Ca<Superscript>2+</Superscript> signals,cell membrane disintegration,and activation of TMEM16F during necroptosis

Activated receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like (MLKL) are essential components of the necroptotic pathway. Phosphorylated MLKL (pMLKL) is thought to induce membrane leakage, leading to cell swelling and disintegration of the cell membrane. However, the molecular identity of the necroptotic membrane pore remains unclear, and the role of pMLKL for membrane permeabilization is currently disputed. We observed earlier that the phospholipid scramblase and ion channel TMEM16F/anoctamin 6 cause large membrane currents, cell swelling, and cell death when activated by a strong increase in intracellular Ca²⁺. We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNFα, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT₂₉, 16HBE, H441, and L929. Time-dependent changes in intracellular Ca²⁺, cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca²⁺, which was paralleled by the activation of outwardly rectifying Cl^? currents, which were typical for TMEM16F/ANO6. Ca²⁺ oscillations were due to Ca²⁺ release from endoplasmic reticulum, and were independent of extracellular Ca²⁺. The initial TSZ-induced cell swelling was followed by cell shrinkage. Using typical channel blockers and siRNA-knockdown, the Cl^? currents were shown to be due to the activation of ANO6. However, the knockdown of ANO6 or inhibitors of ANO6 did not inhibit necroptotic cell death. The present data demonstrate the activation of ANO6 during necroptosis, which, however, is not essential for cell death.     

A new parasitoid (Hymenoptera: Braconidae: Aphidiinae) of the invasive bamboo aphids Takecallis spp. (Hemiptera: Aphididae) from Western Europe

A long-term survey of tritrophic (plant–aphid–parasitoid) associations in the urban ecosystems of Lleida (Catalonia) and Paris (France) resulted in the detection of associations of two bamboo aphids, Takecallis arundinariae (Essig) and Takecallis taiwanus (Takahashi), respectively, with a new aphid parasitoid species. Trioxys remaudierei Starý & Rakhshani sp. nov. is described and illustrated as a unique parasitoid of Takecallis aphids outside the area of their origin. The new species is easily distinguishable from its congeners in having the ventral prongs of the abdomen fused over two-thirds of their length, then bifurcated towards the tip. The only morphologically similar species is Trioxys betulae (Marshall), which exhibits a clearly different prong shape (and has a different host range, Symydobius Mordvilko and Clethrobius Mordvilko). The new species is compared with allied taxa associated with bamboo aphids. The occurrence of Takecallis taiwanus on bamboo is recorded in France for the first time.

www.zoobank.org/urn:lsid:zoobank.org:pub:ED15BA16-E8A9-4CEA-BDA7-FBAB02FEB091     

Microhabitat selection in the sand recluse spider (Sicarius thomisoides): the effect of rock size and temperature

In spiders, temperature is considered an important environmental variable for microhabitat selection. In this study, we evaluated the effect of temperature and rock size on the presence of the sand recluse spider Sicarius thomisoides and the degree of selectivity in different locations. This species is a large spider that lives under rocks in desert and semi-desert climates and is particularly active during the summer. In Chile, these spiders can be found at both coastal and inland locations under different thermal conditions, where usually the temperatures are lower near the coast. If large-scale climatic conditions are important for this species, they may be expected to select lower rock temperatures on the coast than at inland locations. In addition, we would expect that the spiders would choose larger rocks in inland compared to coast locations, which reduce the effect of high temperatures. We found that the probability of finding individuals of this species increased according to rock temperature and rock size in the field. Our results suggest that S. thomisoides prefers larger and warmer rocks to shelter under during the day, this selectivity being similar at both coastal and inland locations. Thus, this species tends to select rocks with the same thermal and structural conditions, independent of the climatic conditions.     

Activation of ataxia telangiectasia muted under experimental models and human Parkinson’s disease

In the present study we demonstrated that neurotoxin MPP⁺-induced DNA damage is followed by ataxia telangiectasia muted (ATM) activation either in cerebellar granule cells (CGC) or in B65 cell line. In CGC, the selective ATM inhibitor KU-55933 showed neuroprotective effects against MPP⁺-induced neuronal cell loss and apoptosis, lending support to the key role of ATM in experimental models of Parkinson’s disease. Likewise, we showed that knockdown of ATM levels in neuroblastoma B65 cells using an ATM-specific siRNA attenuates the phosphorylation of retinoblastoma protein without affecting other cell-cycle proteins involved in the G₀/G₁ cell-cycle phase. Moreover, we demonstrated DNA damage, in human brain samples of PD patients. These findings support a model in which MPP⁺ leads to ATM activation with a subsequent DNA damage response and activation of pRb. Therefore, this study demonstrates a new link between DNA damage by MPP⁺ and cell-cycle re-entry through retinoblastoma protein phosphorylation.     

Common variants at TRAF3IP2 are associated with susceptibility to psoriatic arthritis and psoriasis

Psoriatic arthritis (PsA) is an inflammatory joint disease that is distinct from other chronic arthritides and which is frequently accompanied by psoriasis vulgaris (PsV) and seronegativity for rheumatoid factor. We conducted a genome-wide association study in 609 German individuals with PsA (cases) and 990 controls with replication in 6 European cohorts including a total of 5,488 individuals. We replicated PsA associations at HLA-C and IL12B and identified a new association at TRAF3IP2 (rs13190932, P = 8.56 × 10?1?). TRAF3IP2 was also associated with PsV in a German cohort including 2,040 individuals (rs13190932, P = 1.95 × 10?3). Sequencing of the exons of TRAF3IP2 identified a coding variant (p.Asp10Asn, rs33980500) as the most significantly associated SNP (P = 1.13 × 10?2?, odds ratio = 1.95). Functional assays showed reduced binding of this TRAF3IP2 variant to TRAF6, suggesting altered modulation of immunoregulatory signals through altered TRAF interactions as a new and shared pathway for PsA and PsV.     

Hormonal control of Sertoli cell metabolism regulates spermatogenesis

Hormonal regulation is essential to spermatogenesis. Sertoli cells (SCs) have functions that reach far beyond the physical support of germ cells, as they are responsible for creating the adequate ionic and metabolic environment for germ cell development. Thus, much attention has been given to the metabolic functioning of SCs. During spermatogenesis, germ cells are provided with suitable metabolic substrates, in a set of events mediated by SCs. Multiple signaling cascades regulate SC function and several of these signaling pathways are hormone-dependent and cell-specific. Within the seminiferous tubules, only SCs possess receptors for some hormones rendering them major targets for the hormonal signaling that regulates spermatogenesis. Although the mechanisms by which SCs fulfill their own and germ cells metabolic needs are mostly studied in vitro, SC metabolism is unquestionably a regulation point for germ cell development and the hormonal control of these processes is required for a normal spermatogenesis.     

Extensive characterization of sphere models established from colorectal cancer cell lines

Links between cancer and stem cells have been proposed for many years. As the cancer stem cell (CSC) theory became widely studied, new methods were developed to culture and expand cancer cells with conserved determinants of “stemness”. These cells show increased ability to grow in suspension as spheres in serum-free medium supplemented with growth factors and chemicals. The physiological relevance of this phenomenon in established cancer cell lines remains unclear. Cell lines have traditionally been used to explore tumor biology and serve as preclinical models for the screening of potential therapeutic agents. Here, we grew cell-forming spheres (CFS) from 25 established colorectal cancer cell lines. The molecular and cellular characteristics of CFS were compared to the bulk of tumor cells. CFS could be isolated from 72 % of the cell lines. Both CFS and their parental CRC cell lines were highly tumorigenic. Compared to their parental cells, they showed similar expression of putative CSC markers. The ability of CRC cells to grow as CFS was greatly enhanced by prior treatment with 5-fluorouracil. At the molecular level, CFS and parental CRC cells showed identical gene mutations and very similar genomic profiles, although microarray analysis revealed changes in CFS gene expression that were independent of DNA copy-number. We identified a CFS gene expression signature common to CFS from all CRC cell lines, which was predictive of disease relapse in CRC patients. In conclusion, CFS models derived from CRC cell lines possess interesting phenotypic features that may have clinical relevance for drug resistance and disease relapse.     

Role of the ubiquitin–proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake

The protective effect of high density lipoproteins (HDL) against atherosclerosis is mainly attributed to their capacity to transport excess cholesterol from peripheral tissues back to the liver for further elimination into the bile, a process called reverse cholesterol transport (RCT). Recently, the importance of the P2Y₁₃ receptor (P2Y₁₃-R) was highlighted in HDL metabolism since HDL uptake by the liver was decreased in P2Y₁₃-R deficient mice, which translated into impaired RCT. Here, we investigated for the first time the molecular mechanisms regulating cell surface expression of P2Y₁₃-R. When transiently expressed, P2Y₁₃-R was mainly detected in the endoplasmic reticulum (ER) and strongly subjected to proteasome degradation while its homologous P2Y₁₂ receptor (P2Y₁₂-R) was efficiently targeted to the plasma membrane. We observed an inverse correlation between cell surface expression and ubiquitination level of P2Y₁₃-R in the ER, suggesting a close link between ubiquitination of P2Y₁₃-R and its efficient targeting to the plasma membrane. The C-terminus tail exchange between P2Y₁₃-R and P2Y₁₂-R strongly restored plasma membrane expression of P2Y₁₃-R, suggesting the involvement of the intra-cytoplasmic tail of P2Y₁₃-R in expression defect. Accordingly, proteasomal inhibition increased plasma membrane expression of functionally active P2Y₁₃-R in hepatocytes, and consequently stimulated P2Y₁₃-R-mediated HDL endocytosis. Importantly, proteasomal inhibition strongly potentiated HDL hepatic uptake (>200 %) in wild-type but not in P2Y₁₃-R-deficient mice, thus reinforcing the role of P2Y₁₃-R expression in regulating HDL metabolism. Therefore, specific inhibition of the ubiquitin–proteasome system might be a novel powerful HDL therapy to enhance P2Y₁₃-R expression and consequently promote the overall RCT.     

Critical role of extracellular vesicles in modulating the cellular effects of cytokines

Under physiological and pathological conditions, extracellular vesicles (EVs) are present in the extracellular compartment simultaneously with soluble mediators. We hypothesized that cytokine effects may be modulated by EVs, the recently recognized conveyors of intercellular messages. In order to test this hypothesis, human monocyte cells were incubated with CCRF acute lymphoblastic leukemia cell line-derived EVs with or without the addition of recombinant human TNF, and global gene expression changes were analyzed. EVs alone regulated the expression of numerous genes related to inflammation and signaling. In combination, the effects of EVs and TNF were additive, antagonistic, or independent. The differential effects of EVs and TNF or their simultaneous presence were also validated by Taqman assays and ELISA, and by testing different populations of purified EVs. In the case of the paramount chemokine IL-8, we were able to demonstrate a synergistic upregulation by purified EVs and TNF. Our data suggest that neglecting the modulating role of EVs on the effects of soluble mediators may skew experimental results. On the other hand, considering the combined effects of cytokines and EVs may prove therapeutically useful by targeting both compartments at the same time.