking of the T cell receptor has been proposed to be a prerequisite for T cell activation. Although the evidence supports this notion for CD4 T cells, the situation for CD8 T cells is less clear. Soluble class I monomers have been used to determine activation requirementsin vitrowith contradictory results. The possibility of transfer of peptide from soluble class I molecules onto class I molecules present on the surface of CD8 T cells, with ensuing presentation to other CD8 T cells, has been widely ignored. We show that monomeric and tetrameric class I molecules as well as free peptide can stimulate naïve CD8 T cellsin vitro. We generate and characterize CD8 T cells that express the OT-I T cell receptor (for Kb/SIINFEKL) yet lack Kband Dbmolecules, and show that their activation requirements differ from their class I positive counterparts when stimulated with soluble Kbmolecules. By eliminating the confounding effect of peptide transfer, we unmask the true activation requirements for naïve CD8 T cells and show that multivalent engagement of T cell receptors, as well as costimulation, is required for optimal stimulation.

Wnt/β-catenin signalling controls development and adult tissue homeostasis and causes cancer when inappropriately activated. In unstimulated cells, an Axin1-centred multi-protein complex phosphorylates the transcriptional co-activator β-catenin, marking it for degradation. Wnt signalling antagonizes β-catenin proteolysis, leading to its accumulation and target gene expression. How Wnt stimulation alters the size distribution, composition and activity of endogenous Axin1 complexes remains poorly understood. Here, we employed two-dimensional blue native/SDS-PAGE to analyse endogenous Axin1 and β-catenin complexes during Wnt signalling. We show that the size range of Axin1 complexes is conserved between species and remains largely unaffected by Wnt stimulation. We detect a striking Wnt-dependent, cytosolic accumulation of both non-phosphorylated and phosphorylated β-catenin within a 450 kDa Axin1-based complex and in a distinct, Axin1-free complex of 200 kDa. These results argue that during Wnt stimulation, phosphorylated β-catenin is released from the Axin1 complex but fails to undergo immediate degradation. Importantly, in APC-mutant cancer cells, the distribution of Axin1 and β-catenin complexes strongly resembles that of Wnt-stimulated cells. Our findings argue that Wnt signals and APC mutations interfere with the turnover of phosphorylated β-catenin. Furthermore, our results suggest that the accumulation of small-sized β-catenin complexes may serve as an indicator of Wnt pathway activity in primary cancer cells.

The H-2Ldalloreactive 2C T cell receptor (TCR) is commonly considered as being positively selected on the H-2Kbmolecule. Surprisingly, 2C TCR+CD8+single-positive T cells emerge in massive numbers in fetal thymic organ culture originating from 2C transgenic, H-2KbDb−/−(2C+KbDb−/−) but not in fetal thymic organ culture from β2-microglobulin−/−2C transgenic animals. Mature CD8+T cells are observed in newborn but not in adult 2C+KbDb−/−mice. These CD8+T cells express the α4β7integrin, which allows them to populate the intestine, a pattern of migration visualized by intrathymic injection of FITC and subsequent accrual of FITC-labeled lymphocytes in the gut. We conclude that the 2C TCR is reactive not only with H-2Ldand H-2Kb, but also with nonclassical MHC class I products to enable positive selection of 2C+T cells in the fetal and newborn thymus and to support their maintenance in the intestine.

Objective To examine the expression of the thioredoxin (TRX)–thioredoxin reductase (TR) system in patients with rheumatoid arthritis (RA) and patients with other rheumatic diseases. Methods Levels of TRX in plasma and synovial fluid (SF) were measured using enzyme-linked immunosorbent assay. Cellular distribution of TRX was determined by flow cytometry and histochemistry. Cellular expression of TR was studied by in situ messenger RNA (mRNA) hybridization. The effect of oxidative stress and tumor necrosis factor α (TNFα) on TRX expression by cultured rheumatoid fibroblast-like synoviocytes was studied. Results Significantly increased TRX levels were found in the SF from 22 patients with RA, when compared with plasma levels in the same patients (P < 0.001) and compared with SF TRX levels in 15 patients with osteoarthritis (P < 0.001), 13 patients with gout (P < 0.05), and 9 patients with reactive arthritis (P < 0.0001). The presence of TRX could be demonstrated within the SF-derived mononuclear cells and synovial tissue (ST) of RA patients. Concordantly, expression of TR mRNA was observed in the ST of these patients. Stimulation of synovial fibroblast-like synoviocytes with either H2O2 or TNFα induced an increase in the production of TRX. Conclusion The data demonstrate significantly increased concentrations of TRX in the SF and ST of RA patients when compared with the levels in patients with other joint diseases. Evidence is presented that the local environment in the rheumatic joint contributes to increased TRX production. Based on its growth-promoting and cytokine-like properties, it is proposed that increased expression of TRX contributes to the disease activity in RA.

We found that the magnesium salt of ilimaquinone, named 201-F, specifically disassembled dynamically unstable microtubules in fibroblasts and various epithelial cell lines. Unlike classical tubulin- interacting drugs such as nocodazole or colchicine which affect all classes of microtubules, 201-F did not depolymerize stable microtubules. In WIF-B–polarized hepatic cells, 201-F disrupted the Golgi complex and inhibited albumin and alpha1-antitrypsin secretion to the same extent as nocodazole. By contrast, 201-F did not impair the transport of membrane proteins to the basolateral surface, which was only affected by the total disassembly of cellular microtubules. Transcytosis of two apical membrane proteins—the alkaline phosphodiesterase B10 and dipeptidyl peptidase IV—was affected to the same extent by 201-F and nocodazole. Taken together, these results indicate that only dynamically unstable microtubules are involved in the transport of secretory proteins to the plasma membrane, and in the transcytosis of membrane proteins to the apical surface. By contrast, stable microtubules, which are not functionally affected by 201-F treatment, are involved in the transport of membrane proteins to the basolateral surface. By specifically disassembling highly dynamic microtubules, 201-F is an invaluable tool with which to study the functional specialization of stable and dynamic microtubules in living cells.

In rheumatoid arthritis (RA), the functional status of T cells is incompletely understood. Synovial T cells display phenotypic evidence of former activation, but there is poor production of T cell-derived cytokines in the synovium. In addition, synovial T cell proliferation upon mitogenic and antigenic stimulation was decreased compared with that in peripheral blood T cells. Moreover, previous reports revealed that early Ca2+ rises induced by TCR/CD3 stimulation were decreased in RA T cells compared with those in healthy controls. To investigate the molecular mechanisms of RA synovial T cell hyporesponsiveness, we analyzed the TCR/CD3-mediated protein tyrosine phosphorylation in RA peripheral blood and synovial fluid (SF) T cells. SF T cells exhibited a decreased overall tyrosine phosphorylation pattern upon stimulation. Most notably, the induction of phosphorylation of p38 was virtually absent. Moreover, we found that tyrosine phosphorylation of the TCR zeta-chain, one of the most proximal events in TCR signaling, is clearly diminished in RA SF T cells. The decrease in tyrosine phosphorylation was accompanied by a decrease in detectable levels of zeta-protein within synovial T cells. These results suggest that a defective TCR signaling underlies the hyporesponsiveness of synovial T cells in RA.

A method for purification by countercurrent chromatography (CCC) of phosphatidylcholine with high content of DHA is described. Phospholipids were extracted from squid Illex argentinus characterized by high performance liquid chromatography and their fatty acids profile determined by gas chromatography. Phosphatidylcholine was primarily represented with 48.3 % (w/w) of total phospholipids and contained 39.7 % of DHA (w/w total fatty acids). The analytical separation of phosphatidylcholine, was achieved on a Kromaton II apparatus, in the systems heptane—ethyl acetate—acetonitrile (1:0.65:1, v/v/v) and isooctane—ethyl acetate—acetonitrile (1:0.65:1, v/v/v), at 550 rpm and 1.5 mL/min flow rate using normal ascending and reversed descending elution modes respectively. The fractions collected were further analysed by high performance thin layer chromatography. Phosphatidylcholine was separated from the other phospholipids of the crude extract and was recovered pure in the lower phase with 95 % yield. P...