Piperitone (p-Menth-1-En-3-One): A New Repellent for Tea Shot Hole Borer (Coleoptera: Curculionidae) in Florida Avocado Groves.

The tea shot hole borer, Euwallacea perbrevis, has been recently established in Florida, USA, where it vectors fungal pathogens that cause Fusarium dieback in avocado. Pest monitoring uses a two-component lure containing quercivorol and α-copaene. Incorporation of a repellent into IPM programs may reduce the incidence of dieback in avocado groves, particularly if combined with lures in a push-pull system. This study evaluated piperitone and α-farnesene as potential repellents for E. perbrevis, comparing their efficacy to that of verbenone. Replicate 12-week field tests were conducted in commercial avocado groves. Each test compared beetle captures in traps baited with two-component lures versus captures in traps containing lures plus repellent. To complement field trials, Super-Q collections followed by GC analyses were performed to quantify emissions from repellent dispensers field-aged for 12 weeks. Electroantennography (EAG) was also used to measure beetle olfactory response to each repellent. Results indicated that α-farnesene was ineffective; however, piperitone and verbenone were comparable in repellency, achieving 50-70% reduction in captures, with longevity of 10-12 weeks. EAG responses to piperitone and verbenone were equivalent, and significantly greater than response to α-farnesene. Since piperitone is less expensive than verbenone, this study identifies a potential new E. perbrevis repellent.

HPTLC Analysis and Chemical Composition of Selected Melaleuca Essential Oils.

Tea tree oil (TTO) is a volatile essential oil obtained by distillation, mainly from the Australian native plant Melaleuca alternifolia (Maiden & Betche) Cheel (Myrtaceae). In this study, a comparative analysis of the chemical constituents of seven tea tree oils (M. alternifolia) and four other Melaleuca spp. oils (M. cajuputi, (MCa), two chemotypes of M. quinquenervia, (MNe and MNi), and M. ericifolia (MRo)) was carried out using gas chromatography-mass spectrometry (GC-MS) and high-performance thin-layer chromatography (HPTLC). Among the seven TTOs, terpinen-4-ol (37.66-44.28%), γ-terpinene (16.42-20.75%), α-terpinene (3.47-12.62%), α-terpineol (3.11-4.66%), and terpinolene (2.75-4.19%) were the most abundant compounds. On the other hand, the most abundant compounds of the other Melaleuca oils varied, such as 1,8-cineole (64.63%) in MCa oil, (E)-nerolidol (48.40%) and linalool (33.30%) in MNe oil, 1,8-cineole (52.20%) in MNi oil, and linalool (38.19%) and 1,8-cineole (27.57%) in MRo oil. HPTLC fingerprinting of Melaleuca oils enabled the discrimination of TTO oils from other Melaleuca spp. oils. Variation was observed in the profile of the Rf values among EOs. The present study shows that HPTLC is one of the best ways to identify and evaluate the quality control in authenticating TTOs, other Melaleuca EOs, or EOs from other species within the Myrtaceae.

Attraction and Longevity of 2- and 3-Component Food Cone Lures for the Caribbean Fruit Fly, Anastrepha suspensa (Diptera: Tephritidae).

The Caribbean fruit fly, Anastrepha suspensa (Loew) (Diptera: Tephritidae), is a quarantine pest of Citrus spp. and a production pest of guava and other specialty fruits in Florida. Effective monitoring lures and traps are needed for early pest detection and timely initiation of control measures. As part of a continued effort to identify attractive synthetic lures for the Caribbean fruit fly, we conducted field tests in Homestead, Florida to compare the efficacy and longevity of commercial 2- and 3-component cone lures (2C [ammonium acetate and putrescine], 3C [ammonium acetate, putrescine, and trimethylamine]), the current standards used by regulatory agencies, versus the traditional liquid protein bait consisting of hydrolyzed torula yeast and borax as a positive control. Additional lures were also field-aged and periodically brought into the laboratory to quantify residual chemical contents. Traps baited with the torula yeast-borax mixture captured the highest mean number of A. suspensa, and traps baited with the commercial 2C lures captured more flies than the 3C lures. Traps baited with torula yeast-borax also captured the highest number of nontarget Diptera. Captures with all three treatments were significantly biased toward females. Attractiveness of the 2C lure began to drop after 6-8 wk, and the 3C lure after 5-6 wk. Overall, these data suggest that the 2C cone lure is more attractive to A. suspensa than the 3C cone lure under field conditions in south Florida, and that the 2C lures are attractive for up to 8 wk.

Sap flow, xylem anatomy and photosynthetic variables of three Persea species in response to laurel wilt.

Laurel wilt, a lethal vascular wilt disease caused by the fungus Raffaelea lauricola, affects several tree species in the Lauraceae, including three Persea species. The susceptibility to laurel wilt of two forest tree species native to the southern USA, Persea borbonia and Persea palustris, [(Raf.) Sarg.] and avocado, Persea americana (Mill.) cv Waldin, was examined and related to tree physiology and xylem anatomy. Net CO2 assimilation (A), stomatal conductance (gs), leaf chlorophyll index (LCI), leaf chlorophyll fluorescence (Fv/Fm), xylem sap flow, theoretical stem hydraulic conductivity (Kh) and xylem vessel anatomy were assessed in trees of each species that were inoculated with R. lauricola and in control trees. Laurel wilt caused a reduction in A, gs, LCI, Fv/Fm and blockage of xylem vessels by tyloses formation that negatively impacted Kh and sap flow in all Persea species. However, disease susceptibility as indicated by canopy wilting and sapwood discoloration was less pronounced in P. americana cv Waldin than in the two forest species. Xylem vessel diameter was significantly smaller in P. borbonia and P. palustris than in P. americana cv Waldin. Differences in laurel wilt susceptibility among species appear to be influenced by physiological and anatomical tree responses.

Circulating CASK is associated with recurrent focal segmental glomerulosclerosis after transplantation.

INTRODUCTION: Focal and Segmental GlomeruloSclerosis (FSGS) can cause nephrotic syndrome with a risk of progression to end-stage renal disease. The idiopathic form has a high rate of recurrence after transplantation, suggesting the presence of a systemic circulating factor that causes glomerular permeability and can be removed by plasmapheresis or protein-A immunoadsorption. RESULTS: To identify this circulating factor, the eluate proteins bound on therapeutic immunoadsorption with protein-A columns were analyzed by comparative electrophoresis and mass spectrometry. A soluble form of calcium/calmodulin-dependent serine protein kinase (CASK) was identified. CASK was immunoprecipitated only in the sera of patients with recurrent FSGS after transplantation and not in control patients. Recombinant-CASK (rCASK) induced the reorganization of the actin cytoskeleton in immortalized podocytes, a redistribution of synaptopodin, ZO-1,vinculin and ENA. rCASK also induced alterations in the permeability of a monolayer of podocytes and increased the motility of pdodocytes in vitro. The extracellular domain of CD98, a transmembrane receptor expressed on renal epithelial cells, has been found to co-immunoprecipitated with rCASK. The invalidation of CD98 with siRNA avoided the structural changes of rCask treated cells suggesting its involvement in physiopathology of the disease. In mice, recombinant CASK induced proteinuria and foot process effacement in podocytes. CONCLUSION: Our results suggest that CASK can induce the recurrence of FSGS after renal transplantation.

SYK Inhibition Induces Apoptosis in Germinal Center-Like B Cells by Modulating the Antiapoptotic Protein Myeloid Cell Leukemia-1, Affecting B-Cell Activation and Antibody Production.

B cells play a major role in the antibody-mediated rejection (AMR) of solid organ transplants, a major public health concern. The germinal center (GC) is involved in the generation of donor-specific antibody-producing plasma cells and memory B cells, which are often poorly controlled by current treatments. Myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the B-cell lymphoma-2 family, is essential for maintenance of the GC reaction and B-cell differentiation. During chronic AMR (cAMR), tertiary lymphoid structures resembling GCs appear in the rejected organ, suggesting local lymphoid neogenesis. We report the infiltration of the kidneys with B cells expressing Mcl-1 in patients with cAMR. We modulated GC viability by impairing B-cell receptor signaling, by spleen tyrosine kinase (SYK) inhibition. SYK inhibition lowers viability and Mcl-1 protein levels in Burkitt's lymphoma cell lines. This downregulation of Mcl-1 is coordinated at the transcriptional level, possibly by signal transducer and activator of transcription 3 (STAT3), as shown by (1) the impaired translocation of STAT3 to the nucleus following SYK inhibition, and (2) the lower levels of Mcl-1 transcription upon STAT3 inhibition. Mcl-1 overproduction prevented cells from entering apoptosis following SYK inhibition. In vitro studies with primary tonsillar B cells confirmed that SYK inhibition impaired cell survival and decreased Mcl-1 protein levels. It also impaired B-cell activation and immunoglobulin G secretion by tonsillar B cells. These findings suggest that the SYK-Mcl-1 pathway could be targeted, to improve graft survival by manipulating the humoral immune response.

IGF-1 contributes to the expansion of melanoma-initiating cells through an epithelial-mesenchymal transition process.

Melanoma is a particularly virulent human cancer, due to its resistance to conventional treatments and high frequency of metastasis. Melanomas contain a fraction of cells, the melanoma-initiating cells (MICs), responsible for tumor propagation and relapse. Identification of the molecular pathways supporting MICs is, therefore, vital for the development of targeted treatments. One factor produced by melanoma cells and their microenvironment, insulin-like growth factor-1 (IGF- 1), is linked to epithelial-mesenchymal transition (EMT) and stemness features in several cancers.We evaluated the effect of IGF-1 on the phenotype and chemoresistance of B16-F10 cells. IGF-1 inhibition in these cells prevented malignant cell proliferation, migration and invasion, and lung colony formation in immunodeficient mice. IGF-1 downregulation also markedly inhibited EMT, with low levels of ZEB1 and mesenchymal markers (N-cadherin, CD44, CD29, CD105) associated with high levels of E-cadherin and MITF, the major regulator of melanocyte differentiation. IGF-1 inhibition greatly reduced stemness features, including the expression of key stem markers (SOX2, Oct-3/4, CD24 and CD133), and the functional characteristics of MICs (melanosphere formation, aldehyde dehydrogenase activity, side population). These features were associated with a high degree of sensitivity to mitoxantrone treatment.In this study, we deciphered new connections between IGF-1 and stemness features and identified IGF-1 as instrumental for maintaining the MIC phenotype. The IGF1/IGF1-R nexus could be targeted for the development of more efficient anti-melanoma treatments. Blocking the IGF-1 pathway would improve the immune response, decrease the metastatic potential of tumor cells and sensitize melanoma cells to conventional treatments.

The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing.

BACKGROUND: After viral infection and the stimulation of some pattern-recognition receptors, TANK-binding kinase I (TBK1) is activated by K63-linked polyubiquitination followed by trans-autophosphorylation. While the activated TBK1 induces type I interferon production by phosphorylating the transcription factor IRF3, the precise molecular mechanisms underlying TBK1 activation remain unclear. RESULTS: We report here the localization of the ubiquitinated and phosphorylated active form of TBK1 to the Golgi apparatus after the stimulation of RIG-I-like receptors (RLRs) or Toll-like receptor-3 (TLR3), due to TBK1 K63-linked ubiquitination on lysine residues 30 and 401. The ubiquitin-binding protein optineurin (OPTN) recruits ubiquitinated TBK1 to the Golgi apparatus, leading to the formation of complexes in which TBK1 is activated by trans-autophosphorylation. Indeed, OPTN deficiency in various cell lines and primary cells impairs TBK1 targeting to the Golgi apparatus and its activation following RLR or TLR3 stimulation. Interestingly, the Bluetongue virus NS3 protein binds OPTN at the Golgi apparatus, neutralizing its activity and thereby decreasing TBK1 activation and downstream signaling. CONCLUSIONS: Our results highlight an unexpected role of the Golgi apparatus in innate immunity as a key subcellular gateway for TBK1 activation after RNA virus infection.

Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells.

The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Burkitt's lymphoma (BL) were associated with cell death. We show that PUMA is localized to the cytosol in these cells. By contrast, various apoptosis-triggering signals were found to promote the translocation of PUMA to the mitochondria in these cells, leading to their death by apoptosis. This apoptosis was associated with the binding of mitochondrial PUMA to anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Mcl-1. This translocation was caspase-independent but was prevented by inhibiting or knocking down the expression of the MAPK kinase p38. Our data suggest that the accumulation of PUMA in the cytosol may be important for the participation of this protein in apoptosis without the need for prior transcription. This regulatory pathway may be an important feature of differentiation and tumorigenic processes.

Exploiting the potential of autophagy in cisplatin therapy: A new strategy to overcome resistance.

Resistance to cisplatin is a major challenge in the current cancer therapy. In order to explore new therapeutic strategies to cisplatin resistance, we evaluated, in a model of lung cancer (H1299 and H460 cell lines), the nature of the pathways leading to cell death. We observed that H1299 displayed a natural resistance to cisplatin due to an inability to trigger an apoptotic response that correlates with the induction of autophagy. However, pharmacological and genetic approaches showed how autophagy was a mechanism associated to cell death rather than to resistance. Indeed, pro-autophagic stimuli such as mTOR or Akt inhibition mediate cell death in both cell lines to a similar extent. We next evaluated the response to a novel platinum compound, monoplatin, able to promote cell death in an exclusive autophagy-dependent manner. In this case, no differences were observed between both cell lines. Furthermore, in response to monoplatin, two molecular hallmarks of cisplatin response (p53 and MAPKs) were not implicated, indicating the ability of this pro-autophagic compound to overcome cisplatin resistance. In summary, our data highlight how induction of autophagy could be used in cisplatin resistant tumours and an alternative treatment for p53 mutated patient in a synthetic lethally approach.

Cannabinoid receptor 1 is a major mediator of renal fibrosis.

Chronic kidney disease, secondary to renal fibrogenesis, is a burden on public health. There is a need to explore new therapeutic pathways to reduce renal fibrogenesis. To study this, we used unilateral ureteral obstruction (UUO) in mice as an experimental model of renal fibrosis and microarray analysis to compare gene expression in fibrotic and normal kidneys. The cannabinoid receptor 1 (CB1) was among the most upregulated genes in mice, and the main endogenous CB1 ligand (2-arachidonoylglycerol) was significantly increased in the fibrotic kidney. Interestingly, CB1 expression was highly increased in kidney biopsies of patients with IgA nephropathy, diabetes, and acute interstitial nephritis. Both genetic and pharmacological knockout of CB1 induced a profound reduction in renal fibrosis during UUO. While CB2 is also involved in renal fibrogenesis, it did not potentiate the role of CB1. CB1 expression was significantly increased in myofibroblasts, the main effector cells in renal fibrogenesis, upon TGF-ß1 stimulation. The decrease in renal fibrosis during CB1 blockade could be explained by a direct action on myofibroblasts. CB1 blockade reduced collagen expression in vitro. Rimonabant, a selective CB1 endocannabinoid receptor antagonist, modulated the macrophage infiltrate responsible for renal fibrosis in UUO through a decrease in monocyte chemoattractant protein-1 synthesis. Thus, CB1 has a major role in the activation of myofibroblasts and may be a new target for treating chronic kidney disease.

The E3 ubiquitin ligase RNF121 is a positive regulator of NF-κB activation.

BACKGROUND: The nuclear factor κB (NF-κB) family members regulate several biological processes as cell proliferation and differentiation, inflammation, immunity and tumor progression. Ubiquitination plays a key role in NF-κB activation and the ubiquitylated transmitters of the NF-κB signaling cascade accumulate in close proximity to endomembranes. FINDINGS: We performed an unbiased siRNA library screen targeting the 46 E3 ubiquitin ligases bearing transmembrane domains to uncover new modulators of NF-κB activation, using tumor necrosis factor-α (TNF-α) receptor (TNFR) stimulation as a model. We report here the identification of a new Golgi Apparatus-resident protein, RNF121, as an enhancer of NF-κB promoter activity through the catalytic function of its RING domain. From a molecular standpoint, while knocking down RNF121 did not alter RIP1 ubiquitination and IKK activation, the proteasomal degradation of IκBα was impaired suggesting that this E3 ubiquitin ligase regulates this process. However, RNF121 did not directly ubiquitinate IκBα While they were found in the same complex. Finally, we discovered that RNF121 acts as a broad regulator of NF-κB signaling since its silencing also dampens NF-κB activation following stimulation of Toll-Like Receptors (TLRs), Nod-Like Receptors (NLRs), RIG-I-Like Receptors (RLRs) or after DNA damages. CONCLUSIONS: These results unveil an unexpected role of Golgi Apparatus and reveal RNF121 as a new player involved in the signaling leading to NF-κB activation.

Mitochondrial hyperfusion promotes NF-κB activation via the mitochondrial E3 ligase MULAN.

Mitochondria are dynamic organelles with a morphology resulting from the balance between two opposing processes: fusion and fission. Little is known about the function of mitochondrial fusion, beside its role in the maintenance of mitochondrial DNA. We report here that enforced mitochondrial hyperfusion, due to the expression of a dominant-negative mutant of Drp1 or of MARCH5, promotes NF-κB activation in a TAK1- and IKK-dependent manner, through the mitochondrial E3 ubiquitin ligase MULAN. The capability of MULAN to activate NF-κB depends on its RING domain and on the E3 ubiquitin ligase TRAF2. Under physiological conditions, stress-induced mitochondrial hyperfusion (SIMH) is also accompanied by NF-κB activation, and the prevention of SIMH or the knockdown of MULAN impairs NF-κB activation. During SIMH, MULAN forms a complex with TRAF2 and modulates its ubiquitylation, signifying that TRAF2 may serve as an ubiquitylated transmitter of NF-κB signaling in this pathway. Our results suggest that mitochondria, through their dynamics, convert stress signals into a cell response leading to NF-κB activation.

Protein A immunoadsorption cannot significantly remove the soluble receptor of urokinase from sera of patients with recurrent focal segmental glomerulosclerosis.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a serious disease, the pathogenesis of which is unknown. Its recurrence after transplantation (Tx) and its partial remission after treatment with immunoadsorption (IA) on a protein A column indicate the existence of a circulating factor responsible for the disease that is able to bind to a protein A column. Recently, the soluble receptor of urokinase (suPAR) was described as the factor responsible for FSGS. We tested the capacity of suPAR to bind to protein A and to be eliminated by IA. METHODS: We measured suPAR in eluates of protein A columns from seven patients with recurrent FSGS after Tx (rFSGS) treated with IA, and in the serum of 13 patients with rFSGS and 11 healthy donors (HDs). Additionally, the plasma of these patients was immunoadsorbed in vitro on a protein A Sepharose column, and we quantified suPAR in the eluates and in pre- and post-column samples. RESULTS: The concentration of suPAR was higher in the plasma of patients with rFSGS than that of HD patients. However, the concentration of suPAR was similar before and after IA on protein A for the rFSGS and HD samples. The suPAR concentration was very low in the eluates from protein A columns incubated with plasma from HD or rFSGS patients. However, 85% of rFSGS patients showed a decrease in immunoglobulin G and proteinuria. CONCLUSIONS: Thus, suPAR does not significantly bind to protein A in vitro or in vivo.

The endoplasmic reticulum acts as a platform for ubiquitylated components of nuclear factor κB signaling.

The innate and adaptive immune responses involve the stimulation of nuclear factor κB (NF-κB) transcription factors through the Lys(63) (K(63))-linked ubiquitylation of specific components of NF-κB signaling pathways. We found that ubiquitylated components of the NF-κB pathway accumulated on the cytosolic leaflet of the endoplasmic reticulum (ER) membrane after the engagement of cell-surface, proinflammatory cytokine receptors or antigen receptors. Through mass spectrometric analysis, we found that the ER-anchored protein metadherin (MTDH) was a partner for these ubiquitylated activators of NF-κB and that it directly bound to K(63)-linked polyubiquitin chains. Knockdown of MTDH inhibited the accumulation of ubiquitylated NF-κB signaling components at the ER, reduced the extent of NF-κB activation, and decreased the amount of proinflammatory cytokines produced. Our observations highlight an unexpected facet of the ER as a key subcellular gateway for NF-κB activation.

Negative regulation of NF-κB signaling in T lymphocytes by the ubiquitin-specific protease USP34.

BACKGROUND: NF-κB is a master gene regulator involved in plethora of biological processes, including lymphocyte activation and proliferation. Reversible ubiquitinylation of key adaptors is required to convey the optimal activation of NF-κB. However the deubiquitinylases (DUBs), which catalyze the removal of these post-translational modifications and participate to reset the system to basal level following T-Cell receptor (TCR) engagement continue to be elucidated. FINDINGS: Here, we performed an unbiased siRNA library screen targeting the DUBs encoded by the human genome to uncover new regulators of TCR-mediated NF-κB activation. We present evidence that knockdown of Ubiquitin-Specific Protease 34 (USP34) selectively enhanced NF-κB activation driven by TCR engagement, similarly to siRNA against the well-characterized DUB cylindromatosis (CYLD). From a molecular standpoint, USP34 silencing spared upstream signaling but led to a more pronounced degradation of the NF-κB inhibitor IκBα, and culminated with an increased DNA binding activity of the transcription factor. CONCLUSIONS: Collectively, our data unveils USP34 as a new player involved in the fine-tuning of NF-κB upon TCR stimulation.

MAVS ubiquitination by the E3 ligase TRIM25 and degradation by the proteasome is involved in type I interferon production after activation of the antiviral RIG-I-like receptors.

BACKGROUND: During a viral infection, the intracellular RIG-I-like receptors (RLRs) sense viral RNA and signal through the mitochondrial antiviral signaling adaptor MAVS (also known as IPS-1, Cardif and VISA) whose activation triggers a rapid production of type I interferons (IFN) and of pro-inflammatory cytokines through the transcription factors IRF3/IRF7 and NF-κB, respectively. While MAVS is essential for this signaling and known to operate through the scaffold protein NEMO and the protein kinase TBK1 that phosphorylates IRF3, its mechanism of action and regulation remain unclear. RESULTS: We report here that RLR activation triggers MAVS ubiquitination on lysine 7 and 10 by the E3 ubiquitin ligase TRIM25 and marks it for proteasomal degradation concomitantly with downstream signaling. Inhibition of this MAVS degradation with a proteasome inhibitor does not affect NF-κB signaling but it hampers IRF3 activation, and NEMO and TBK1, two essential mediators in type I IFN production, are retained at the mitochondria. CONCLUSIONS: These results suggest that MAVS functions as a recruitment platform that assembles a signaling complex involving NEMO and TBK1, and that the proteasome-mediated MAVS degradation is required to release the signaling complex into the cytosol, allowing IRF3 phosphorylation by TBK1.

Differentiation therapy: targeting human renal cancer stem cells with interleukin 15.

BACKGROUND: Many renal cancer patients experience disease recurrence after immunotherapy or combined treatments due to persistence of cancer stem cells (CSCs). The identification of reliable inducers of CSC differentiation may facilitate the development of efficient strategies for eliminating CSCs. We investigated whether interleukin 15 (IL-15), a regulator of kidney homeostasis, induces the differentiation of CD105-positive (CD105(+)) CSCs from human renal cancers. METHODS: CD105(+) CSCs were cultured to preserve their stem cell properties and treated with recombinant human IL-15 (rhIL-15) to evaluate their ability to differentiate, to acquire sensitivity to chemotherapeutic drugs, and to form spheroids in vitro and tumors in vivo. Expression of stem cell and epithelial markers were studied by flow cytometry, immunocytochemistry, and immunoblotting. Identification of a CSC side population fraction and its sensitivity to chemotherapy drugs and expression of ATP-binding cassette (ABC) transporters and aldehyde dehydrogenase (ALDH) activities were determined by flow cytometry. Spheroid formation was determined in limiting dilution assay. Xenograft tumors were generated in severe combined immunodeficient mice (n = 12-18 mice per group). All statistical tests were two-sided. RESULTS: CD105(+) CSCs treated with rhIL-15 at 10 pg/mL differentiated into cells expressing epithelial markers. rhIL-15 induced epithelial differentiation of all CD105(+) CSCs subsets and blocked CSC self-renewal (sphere-forming ability) and their tumorigenic properties in severe combined immunodeficient mice. Vinblastine and paclitaxel induced statistically significant higher levels of apoptosis in rhIL-15-differentiated epithelial cells compared with CD105(+) CSCs (mean percentage of apoptotic cells, vinblastine: 33% vs 16.5%, difference = 16.5%, 95% confidence interval = 12.25% to 20.74%, P = .0025; paclitaxel: 35% vs 11.6%, difference = 23.4%, 95% confidence interval = 22.5% to 24.24%, P = .0015). The higher sensitivity of rhIL-15-differentiated epithelial cells to chemotherapeutic drugs was associated with loss of detoxifying mechanisms such as ALDH and ABC transporter activities. CONCLUSION: IL-15 directs the epithelial differentiation of renal CSCs and meets the criteria for a treatment strategy: CSC pool depletion and generation of differentiated nontumorigenic cells that are sensitive to chemotherapeutic agents.

Regulation of memory B-cell survival by the BH3-only protein Puma.

Apoptosis is crucial for immune system homeostasis, including selection and survival of long-lived antibody-forming cells and memory cells. The interactions between proapoptotic and pro-survival proteins of the Bcl-2 family are critical for this process. In this report, we show that expression of the proapoptotic BH3-only Bcl-2 family member Puma was selectively up-regulated on in vitro activation with antigens or mitogens of both human and mouse B cells. Puma expression coincided in vivo, with the prosurvival Bcl-2 family member Mcl-1 within the germinal centers and its expression correlates with the germinal center like phenotype of Burkitt lymphoma. Experiments performed in Puma-deficient mice revealed that Puma is essential for apoptosis of mitogen-activated B cells in vitro and for the control of memory B-cell survival. In conclusion, using both human and murine models, our data show that Puma has a major role in the T cell- dependent B-cell immune response. These data demonstrate that Puma is a major regulator of memory B lymphocyte survival and therefore a key molecule in the control of the immune response.

Participation of the cell polarity protein PALS1 to T-cell receptor-mediated NF-κB activation.

BACKGROUND: Beside their established function in shaping cell architecture, some cell polarity proteins were proposed to participate to lymphocyte migration, homing, scanning, as well as activation following antigen receptor stimulation. Although PALS1 is a central component of the cell polarity network, its expression and function in lymphocytes remains unknown. Here we investigated whether PALS1 is present in T cells and whether it contributes to T Cell-Receptor (TCR)-mediated activation. METHODOLOGY/PRINCIPAL FINDINGS: By combining RT-PCR and immunoblot assays, we found that PALS1 is constitutively expressed in human T lymphocytes as well as in Jurkat T cells. siRNA-based knockdown of PALS1 hampered TCR-induced activation and optimal proliferation of lymphocyte. We further provide evidence that PALS1 depletion selectively hindered TCR-driven activation of the transcription factor NF-κB. CONCLUSIONS: The cell polarity protein PALS1 is expressed in T lymphocytes and participates to the optimal activation of NF-κB following TCR stimulation.