APRF1 Interactome Reveals HSP90 as a New Player in the Complex That Epigenetically Regulates Flowering Time in Arabidopsis thaliana.

WD40 repeat proteins (WDRs) are present in all eukaryotes and include members that are implicated in numerous cellular activities. They act as scaffold proteins and thus as molecular "hubs" for protein-protein interactions, which mediate the assembly of multifunctional complexes that regulate key developmental processes in Arabidopsis thaliana, such as flowering time, hormonal signaling, and stress responses. Despite their importance, many aspects of their putative functions have not been elucidated yet. Here, we show that the late-flowering phenotype of the anthesis promoting factor 1 (aprf1) mutants is temperature-dependent and can be suppressed when plants are grown under mild heat stress conditions. To gain further insight into the mechanism of APRF1 function, we employed a co-immunoprecipitation (Co-IP) approach to identify its interaction partners. We provide the first interactome of APRF1, which includes proteins that are localized in several subcellular compartments and are implicated in diverse cellular functions. The dual nucleocytoplasmic localization of ARRF1, which was validated through the interaction of APRF1 with HEAT SHOCK PROTEIN 1 (HSP90.1) in the nucleus and with HSP90.2 in the cytoplasm, indicates a dynamic and versatile involvement of APRF1 in multiple biological processes. The specific interaction of APRF1 with the chaperon HSP90.1 in the nucleus expands our knowledge regarding the epigenetic regulation of flowering time in A. thaliana and further suggests the existence of a delicate thermoregulated mechanism during anthesis.

Genetic Targeting of dSAMTOR, A Negative dTORC1 Regulator, during Drosophila Aging: A Tissue-Specific Pathology.

mTORC1 regulates mammalian cell metabolism and growth in response to diverse environmental stimuli. Nutrient signals control the localization of mTORC1 onto lysosome surface scaffolds that are critically implicated in its amino acid-dependent activation. Arginine, leucine and S-adenosyl-methionine (SAM) can serve as major mTORC1-signaling activators, with SAM binding to SAMTOR (SAM + TOR), a fundamental SAM sensor, preventing the protein's (SAMTOR's) inhibitory action(s) against mTORC1, thereby triggering its (mTORC1) kinase activity. Given the lack of knowledge regarding the role of SAMTOR in invertebrates, we have identified the Drosophila SAMTOR homologue (dSAMTOR) in silico and have, herein, genetically targeted it through the utilization of the GAL4/UAS transgenic tool. Survival profiles and negative geotaxis patterns were examined in both control and dSAMTOR-downregulated adult flies during aging. One of the two gene-targeted schemes resulted in lethal phenotypes, whereas the other one caused rather moderate pathologies in most tissues. The screening of head-specific kinase activities, via PamGene technology application, unveiled the significant upregulation of several kinases, including the dTORC1 characteristic substrate dp70S6K, in dSAMTOR-downregulated flies, thus strongly supporting the inhibitory dSAMTOR action(s) upon the dTORC1/dp70S6K signaling axis in Drosophila brain settings. Importantly, genetic targeting of the Drosophila BHMT bioinformatics counterpart (dBHMT), an enzyme that catabolizes betaine to produce methionine (the SAM precursor), led to severe compromises in terms of fly longevity, with glia-, motor neuron- and muscle-specific dBHMT downregulations exhibiting the strongest effects. Abnormalities in wing vein architectures were also detected in dBHMT-targeted flies, thereby justifying their notably reduced negative geotaxis capacities herein observed mainly in the brain-(mid)gut axis. In vivo adult fly exposure to clinically relevant doses of methionine revealed the mechanistic synergism of decreased dSAMTOR and increased methionine levels in pathogenic longevity, thus rendering (d)SAMTOR an important component in methionine-associated disorders, including homocystinuria(s).

Effect of Cord Blood Platelet Gel on wound healing capacity of human Mesenchymal Stromal Cells.

BACKGROUND: Wound healing is a dynamic process, involving the recruitment of growth factors, cytokines, chemokines and cellular populations. Recently, the Cord Blood Platelet Gel (CBPG) has been applied successfully in wound closure and tissue regeneration. Moreover, its proper combination with stem cell populations such as Mesenchymal Stromal Cells (MSCs) may positively improve the wound healing process. Based on the above data, this study aimed to the evaluation of wound healing capacity of MSCs combined with CBPG under in vitro conditions. METHODS: Initially, CBPG was developed from Cord Blood Units (CBUs). The determination of wound healing ability of MSCs was performed using the scratch wound assay. In addition, the morphological features, immunophenotypical characteristics and differentiation capacity of MSCs were evaluated. RESULTS: Scratch wound assay results showed, that CBPG could positively stimulate the MSCs migration. Moreover, MSCs cultured in presence of CBPG were characterized by elongated shape and improved stemness properties as it was indicated by flow cytometric analysis and differentiation process. CONCLUSION: These results clearly showed the beneficial effect of CBPG in combination with MSCs in wound healing. The proper combination of CBPG with stem cells strategy may enhance the healing process in patients with skin erosions.

Human Melanoma-Cell Metabolic Profiling: Identification of Novel Biomarkers Indicating Metastasis.

Melanoma is the most aggressive type of skin cancer, leading to metabolic rewiring and enhancement of metastatic transformation. Efforts to improve its early and accurate diagnosis are largely based on preclinical models and especially cell lines. Hence, we herein present a combinational Nuclear Magnetic Resonance (NMR)- and Ultra High Performance Liquid Chromatography-High-Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS)-mediated untargeted metabolomic profiling of melanoma cells, to landscape metabolic alterations likely controlling metastasis. The cell lines WM115 and WM2664, which belong to the same patient, were examined, with WM115 being derived from a primary, pre-metastatic, tumor and WM2664 clonally expanded from lymph-node metastases. Metabolite samples were analyzed using NMR and UHPLC-HRMS. Multivariate statistical analysis of high resolution NMR and MS (positive and negative ionization) results was performed by Principal Component Analysis (PCA), Partial Least Squares-Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA), while metastasis-related biomarkers were determined on the basis of VIP lists, S-plots and Student's t-tests. Receiver Operating Characteristic (ROC) curves of NMR and MS data revealed significantly differentiated metabolite profiles for each cell line, with WM115 being mainly characterized by upregulated levels of phosphocholine, choline, guanosine and inosine. Interestingly, WM2664 showed notably increased contents of hypoxanthine, myo-inositol, glutamic acid, organic acids, purines, pyrimidines, AMP, ADP, ATP and UDP(s), thus indicating the critical roles of purine, pyrimidine and amino acid metabolism during human melanoma metastasis.

Malignancy Grade-Dependent Mapping of Metabolic Landscapes in Human Urothelial Bladder Cancer: Identification of Novel, Diagnostic, and Druggable Biomarkers.

BACKGROUND: Urothelial bladder cancer (UBC) is one of the cancers with the highest mortality rate and prevalence worldwide; however, the clinical management of the disease remains challenging. Metabolomics has emerged as a powerful tool with beneficial applications in cancer biology and thus can provide new insights on the underlying mechanisms of UBC progression and/or reveal novel diagnostic and therapeutic schemes. METHODS: A collection of four human UBC cell lines that critically reflect the different malignancy grades of UBC was employed; RT4 (grade I), RT112 (grade II), T24 (grade III), and TCCSUP (grade IV). They were examined using Nuclear Magnetic Resonance, Mass Spectrometry, and advanced statistical approaches, with the goal of creating new metabolic profiles that are mechanistically associated with UBC progression toward metastasis. RESULTS: Distinct metabolic profiles were observed for each cell line group, with T24 (grade III) cells exhibiting the most abundant metabolite contents. AMP and creatine phosphate were highly increased in the T24 cell line compared to the RT4 (grade I) cell line, indicating the major energetic transformation to which UBC cells are being subjected during metastasis. Thymosin ß4 and ß10 were also profiled with grade-specific patterns of expression, strongly suggesting the importance of actin-cytoskeleton dynamics for UBC advancement to metastatic and drug-tolerant forms. CONCLUSIONS: The present study unveils a novel and putatively druggable metabolic signature that holds strong promise for early diagnosis and the successful chemotherapy of UBC disease.

Recipient's effects on stored red blood cell performance: the case of uremic plasma.

BACKGROUND: Despite universal administration of erythropoiesis-stimulating agents, patients with end-stage renal disease (ESRD) are at high risk for presenting persistent anemia. Due to ambiguities in optimal hemoglobin targets and evidence of recombinant human erythropoietin (EPO)-related toxicity, an increase in blood transfusions has been observed in chronic renal disease over the past years. The probable effects of uremic plasma on the performance of stored red blood cells (RBCs) after transfusion have not been investigated. STUDY DESIGN AND METHODS: Leukoreduced RBCs after short or long storage in CPD-SAGM (n = 5) were assessed for hemolysis, surface removal signaling, reactive oxygen species (ROS) accumulation, and shape distortions before and after reconstitution with healthy (n = 10) or uremic plasma from ESRD patients (n = 20) for 24 hours at physiologic temperature, by using a previously reported in vitro model of transfusion. RESULTS: Temperature and cell environment shifts from blood bag to plasma independently and in synergy affected the RBC physiology. Outcome measures at transfusion-simulating conditions might not be analogous to timing of storage lesion. The uremic plasma ameliorated the susceptibility of stored RBCs to hemolysis, phosphatidylserine externalization, and ROS generation after stimulation by oxidants, but negatively affected shape homeostasis versus healthy plasma. Creatinine, uric acid, and EPO levels had correlations with the performance of stored RBCs in ESRD plasma. CONCLUSION: Renal insufficiency and EPO supplementation likely affect the recovery of donor RBCs and the reactivity of RBCs after transfusion by exerting both toxic and cytoprotective influences on them. ESRD patients constitute a specific recipient group that deserves further examination.

Revisiting Histone Deacetylases in Human Tumorigenesis: The Paradigm of Urothelial Bladder Cancer.

Urinary bladder cancer is a common malignancy, being characterized by substantial patient mortality and management cost. Its high somatic-mutation frequency and molecular heterogeneity usually renders tumors refractory to the applied regimens. Hitherto, methotrexate-vinblastine-adriamycin-cisplatin and gemcitabine-cisplatin represent the backbone of systemic chemotherapy. However, despite the initial chemosensitivity, the majority of treated patients will eventually develop chemoresistance, which severely reduces their survival expectancy. Since chromatin regulation genes are more frequently mutated in muscle-invasive bladder cancer, as compared to other epithelial tumors, targeted therapies against chromatin aberrations in chemoresistant clones may prove beneficial for the disease. "Acetyl-chromatin" homeostasis is regulated by the opposing functions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The HDAC/SIRT (super-)family contains 18 members, which are divided in five classes, with each family member being differentially expressed in normal urinary bladder tissues. Since a strong association between irregular HDAC expression/activity and tumorigenesis has been previously demonstrated, we herein attempt to review the accumulated published evidences that implicate HDACs/SIRTs as critical regulators in urothelial bladder cancer. Moreover, the most extensively investigated HDAC inhibitors (HDACis) are also analyzed, and the respective clinical trials are also described. Interestingly, it seems that HDACis should be preferably used in drug-combination therapeutic schemes, including radiation.

Gene-Specific Intron Retention Serves as Molecular Signature that Distinguishes Melanoma from Non-Melanoma Cancer Cells in Greek Patients.

BACKGROUND: Skin cancer represents the most common human malignancy, and it includes BCC, SCC, and melanoma. Since melanoma is one of the most aggressive types of cancer, we have herein attempted to develop a gene-specific intron retention signature that can distinguish BCC and SCC from melanoma biopsy tumors. METHODS: Intron retention events were examined through RT-sqPCR protocols, using total RNA preparations derived from BCC, SCC, and melanoma Greek biopsy specimens. Intron-hosted miRNA species and their target transcripts were predicted via the miRbase and miRDB bioinformatics platforms, respectively. Ιntronic ORFs were recognized through the ORF Finder application. Generation and visualization of protein interactomes were achieved by the IntAct and Cytoscape softwares, while tertiary protein structures were produced by using the I-TASSER online server. RESULTS: c-MYC and Sestrin-1 genes proved to undergo intron retention specifically in melanoma. Interaction maps of proteins encoded by genes being potentially targeted by retained intron-accommodated miRNAs were generated and SRPX2 was additionally delivered to our melanoma-specific signature. Novel ORFs were identified in MCT4 and Sestrin-1 introns, with potentially critical roles in melanoma development. CONCLUSIONS: The property of c-MYC, Sestrin-1, and SRPX2 genes to retain specific introns could be clinically used to molecularly differentiate non-melanoma from melanoma tumors.

Donor-specific individuality of red blood cell performance during storage is partly a function of serum uric acid levels.

BACKGROUND: Previous investigations in leukoreduced units of red blood cells (RBCs) in mannitol additive solution revealed the close association of uric acid (UA) levels in vivo with the susceptibility of RBCs to storage lesion markers. In this study, we examined whether UA has a similar correlation with the capability of RBCs to cope with the oxidative provocations of storage under different conditions, namely, in CPDA-1 and in the absence of leukoreduction. STUDY DESIGN AND METHODS: The UA-dependent antioxidant capacity of the supernatant was measured in nonleukoreduced units of RBCs in CPDA (n = 47). The possible effect of UA variability on the storage lesion profile was assessed by monitoring several physiologic properties of RBCs and supernatant, including cell shape, reactive oxygen species, and size distribution of extracellular vesicles, in units exhibiting the lowest or highest levels of UA activity (n = 16) among donors, throughout the storage period. RESULTS: In stored RBC units, the UA-dependent antioxidant activity of the supernatant declined as a function of storage duration but always in strong relation to the UA levels in fresh blood. Contrary to units of poor-UA activity, RBCs with the highest levels of UA activity exhibited better profile of calcium- and oxidative stress-driven modifications, including a significant decrease in the percentages of spherocytes and of 100- to 300-nm-sized vesicles, typically associated with the exovesiculation of stored RBCs. CONCLUSION: The antioxidant activity of UA is associated with donor-specific differences in the performance of RBCs under storage in nonleukoreduced CPDA units.

Short-term effects of hemodiafiltration versus conventional hemodialysis on erythrocyte performance.

Hemodiafiltration (HDF) is a renal replacement therapy that is based on the principles of diffusion and convection for the elimination of uremic toxins. A significant and increasing number of end-stage renal disease (ESRD) patients are treated with HDF, even in the absence of definite and conclusive survival and anemia treatment data. However, its effects on red blood cell (RBC) physiological features have not been examined in depth. In this study, ESRD patients under regular HDF or conventional hemodialysis (cHD) treatment were examined for RBC-related parameters, including anemia, hemolysis, cell shape, redox status, removal signaling, membrane protein composition, and microvesiculation, in repeated paired measurements accomplished before and right after each dialysis session. The HDF group was characterized by better redox potential and suppressed exovesiculation of blood cells compared with the cHD group pre-dialysis. However, HDF was associated with a temporary but acute, oxidative-stress-driven increase in hemolysis, RBC removal signaling, and stomatocytosis, probably associated with the effective clearance of dialyzable natural antioxidant components, including uric acid, from the uremic plasma. The nature of these adverse short-term effects of HDF on post-dialysis plasma and RBCs strongly suggests the use of a parallel antioxidant therapy during the HDF session.

Pathophysiological aspects of red blood cells in end-stage renal disease patients resistant to recombinant human erythropoietin therapy.

OBJECTIVE: Modified, bioreactive red blood cells (RBCs) and RBC-derived microvesicles (MVs) likely contribute to the hematological and cardiovascular complications in end-stage renal disease (ESRD). This study assesses the physiological profile of RBCs in patients with ESRD receiving standard or high doses of recombinant human erythropoietin (rhEPO). METHOD: Blood samples from twenty-eight patients under sustained hemodialysis, responsive, or not to standard rhEPO administration were examined for RBC morphology, fragility, hemolysis, redox status, removal signaling, membrane protein composition, and microvesiculation before and after dialysis. Acute effects of uremic plasma on RBC features were examined in vitro through reconstitution experiments. RESULTS: Overall, the ESRD RBCs were characterized by pathological levels of shape distortions, surface removal signaling, and membrane exovesiculation, but reduced fragility compared to healthy RBCs. Irreversible transformation of RBCs was found to be a function of baseline Hb concentration. The more toxic uremic context in non-responsive patients compared to rhEPO responders was blunted in part by the antioxidant, antihemolytic, and anti-apoptotic effects of high rhEPO doses, and probably, of serum uric acid. A selective lower expression of RBC membrane in complement regulators (CD59, clusterin) and of CD47 "marker-of-self" was detected in non-responders and responders, respectively. Evidence for different short-term dialysis effects and probably for a different erythrocyte vesiculation mechanism in rhEPO responsive compared to non-responsive patients was also revealed. CONCLUSION: Deregulation of RBC homeostasis might involve diverse molecular pathways driving erythrocyte signaling and removal in rhEPO non-responders compared to responsive patients.

3-BrPA eliminates human bladder cancer cells with highly oncogenic signatures via engagement of specific death programs and perturbation of multiple signaling and metabolic determinants.

BACKGROUND: Urinary bladder cancer is one of the most fatal and expensive diseases of industrialized world. Despite the strenuous efforts, no seminal advances have been achieved for its clinical management. Given the importance of metabolic reprogramming in cancer cell survival and growth, we have herein employed 3-BrPA, a halogenated derivative of pyruvate and historically considered inhibitor of glycolysis, to eliminate bladder cancer cells with highly oncogenic molecular signatures. METHODS: Bladder cancer cells were exposed to 3-BrPA in the absence or presence of several specific inhibitors. Cell viability was determined by MTT and flow-cytometry assays; cell death, signaling activity and metabolic integrity by Western blotting and immunofluorescence; mutant-gene profiling by DNA sequencing; and gene expression by RT-sqPCR. RESULTS: 3-BrPA could activate dose-dependent apoptosis (type 1 PCD) and regulated necrosis (type 3 PCD) of T24 (grade III; H-Ras(G12V); p53(ΔY126)), but not RT4 (grade I), cells, with PARP, MLKL, Drp1 and Nec-7-targeted components critically orchestrating necrotic death. However, similarly to RIPK1 and CypD, p53 presented with non-essential contribution to 3-BrPA-induced cellular collapse, while reactivation of mutant p53 with PRIMA-1 resulted in strong synergism of the two agents. Given the reduced expression of MPC components (likely imposing mitochondrial dysfunction) in T24 cells, the suppression of constitutive autophagy (required by cells carrying oncogenic Ras; also, type 2 PCD) and derangement of glucose-homeostasis determinants by 3-BrPA critically contribute to drug-directed depletion of ATP cellular stores. This bioenergetic crisis is translated to severe dysregulation of Akt/FoxO/GSK-3, mTOR/S6, AMPK and MAPK (p44/42, p38 and SAPK/JNK) signaling pathways in 3-BrPA-treated T24 cells. Sensitivity to 3-BrPA (and tolerance to glucose deprivation) does not rely on B-Raf(V600E) or K-Ras(G13D) mutant oncogenic proteins, but partly depends on aberrant signaling activities of Akt, MAPK and AMPK kinases. Interestingly, MCT1- and macropinocytosis-mediated influx of 3-BrPA in T24 represents the principal mechanism that regulates cellular responsiveness to the drug. Besides its capacity to affect transcription in gene-dependent manner, 3-BrPA can also induce GLUT4-specific splicing silencing in both sensitive and resistant cells, thus dictating alternative routes of drug trafficking. CONCLUSIONS: Altogether, it seems that 3-BrPA represents a promising agent for bladder cancer targeted therapy.

Detrimental effects of proteasome inhibition activity in Drosophila melanogaster: implication of ER stress, autophagy, and apoptosis.

In eukaryotes, the ubiquitin-proteasome machinery regulates a number of fundamental cellular processes through accurate and tightly controlled protein degradation pathways. We have, herein, examined the effects of proteasome functional disruption in Dmp53 (+/+) (wild-type) and Dmp53 (-/-) Drosophila melanogaster fly strains through utilization of Bortezomib, a proteasome-specific inhibitor. We report that proteasome inhibition drastically shortens fly life-span and impairs climbing performance, while it also causes larval lethality and activates developmentally irregular cell death programs during oogenesis. Interestingly, Dmp53 gene seems to play a role in fly longevity and climbing ability. Moreover, Bortezomib proved to induce endoplasmic reticulum (ER) stress that was able to result in the engagement of unfolded protein response (UPR) signaling pathway, as respectively indicated by fly Xbp1 activation and Ref(2)P-containing protein aggregate formation. Larva salivary gland and adult brain both underwent strong ER stress in response to Bortezomib, thus underscoring the detrimental role of proteasome inhibition in larval development and brain function. We also propose that the observed upregulation of autophagy operates as a protective mechanism to "counterbalance" Bortezomib-induced systemic toxicity, which is tightly associated, besides ER stress, with activation of apoptosis, mainly mediated by functional Drice caspase and deregulated dAkt kinase. The reduced life-span of exposed to Bortezomib flies overexpressing Atg1_RNAi or Atg18_RNAi supports the protective nature of autophagy against proteasome inhibition-induced stress. Our data reveal the in vivo significance of proteasome functional integrity as a major defensive system against cellular toxicity likely occurring during critical biological processes and morphogenetic courses.

Microtubule Dynamics Deregulation Induces Apoptosis in Human Urothelial Bladder Cancer Cells via a p53-Independent Pathway.

Bladder cancer (BLCA) is the sixth most common type of cancer and has a dismal prognosis if diagnosed late. To identify treatment options for BLCA, we systematically evaluated data from the Broad Institute DepMap project. We found that urothelial BLCA cell lines are among the most sensitive to microtubule assembly inhibition by paclitaxel treatment. Strikingly, we revealed that the top dependencies in BLCA cell lines include genes encoding proteins involved in microtubule assembly. This highlights the importance of microtubule network dynamics as a major vulnerability in human BLCA. In cancers such as ovarian and breast, where paclitaxel is the gold standard of care, resistance to paclitaxel treatment has been linked to p53-inactivating mutations. To study the response of BLCA to microtubule assembly inhibition and its mechanistic link with the mutational status of the p53 protein, we treated a collection of BLCA cell lines with a dose range of paclitaxel and performed a detailed characterization of the response. We discovered that BLCA cell lines are significantly sensitive to low concentrations of paclitaxel, independently of their p53 status. Paclitaxel induced a G2/M cell cycle arrest and growth inhibition, followed by robust activation of apoptosis. Most importantly, we revealed that paclitaxel triggered a robust DNA-damage response and apoptosis program without activating the p53 pathway. Integration of transcriptomics, epigenetic, and dependency data demonstrated that the response of BLCA to paclitaxel is independent of p53 mutational signatures but strongly depends on the expression of DNA repair genes. Our work highlights urothelial BLCA as an exceptional candidate for paclitaxel treatment. It paves the way for the rational use of a combination of paclitaxel and DNA repair inhibitors as an effective, novel therapeutic strategy.

Early and Late-Phase 24 h Responses of Stored Red Blood Cells to Recipient-Mimicking Conditions.

The 24-hour (24 h) post-transfusion survival of donor red blood cells (RBCs) is an important marker of transfusion efficacy. Nonetheless, within that period, donated RBCs may encounter challenges able to evoke rapid stress-responses. The aim of the present study was to assess the effect of exposure to plasma and body temperature upon stored RBCs under recipient-mimicking conditions in vitro from the first hours "post-transfusion" up to 24 h. For this purpose, packed RBCs from seven leukoreduced CPD/SAGM units were reconstituted with plasma of twenty-seven healthy individuals and incubated for 24 h at 37oC. Three units were additionally used to examine stress-responses in 3-hour intervals post mixing with plasma (n = 5) until 24 h. All experiments were performed in shortly-, medium-, and long-stored RBCs. Hemolysis, redox, morphology, membrane protein binding and vesiculation parameters were assessed. Even though spontaneous hemolysis was minimal post-reconstitution, it presented a time-dependent increase. A similar time-course profile was evident for the concentration of procoagulant extracellular vesicles and the osmotic fragility (shortly-stored RBCs). On the contrary, mechanical fragility and reactive oxygen species accumulation were characterized by increases in medium-stored RBCs, evident even from the first hours in the recipient-mimicking environment. Finally, exposure to plasma resulted in rapid improvement of morphology, especially in medium-stored RBCs. Overall, some RBC properties vary significantly during the first 24 h post-mixing, at levels different from both the storage ones and the standard end-of-24 h. Such findings may be useful for understanding the performance of RBCs and their possible clinical effects -especially on susceptible recipients- during the first hours post-transfusion.

Programmed cell death of the ovarian nurse cells during oogenesis of the ladybird beetle Adalia bipunctata (Coleoptera: Coccinellidae).

Programmed cell death (PCD) is an evolutionary conserved and genetically regulated form of cell death, in which the cell plays an active role in its own demise. It is widely recognized that PCD can be morphologically classified into three major types: type I, known as apoptosis, type II, called autophagy, and type III, specified as cytoplasmic cell death. So far, PCD has been morphologically analyzed in certain model insect species of the meroistic polytrophic ovary-type, but has never been examined before in insects carrying meroistic telotrophic ovaries. In the present study, we attempted to thoroughly describe the three different types (I, II and III) of PCD occurring during oogenesis in the meroistic telotrophic ovary of the Coleoptera species Adalia bipunctata, at different developmental ages of the adult female insects. We reveal that in the ladybird beetle A. bipunctata, the ovarian tropharia undergo age-dependent forms of apoptotic, autophagic and cytoplasmic (paraptotic-like) cell death, which seem to operate in a rather synergistic fashion, in accordance with previous observations in Diptera and Lepidoptera species. Furthermore, we herein demonstrate the occurrence of morphogenetically abnormal ovarioles in A. bipunctata female insects. These atretic ovarioles collapse and die through a PCD-mediated process that is characterized by the combined activation of all three types of PCD. Conclusively, the distinct cell death programs (I, II and III) specifically engaged during oogenesis of A. bipunctata provide strong evidence for the structural and functional conserved nature of PCD during insect evolution among meroistic telotrophic and meroistic polytrophic ovary-type insects.

Proteasome inhibition induces developmentally deregulated programs of apoptotic and autophagic cell death during Drosophila melanogaster oogenesis.

Ubiquitin/proteasome-mediated degradation of eukaryotic proteins is critically implicated in a number of signalling pathways and cellular processes. To specifically impair proteasome activities, in vitro developing Drosophila melanogaster egg chambers were exposed to the MG132 or epoxomicin proteasome inhibitors, while a GAL4/UAS binary genetic system was employed to generate double transgenic flies overexpressing ß2 and ß6 conditional mutant proteasome subunits in a cell type-specific manner. MG132 and epoxomicin administration resulted in severe deregulation of in vitro developing egg chambers, which was tightly associated with precocious induction of nurse cell-specific apoptotic and autophagic death programmes, featured by actin cytoskeleton disorganization, nuclear chromatin condensation, DRICE caspase activation and autophagosome accumulation. In vivo targeted overexpression of ß2 and ß6 conditional mutants, specifically in the nurse cell compartment, led to a notable up-regulation of sporadic apoptosis potency during early and mid-oogenesis 'checkpoints', thus reasonably justifying the observed reduction in eclosion efficiency. Furthermore, in response to the intracellular abundance of ß2 and ß6 conditional mutant forms, specifically in numerous tissues of third instar larval stage, the developmental course was arrested, and lethal phenotypes were obtained at this particular embryonic period, with the double transgenic heterozygote embryos being unable to further proceed to complete maturation to adult flies. Our data demonstrate that physiological proteasome function is required to ensure normal oogenesis and embryogenesis in D. melanogaster, since targeted and cell type-dependent proteasome inactivation initiates developmentally deregulated apoptotic and autophagic mechanisms.

AGO2 localizes to cytokinetic protrusions in a p38-dependent manner and is needed for accurate cell division.

Argonaute 2 (AGO2) is an indispensable component of the RNA-induced silencing complex, operating at the translational or posttranscriptional level. It is compartmentalized into structures such as GW- and P-bodies, stress granules and adherens junctions as well as the midbody. Here we show using immunofluorescence, image and bioinformatic analysis and cytogenetics that AGO2 also resides in membrane protrusions such as open- and close-ended tubes. The latter are cytokinetic bridges where AGO2 colocalizes at the midbody arms with cytoskeletal components such as α-Τubulin and Aurora B, and various kinases. AGO2, phosphorylated on serine 387, is located together with Dicer at the midbody ring in a manner dependent on p38 MAPK activity. We further show that AGO2 is stress sensitive and important to ensure the proper chromosome segregation and cytokinetic fidelity. We suggest that AGO2 is part of a regulatory mechanism triggered by cytokinetic stress to generate the appropriate micro-environment for local transcript homeostasis.

From Proteomic Mapping to Invasion-Metastasis-Cascade Systemic Biomarkering and Targeted Drugging of Mutant BRAF-Dependent Human Cutaneous Melanomagenesis.

Melanoma is classified among the most notoriously aggressive human cancers. Despite the recent progress, due to its propensity for metastasis and resistance to therapy, novel biomarkers and oncogenic molecular drivers need to be promptly identified for metastatic melanoma. Hence, by employing nano liquid chromatography-tandem mass spectrometry deep proteomics technology, advanced bioinformatics algorithms, immunofluorescence, western blotting, wound healing protocols, molecular modeling programs, and MTT assays, we comparatively examined the respective proteomic contents of WM115 primary (n = 3955 proteins) and WM266-4 metastatic (n = 6681 proteins) melanoma cells. It proved that WM115 and WM266-4 cells have engaged hybrid epithelial-to-mesenchymal transition/mesenchymal-to-epithelial transition states, with TGF-ß controlling their motility in vitro. They are characterized by different signatures of SOX-dependent neural crest-like stemness and distinct architectures of the cytoskeleton network. Multiple signaling pathways have already been activated from the primary melanoma stage, whereas HIF1α, the major hypoxia-inducible factor, can be exclusively observed in metastatic melanoma cells. Invasion-metastasis cascade-specific sub-routines of activated Caspase-3-triggered apoptosis and LC3B-II-dependent constitutive autophagy were also unveiled. Importantly, WM115 and WM266-4 cells exhibited diverse drug response profiles, with epirubicin holding considerable promise as a beneficial drug for metastatic melanoma clinical management. It is the proteome navigation that enables systemic biomarkering and targeted drugging to open new therapeutic windows for advanced disease.

Exploitation of Drosophila Choriogenesis Process as a Model Cellular System for Assessment of Compound Toxicity: the Phloroglucinol Paradigm.

Phloroglucinol (1,3,5 tri-hydroxy-benzene) (PGL), a natural phenolic substance, is a peroxidase inhibitor and has anti-oxidant, anti-diabetic, anti-inflammatory, anti-thrombotic, radio-protective, spasmolytic and anti-cancer activities. PGL, as a medicine, is administered to patients to control the symptoms of irritable bowel syndrome and acute renal colic, in clinical trials. PGL, as a phenolic substance, can cause cytotoxic effects. Administration of PGL up to 300 mg/kg (bw) is well tolerated by animals, while in cell lines its toxicity is developed at concentrations above the dose of 10 µg/ml. Furthermore, it seems that tumor or immortalized cells are more susceptible to the toxic power of PGL, than normal cells. However, studies of its cytotoxic potency, at the cellular level, in complex, differentiated and meta-mitotic biological systems, are still missing. In the present work, we have investigated the toxic activity of PGL in somatic epithelial cells, constituting the follicular compartment of a developing egg-chamber (or, follicle), which directs the choriogenesis (i.e. chorion assembly) process, during late oogenesis of Drosophila melanogaster. Our results reveal that treatment of in vitro growing Drosophila follicles with PGL, at a concentration of 0.2 mM (or, 25.2 µg/ml), does not lead to follicle-cell toxicity, since the protein-synthesis program and developmental pattern of choriogenesis are normally completed. Likewise, the 1 mM dose of PGL was also characterized by lack of toxicity, since the chorionic proteins were physiologically synthesized and the chorion structure appeared unaffected, except for a short developmental delay, being observed. In contrast, concentrations of 10, 20 or 40 mM of PGL unveiled a dose-dependent, increasing, toxic effect, being initiated by interruption of protein synthesis and disassembly of cell-secretory machinery, and, next, followed by fragmentation of the granular endoplasmic reticulum (ER) into vesicles, and formation of autophagic vacuoles. Follicle cells enter into an apoptotic process, with autophagosomes and large vacuoles being formed in the cytoplasm, and nucleus showing protrusions, granular nucleolus and condensed chromatin. PGL, also, proved able to induce disruption of nuclear envelope, activation of nucleus autophagy (nucleophagy) and formation of a syncytium-like pattern being produced by fusion of plasma membranes of two or more individual follicle cells. Altogether, follicle cell-dependent choriogenesis in Drosophila has been herein presented as an excellent, powerful and reliable multi-cellular, differentiated, model biological (animal) system for drug-cytotoxicity assessment, with the versatile compound PGL serving as a characteristic paradigm. In conclusion, PGL is a substance that may act beneficially for a variety of pathological conditions and can be safely used for differentiated somatic -epithelial- cells at clinically low concentrations. At relatively high doses, it could potentially induce apoptotic and autophagic cell death, thus being likely exploited as a therapeutic agent against a number of pathologies, including human malignancies.