Three-Dimensional Fractal Analysis of the Interstitial Cells of Cajal Networks of Gastrointestinal Tissue Specimens.

Introduction: Several functional gastrointestinal disorders (FGIDs) have been associated with the degradation or remodeling of the network of interstitial cells of Cajal (ICC). Introducing fractal analysis to the field of gastroenterology as a promising data analytics approach to extract key structural characteristics that may provide insightful features for machine learning applications in disease diagnostics. Fractal geometry has advantages over several physically based parameters (or classical metrics) for analysis of intricate and complex microstructures that could be applied to ICC networks. Methods: In this study, three fractal structural parameters: Fractal Dimension, Lacunarity, and Succolarity were employed to characterize scale-invariant complexity, heterogeneity, and anisotropy; respectively of three types of gastric ICC network structures from a flat-mount transgenic mouse stomach. Results: The Fractal Dimension of ICC in the longitudinal muscle layer was found to be significantly lower than ICC in the myenteric plexus and circumferential muscle in the proximal, and distal antrum, respectively (both p < 0.0001). Conversely, the Lacunarity parameters for ICC-LM and ICC-CM were found to be significantly higher than ICC-MP in the proximal and in the distal antrum, respectively (both p < 0.0001). The Succolarity measures of ICC-LM network in the aboral direction were found to be consistently higher in the proximal than in the distal antrum (p < 0.05). Conclusions: The fractal parameters presented here could go beyond the limitation of classical metrics to provide better understanding of the structural-functional relationship between ICC networks and the conduction of gastric bioelectrical slow waves.

Ferroptosis Induction and YAP Inhibition as New Therapeutic Targets in Gastrointestinal Stromal Tumors (GISTs).

GISTs are sarcomas of the gastrointestinal tract often associated with gain-of-function mutations in KIT or PDGFRA receptor genes. While most GISTs initially respond to tyrosine kinase inhibitors, relapses due to acquired resistance frequently occur. The induction of ferroptosis, an iron-dependent form of non-apoptotic cell death, emerged as a novel therapeutic approach in cancers and remains poorly characterized in GISTs. We studied hallmarks of ferroptosis, i.e., lipid peroxidation, iron and glutathione content, and GPX4 protein expression in imatinib-sensitive (GIST882) and -resistant (GIST48) GIST cell lines. GIST cells were highly sensitive to the induction of ferroptosis by RSL3, which was reversed by liproxstatin and deferoxamine. Lipid peroxidation and ferroptosis were mediated by VP and CA3 in GIST cells through a significant decrease in antioxidant defenses. Moreover, VP, but surprisingly not CA3, inhibited a series of target genes downstream of YAP in GIST cells. The ferroptosis marker TFRC was also investigated by immunohistochemistry in GIST tissue arrays. TFRC expression was observed in all samples. High TFRC expression was positively correlated with high-risk GISTs, elevated mitotic count, and YAP nuclear localization, reflecting YAP activation. This study highlights ferroptosis as a novel cell death mechanism in GISTs, and a potential therapeutic target to overcome resistance to tyrosine kinase inhibitors.

Deciphering Stomach Myoelectrical Slow Wave Conduction Patterns via Confocal Imaging of Gastric Pacemaker Cells and Fractal Geometry.

Interstitial Cells of Cajal (ICC) are specialized gastrointestinal (GI) pacemaker cells that generate and actively propagate slow waves of depolarization (SWs) of the muscularis propria. SWs regulate the motility of the GI tract necessary for digestion, absorption of nutrients, and elimination of waste. Within the gastric wall, there are three main inter-connected layers of ICC networks: longitudinal muscle ICC (ICC-LM), myenteric plexus ICC (ICC-MP) & circumferential muscle (ICC-CM). Fractal structural parameters such as Fractal Dimension (FD), Lacunarity and Succolarity, have many advantages over physically-based parameters when it comes to characterizing the complex architectures of ICC networks. The analysis of networks of ICC throughout the proximal and distal murine gastric antrum with the FD and Lacunarity metrics was previously performed. Although the application of Succolarity is relatively nascent compared to the FD and Lacunarity; nevertheless, numerous studies have demonstrated the capability of this fractal measure to extract information from images associated with flow by which neither the FD nor Lacunarity are capable of discerning. In this study, Succolarity analysis of ICC-MP and ICC-CM networks were performed with confocal images taken across the proximal and distal murine antrum. Our findings demonstrated the Succolarity of ICC-MP and ICC-CM varied with directions and antral regions. The Succolarity of ICC-MP did not vary considerably with direction, however, Succolarity was higher in the aboral direction with 0.2113 ±0.1589, and 0.0637 ±0.0822 in the proximal and distal antrum, respectively. The overall Succolarity of ICC-MP was significantly higher than that of ICC-CM in the proximal antrum ( 0.1580±0.1325 vs [Formula: see text]) and in the distal antrum ( 0.0449 ±0.0409 vs [Formula: see text]). Clinical Relevance-Modeling SWs conduction patterns via image analysis of detailed ICC networks help to facilitate an improved understanding of the mechanisms underpinning GI myoelectric activity and the diseases associated with its dysfunction.

Genome-wide analysis of Brucella melitensis genes required throughout intranasal infection in mice.

Brucellae are facultative intracellular Gram-negative coccobacilli that chronically infect various mammals and cause brucellosis. Human brucellosis is among the most common bacterial zoonoses and the vast majority of cases are attributed to B. melitensis. Using transposon sequencing (Tn-seq) analysis, we showed that among 3369 predicted genes of the B. melitensis genome, 861 are required for optimal growth in rich medium and 186 additional genes appeared necessary for survival of B. melitensis in RAW 264.7 macrophages in vitro. As the mucosal immune system represents the first defense against Brucella infection, we investigated the early phase of pulmonary infection in mice. In situ analysis at the single cell level indicates a succession of killing and growth phases, followed by heterogenous proliferation of B. melitensis in alveolar macrophages during the first 48 hours of infection. Tn-seq analysis identified 94 additional genes that are required for survival in the lung at 48 hours post infection. Among them, 42 genes are common to RAW 264.7 macrophages and the lung conditions, including the T4SS and purine synthesis genes. But 52 genes are not identified in RAW 264.7 macrophages, including genes implicated in lipopolysaccharide (LPS) biosynthesis, methionine transport, tryptophan synthesis as well as fatty acid and carbohydrate metabolism. Interestingly, genes implicated in LPS synthesis and ß oxidation of fatty acids are no longer required in Interleukin (IL)-17RA-/- mice and asthmatic mice, respectively. This demonstrates that the immune status determines which genes are required for optimal survival and growth of B. melitensis in vivo.

Analysis of Regional Variations of the Interstitial Cells of Cajal in the Murine Distal Stomach Informed by Confocal Imaging and Machine Learning Methods.

Introduction: The network of Interstitial Cells of Cajal (ICC) plays a plethora of key roles in maintaining, coordinating, and regulating the contractions of the gastrointestinal (GI) smooth muscles. Several GI functional motility disorders have been associated with ICC degradation. This study extended a previously reported 2D morphological analysis and applied it to 3D spatial quantification of three different types of ICC networks in the distal stomach guided by confocal imaging and machine learning methods. The characterization of the complex changes in spatial structure of the ICC network architecture contributes to our understanding of the roles that different types of ICC may play in post-prandial physiology, pathogenesis, and/or amelioration of GI dsymotility- bridging structure and function. Methods: A validated classification method using Trainable Weka Segmentation was applied to segment the ICC from a confocal dataset of the gastric antrum of a transgenic mouse, followed by structural analysis of the segmented images. Results: The machine learning model performance was compared to manually segmented subfields, achieving an area under the receiver-operating characteristic (AUROC) of 0.973 and 0.995 for myenteric ICC (ICC-MP; n = 6) and intramuscular ICC (ICC-IM; n = 17). The myenteric layer in the distal antrum increased in thickness (from 14.5 to 34 µm) towards the lesser curvature, whereas the thickness decreased towards the lesser curvature in the proximal antrum (17.7 to 9 µm). There was an increase in ICC-MP volume from proximal to distal antrum (406,960 ± 140,040 vs. 559,990 ± 281,000 µm3; p = 0.000145). The % of ICC volume was similar for ICC-LM and for ICC-CM between proximal (3.6 ± 2.3% vs. 3.1 ± 1.2%; p = 0.185) and distal antrum (3.2 ± 3.9% vs. 2.5 ± 2.8%; p = 0.309). The average % volume of ICC-MP was significantly higher than ICC-IM at all points throughout sample (p < 0.0001). Conclusions: The segmentation and analysis methods provide a high-throughput framework of investigating the structural changes in extended ICC networks and their associated physiological functions in animal models.

Antral Variation of Murine Gastric Pacemaker Cells Informed by Confocal Imaging and Machine Learning Methods.

The Interstitial Cells of Cajal (ICC) are specialized gastrointestinal (GI) pacemaker cells that generate and actively propagate electrophysiological events called slow waves. Slow waves regulate the GI motility necessary for digestion. Several functional GI motility disorders have been associated with depletion in the ICC. In this study, a validated Fast Random Forest (FRF) classification method using Trainable WEKA Segmentation for segmenting the networks of ICC was applied to confocal microscopy images of a whole mount tissue from the distal antrum of a mouse stomach (583 × 3,376 × 133 µm3, parcellated into 24 equal image stacks). The FRF model performance was compared to 6 manually segmented subflelds and produced an area under the receiver-operating characteristic (AUROC) of 0.95. Structural variations of ICC network in the longitudinal muscle (ICC-LM) and myenteric plexus (ICC-MP) were quantified. The average volume of ICC-MP was significantly higher than ICC-LM at any point throughout the antral tissue sampled. There was a pronounced decline of up to 80% in ICC-LM (from 3,705 µm3 to 716 µm3) over a distance of 279.3 µm, that eventually diminished towards the distal antrum. However, an inverse relationship was observed in ICC-MP with an overall increase of up to 157% (from 59,100 µm3 to 151,830 µm3) over a distance of approximately 2 mm that proceeds towards the distal antrum.

Inhibition of Phosphodiesterase 3A by Cilostazol Dampens Proinflammatory Platelet Functions.

OBJECTIVE: platelets possess not only haemostatic but also inflammatory properties, which combined are thought to play a detrimental role in thromboinflammatory diseases such as acute coronary syndromes and stroke. Phosphodiesterase (PDE) 3 and -5 inhibitors have demonstrated efficacy in secondary prevention of arterial thrombosis, partially mediated by their antiplatelet action. Yet it is unclear whether such inhibitors also affect platelets' inflammatory functions. Here, we aimed to examine the effect of the PDE3A inhibitor cilostazol and the PDE5 inhibitor tadalafil on platelet function in various aspects of thromboinflammation. Approach and results: cilostazol, but not tadalafil, delayed ex vivo platelet-dependent fibrin formation under whole blood flow over type I collagen at 1000 s-1. Similar results were obtained with blood from Pde3a deficient mice, indicating that cilostazol effects are mediated via PDE3A. Interestingly, cilostazol specifically reduced the release of phosphatidylserine-positive extracellular vesicles (EVs) from human platelets while not affecting total EV release. Both cilostazol and tadalafil reduced the interaction of human platelets with inflamed endothelium under arterial flow and the release of the chemokines CCL5 and CXCL4 from platelets. Moreover, cilostazol, but not tadalafil, reduced monocyte recruitment and platelet-monocyte interaction in vitro. CONCLUSIONS: this study demonstrated yet unrecognised roles for platelet PDE3A and platelet PDE5 in platelet procoagulant and proinflammatory responses.

Supervised Machine Learning Segmentation and Quantification of Gastric Pacemaker Cells.

Interstitial Cells of Cajal (ICC) are specialized pacemaker cells that generate and actively propagate electrophysiological events called slow waves. Slow waves regulate the motility of the gastrointestinal tract necessary for digesting food. Degradation in the ICC network structure has been qualitatively associated to several gastrointestinal motility disorders. ICC network structure can be obtained using confocal microscopy, but the current limitations in imaging and segmentation techniques have hindered an accurate representation of the networks. In this study, supervised machine learning techniques were applied to extract the ICC networks from 3D confocal microscopy images. The results showed that the Fast Random Forest classification method using Trainable WEKA Segmentation outperformed the Decision Table and Naïve Bayes classification methods in sensitivity, accuracy, and F-measure. Using the Fast Random Forest classifier, 12 gastric antrum tissue blocks were segmented and variations in ICC network thickness, density and process width were quantified for the myenteric plexus ICC network (the primary pacemakers). Our findings demonstrated regional variation in ICC network density and thickness along the circumferential and longitudinal axis of the mouse antrum. An inverse relationship was observed in the distal and proximal antrum for density (proximal: 9.8±4.0% vs distal: 7.6±4.6%) and thickness (proximal: 15±3 µm vs distal: 24±10 µm). Limited variation in ICC process width was observed throughout the antrum (5±1 µm).Clinical Relevance- Detailed quantification of regional ICC structural properties will provide insights into the relationship between ICC structure, slow waves and resultant gut motility. This will improve techniques for the diagnosis and treatment of functional GI motility disorders.

Continuum Based Bioelectrical Simulations using Structurally Realistic Gastrointestinal Pacemaker Cell Networks.

Cellular and tissue level bioelectrical activity was simulated over structurally realistic 3D interstitial cell of Cajal (ICC) networks reconstructed from confocal images of a wild type (WT) mouse model with a normal ICC distribution and a Spry4 knockout (KO) mouse model with a mild ICC hyperplasia. First, the ICC pixels within the confocal images were segmented. Then, the segmented images were visually inspected and the 3D surface mesh of the ICC tissue network was created from the 90 slices spanning the myenteric plexus ICC network. After two additional concentric meshes (representing the non-ICC and tissue bath regions) surrounding the ICC region were added, a 3D tetrahedral volume mesh containing the three regions was reconstructed. The electrical propagation through the tissue network was simulated using the bidomain continuum model. The results showed that the ICC network of the WT mouse had a smaller volume than the KO mouse (0.008 vs 0.012 mm3). The simulated bioelectrical activity for both mice showed an isotropic propagation from the initial activation region. Mean velocities of 4.2±1.5 and 4.1±1.3 mm/s were reported for the WT and KO mice, respectively. The velocity in the x-direction was higher than the y-direction for the WT mouse with a percent difference of 14.8%. On the other hand, the velocity in the y-direction was higher for the KO mouse with a percent difference of 9.5%. For both cases, there was no propagation in the z-direction as all the solution points along the same z-depth were simultaneously activated.

Route of Infection Strongly Impacts the Host-Pathogen Relationship.

Live attenuated vaccines play a key role in the control of many human and animal pathogens. Their rational development is usually helped by identification of the reservoir of infection, the lymphoid subpopulations associated with protective immunity as well as the virulence genes involved in pathogen persistence. Here, we compared the course of Brucella melitensis infection in C57BL/6 mice infected via intraperitoneal (i.p.), intranasal (i.n.) and intradermal (i.d.) route and demonstrated that the route of infection strongly impacts all of these parameters. Following i.p. and i.n. infection, most infected cells observed in the spleen or lung were F4/80+ myeloid cells. In striking contrast, infected Ly6G+ neutrophils and CD140a+ fibroblasts were also observed in the skin after i.d. infection. The virB operon encoding for the type IV secretion system is considered essential to deflecting vacuolar trafficking in phagocytic cells and allows Brucella to multiply and persist. Unexpectedly, the ΔvirB Brucella strain, which does not persist in the lung after i.n. infection, persists longer in skin tissues than the wild strain after i.d. infection. While the CD4+ T cell-mediated Th1 response is indispensable to controlling the Brucella challenge in the i.p. model, it is dispensable for the control of Brucella in the i.d. and i.n. models. Similarly, B cells are indispensable in the i.p. and i.d. models but dispensable in the i.n. model. γδ+ T cells appear able to compensate for the absence of αß+ T cells in the i.d. model but not in the other models. Taken together, our results demonstrate the crucial importance of the route of infection for the host pathogen relationship.

Potentiation of imatinib by cilostazol in sensitive and resistant gastrointestinal stromal tumor cell lines involves YAP inhibition.

Despite the introduction of tyrosine kinase inhibitors, gastrointestinal stromal tumors (GIST) resistance remains a major clinical challenge. We previously identified phosphodiesterase 3A (PDE3A) as a potential therapeutic target expressed in most GIST. The PDE3 inhibitor cilostazol reduced cell viability and synergized with the tyrosine kinase inhibitor imatinib (Gleevec™) in the imatinib-sensitive GIST882 cell line. Here, we found that cilostazol potentiated imatinib also in the imatinib-resistant GIST48 cell line. Cilostazol induced nuclear exclusion, hence inactivation, of the transcriptional co-activator YAP, in a cAMP-independent manner. Verteporfin, a YAP/TEAD interaction inhibitor, reduced by 90% the viability of both GIST882 and GIST48 cells. Our results highlight the potential use of compounds targeting PDE3A or YAP in combined multitherapy to tackle GIST resistance.

Allergic Asthma Favors Brucella Growth in the Lungs of Infected Mice.

Allergic asthma is a chronic Th2 inflammatory disease of the lower airways affecting a growing number of people worldwide. The impact of infections and microbiota composition on allergic asthma has been investigated frequently. Until now, however, there have been few attempts to investigate the impact of asthma on the control of infectious microorganisms and the underlying mechanisms. In this work, we characterize the consequences of allergic asthma on intranasal (i.n.) infection by Brucella bacteria in mice. We observed that i.n. sensitization with extracts of the house dust mite Dermatophagoides farinae or the mold Alternaria alternata (Alt) significantly increased the number of Brucella melitensis, Brucella suis, and Brucella abortus in the lungs of infected mice. Microscopic analysis showed dense aggregates of infected cells composed mainly of alveolar macrophages (CD11c+ F4/80+ MHCII+) surrounded by neutrophils (Ly-6G+). Asthma-induced Brucella susceptibility appears to be dependent on CD4+ T cells, the IL-4/STAT6 signaling pathway and IL-10, and is maintained in IL-12- and IFN-γR-deficient mice. The effects of the Alt sensitization protocol were also tested on Streptococcus pneumoniae and Mycobacterium tuberculosis pulmonary infections. Surprisingly, we observed that Alt sensitization strongly increases the survival of S. pneumoniae infected mice by a T cell and STAT6 independent signaling pathway. In contrast, the course of M. tuberculosis infection is not affected in the lungs of sensitized mice. Our work demonstrates that the impact of the same allergic sensitization protocol can be neutral, negative, or positive with regard to the resistance of mice to bacterial infection, depending on the bacterial species.

A framework for simulating gastric electrical propagation in confocal microscopy derived geometries.

Interstitial Cells of Cajal (ICC) initiate and actively propagate electrical events in the gastrointestinal tract known as slow-waves. The slow-waves coordinate the contraction of the gastrointestinal tract necessary for breakdown and mixing of ingested food. Degradation of the ICC numbers has been linked to several gastrointestinal motility disorders. However, limitations in imaging techniques and techniques for the quantification of ICC network structure have hindered our understanding of these disorders. We evaluated different machine learning techniques to segment ICC networks imaged using confocal microscopy. The accuracy the segmented networks were then quantified and compared using numerical metrics. Structurally realistic finite element meshes were constructed and used to simulate the propagation of electrical activation over the tissue blocks. The presented framework provides a system to quantify the structure and function of an ICC tissue sample. These methods are also applicable to other biological tissues and networks.

Phosphodiesterase 3A: a new player in development of interstitial cells of Cajal and a prospective target in gastrointestinal stromal tumors (GIST).

We previously identified phosphodiesterase 3A (PDE3A) as a marker for interstitial cells of Cajal (ICC) in adult mouse gut. However, PDE3A expression and function during gut development and in ICC-derived gastrointestinal stromal tumors (GIST) remained unknown. Here we found that PDE3A was expressed throughout ICC development and that ICC density was halved in PDE3A-deficient mice. In the human imatinib-sensitive GIST882 cell line, the PDE3 inhibitor cilostazol halved cell viability (IC50 0.35 µM) and this effect synergized with imatinib (Chou-Talalay's CI50 0.15). Recently the compound 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP was found to be cytotoxic selectively for cells expressing both PDE3A and Schlafen12 (SLFN12) (de Waal L et al. Nat Chem Bio 2016), identifying a new, non-catalytic, role for PDE3A. 108 out of 117 (92%) of our human GIST samples displayed both PDE3A and SLFN12 immunoreactivity. GIST882 cells express both PDE3A and SLFN12 and DNMDP decreased their viability by 90%. Our results suggest a role for PDE3A during ICC development and open novel perspectives for PDE3A in targeted GIST therapy, on one hand by the synergism between imatinib and cilostazol, a PDE3 inhibitor already in clinical use for other indications, and, on the other hand, by the neomorphic, druggable, PDE3A-SLFN12 cytotoxic interplay.

Thrombospondin 1 protects pancreatic ß-cells from lipotoxicity via the PERK-NRF2 pathway.

The failure of ß-cells has a central role in the pathogenesis of type 2 diabetes, and the identification of novel approaches to improve functional ß-cell mass is essential to prevent/revert the disease. Here we show a critical novel role for thrombospondin 1 (THBS1) in ß-cell survival during lipotoxic stress in rat, mouse and human models. THBS1 acts from within the endoplasmic reticulum to activate PERK and NRF2 and induce a protective antioxidant defense response against palmitate. Prolonged palmitate exposure causes THBS1 degradation, oxidative stress, activation of JNK and upregulation of PUMA, culminating in ß-cell death. These findings shed light on the mechanisms leading to ß-cell failure during metabolic stress and point to THBS1 as an interesting therapeutic target to prevent oxidative stress in type 2 diabetes.

BiDiFuse: a FIJI plugin for fusing bi-directionally recorded microscopic image volumes.

Deep tissue imaging is increasingly used for non-destructive interrogation of intact organs and small model organisms. An intuitive approach to increase the imaging depth by almost a factor of 2 is to record a sample from two sides and fuse both image stacks. However, imperfect three-dimensional alignment of both stacks presents a computational challenge. We have developed a FIJI plugin, called BiDiFuse, which merges bi-directionally recorded image stacks via 3D rigid transformations. The method is broadly applicable, considering it is compatible with all optical sectioning microscopes and it does not rely on fiducial markers for image registration. AVAILABILITY AND IMPLEMENTATION: The method is freely available as a plugin for FIJI from https://github.com/JanDetrez/BiDiFuse/ CONTACT: winnok.devos@uantwerpen.be.

The SHIP2 interactor Myo1c is required for cell migration in 1321 N1 glioblastoma cells.

The phosphoinositide 5-phosphatases consist of several enzymes that have been shown to modulate cell migration and invasion. SHIP2, one family member, is known to interact with growth factor receptors and cytoskeletal proteins. In a human model of glioblastoma 1321 N1 cells, we recently identified Myo1c as a new interactor of SHIP2. This was shown in a complex of proteins also containing filamin A. We show here that SHIP2 localization at lamellipodia and ruffles is impaired in Myo1c depleted cells. In the absence of Myo1c, N1 cells tend to associate to form clusters. Cell migration is very much reduced in Myo1c depleted cells, concomitantly with a decrease in FAK Tyr397 phosphorylation, focal adhesion length and PI(4,5)P2 immunostaining. In N1 cells, Myo1c is thus important for lamellipodia formation to assemble a protein complex containing SHIP2 to facilitate cell migration.

SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration in 1321 N1 glioblastoma cells.

Phosphoinositides, particularly phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], are recognized by SHIP2 (also known as INPPL1) a member of the inositol polyphosphate 5-phosphatase family. SHIP2 dephosphorylates PI(3,4,5)P3 to form PI(3,4)P2; the latter interacts with specific target proteins (e.g. lamellipodin). Although the preferred SHIP2 substrate is PI(3,4,5)P3, PI(4,5)P2 can also be dephosphorylated by this enzyme to phosphatidylinositol 4-phosphate (PI4P). Through depletion of SHIP2 in the glioblastoma cell line 1321 N1, we show that SHIP2 inhibits cell migration. In different glioblastoma cell lines and primary cultures, SHIP2 staining at the plasma membrane partly overlaps with PI(4,5)P2 immunoreactivity. PI(4,5)P2 was upregulated in SHIP2-deficient N1 cells as compared to control cells; in contrast, PI4P was very much decreased in SHIP2-deficient cells. Therefore, SHIP2 controls both PI(3,4,5)P3 and PI(4,5)P2 levels in intact cells. In 1321 N1 cells, the PI(4,5)P2-binding protein myosin-1c was identified as a new interactor of SHIP2. Regulation of PI(4,5)P2 and PI4P content by SHIP2 controls 1321 N1 cell migration through the organization of focal adhesions. Thus, our results reveal a new role of SHIP2 in the control of PI(4,5)P2, PI4P and cell migration in PTEN-deficient glioblastoma 1321 N1 cells.

In Situ Characterization of Splenic Brucella melitensis Reservoir Cells during the Chronic Phase of Infection in Susceptible Mice.

Brucella are facultative intracellular Gram-negative coccobacilli that chronically infect humans as well as domestic and wild-type mammals, and cause brucellosis. Alternatively activated macrophages (M2a) induced by IL-4/IL-13 via STAT6 signaling pathways have been frequently described as a favorable niche for long-term persistence of intracellular pathogens. Based on the observation that M2a-like macrophages are induced in the spleen during the chronic phase of B. abortus infection in mice and are strongly infected in vitro, it has been suggested that M2a macrophages could be a potential in vivo niche for Brucella. In order to test this hypothesis, we used a model in which infected cells can be observed directly in situ and where the differentiation of M2a macrophages is favored by the absence of an IL-12-dependent Th1 response. We performed an in situ analysis by fluorescent microscopy of the phenotype of B. melitensis infected spleen cells from intranasally infected IL-12p40-/- BALB/c mice and the impact of STAT6 deficiency on this phenotype. Most of the infected spleen cells contained high levels of lipids and expressed CD11c and CD205 dendritic cell markers and Arginase1, but were negative for the M2a markers Fizz1 or CD301. Furthermore, STAT6 deficiency had no effect on bacterial growth or the reservoir cell phenotype in vivo, leading us to conclude that, in our model, the infected cells were not Th2-induced M2a macrophages. This characterization of B. melitensis reservoir cells could provide a better understanding of Brucella persistence in the host and lead to the design of more efficient therapeutic strategies.

Sox9 Controls Self-Renewal of Oncogene Targeted Cells and Links Tumor Initiation and Invasion.

Sox9 is a transcription factor expressed in most solid tumors. However, the molecular mechanisms underlying Sox9 function during tumorigenesis remain unclear. Here, using a genetic mouse model of basal cell carcinoma (BCC), the most frequent cancer in humans, we show that Sox9 is expressed from the earliest step of tumor formation in a Wnt/ß-catenin-dependent manner. Deletion of Sox9 together with the constitutive activation of Hedgehog signaling completely prevents BCC formation and leads to a progressive loss of oncogene-expressing cells. Transcriptional profiling of oncogene-expressing cells with Sox9 deletion, combined with in vivo ChIP sequencing, uncovers a cancer-specific gene network regulated by Sox9 that promotes stemness, extracellular matrix deposition, and cytoskeleton remodeling while repressing epidermal differentiation. Our study identifies the molecular mechanisms regulated by Sox9 that link tumor initiation and invasion.