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Background Endoscopic per-oral pyloromyotomy (POP) has emerged as a safe and effective first line option in medically refractory gastroparesis. Determining the appropriate extent of the pyloromyotomy continues to present a challenge as there are no standardized tools for measuring changes in pyloric distensibility during the procedure. The objective of this study was to evaluate the utility of using impedance planimetry with endoscopic functional luminal imaging probe (FLIP) to measure changes in pyloric distensibility after POP, and to compare these changes with improvement in symptoms and objective gastric emptying. Methods Patients with medically refractory gastroparesis underwent POP with FLIP measurements of the pylorus (EndoFLIP®, Medtronic, Fridley MN). FLIP measurements, as well as changes in symptoms measured by the validated gastroparesis cardinal symptom index (GCSI) and scintigraphic gastric emptying studies (GES), were evaluated before and after POP. Results A total of 14 patients underwent measurement with FLIP during POP, 12 of whom had pre- and post-POP measurements. Mean pyloric diameter increased by 1.4 mm, from 13.9 mm to 15.3 mm (p = 0.0012). Mean distensibility index increased from 6.2 mm2/mmHg to 9.1 mm2/mmHg (p = 0.0074). Successful division of the pylorus was achieved in 100% of patients with a mean operative time of 36 min and no perioperative complications. The mean length of stay was 0.7 days (0–3 days). Post-POP mean GCSI score improved from 2.97 to 2.28 at a mean follow-up time of 27 days (p Conclusions FLIP is a safe and feasible tool to provide objective measurements during POP. Larger cohorts with longer follow-up are required to determine if measured improvements in pyloric diameter and distensibility are predictive of sustained improvements in GCSI and GES.

BACKGROUND: Esophagogastric junction outflow obstruction (EGJOO) is a common but non-specific motility pattern identified by esophageal high-resolution manometry (HRM). Functional luminal impedance planimetry (FLIP) provides information regarding lower esophageal sphincter (LES) mechanics, which can identify achalasia-spectrum disorders and is useful in evaluating EGJOO. However, the relationship between HRM and FLIP parameters in EGJOO is not clearly defined. AIMS: To identify predictors of abnormal FLIP findings in patients with non-mechanical EGJOO. METHODS: This is a retrospective cohort study of patients with non-mechanical EGJOO who underwent FLIP between 10/1/16 and 7/1/19. Demographic data including age and gender, exam indication, concomitant medications, HRM parameters, symptom burden, and FLIP metrics of diameter and distensibility index (DI) were collected. DI was categorized as not low (DI>2.8), borderline low (DI 1.1–2.8), and definitely low (DI≤1). Kruskal-Wallis and Fisher exact tests were used to assess the relationship between HRM and FLIP parameters and to identify predictors of abnormal FLIP. RESULTS: Among the 44 patients studied, most were female (n=33, 75%) and the median age was 63. The median IRP was 18.2 and 10 (23%) patients used chronic narcotics. Lower total heartburn and regurgitation scores, and LES diameter by FLIP are associated with definitely low DI. CONCLUSIONS: In patients with non-mechanical EGJOO, reflux burden scores and FLIP diameters can aid in predicting DI. These results may provide useful adjunctive data to help in differentiating which patients have meaningful outflow obstruction.

Recent innovations in esophageal diagnostic testing have enhanced gastroenterology clinical practice by facilitating more nuanced and advanced evaluation of esophageal symptoms. Among these pivotal advances is the FDA-approved functional lumen imaging probe (FLIP), which utilizes impedance planimetry via volumetric distension of a catheter-mounted balloon at the time of sedated upper endoscopy, to acquire esophageal dimensions and pressures. In real time, FLIP can display cross-sectional areas (CSA) and distensibility indices (ratios of CSA to intra-balloon pressures) throughout the esophagus, most notably at the esophagogastric junction, as well as secondary peristaltic esophageal body contractile patterns. As the use of FLIP has progressively spread and permeated into the practice of clinical gastroenterology since its introduction, increasing data on and experiences with its applications have accumulated to guide its utility in clinical practice. In this current review developed for gastroenterologists and foregut surgeons across clinical practice, we provide an introduction to FLIP technology and metrics and discuss the clinical scenarios in which performance of or referral for FLIP may be helpful in the evaluation and management of patients with commonly encountered esophageal symptoms and disorders. Specifically, we discuss the potential applications and limitations of FLIP as a complementary diagnostic modality in patients with non-obstructive dysphagia, established or suspected achalasia spectrum disorders, eosinophilic esophagitis, gastroesophageal reflux disease and those undergoing esophageal surgery.

Esophagogastric junction distensibility index (DI), measured using the functional luminal imaging probe (FLIP), correlates with symptomatic outcomes after interventions for achalasia. The objective of this study was to determine if the intraoperative measurement of DI using FLIP was associated with improved clinical outcomes following per-oral endoscopic myotomy (POEM) for achalasia when compared with procedures in which FLIP was not utilized. Patients undergoing POEM from 2012 to 2017 at a single institution by a single surgeon were studied. Use of FLIP during this time period was based on catheter and technician availability, resulting in two patient cohorts. In patients in whom FLIP was used, operative video recordings were reviewed to determine when DI measurements led to the performance of additional myotomy. Postoperative Eckardt symptom scores (ES) at 12 months and postoperative physiologic studies were compared between patients with and without intraoperative FLIP. Associations were assessed using Mann–Whitney U and Chi-square tests. 143 patients were included in the analysis (61 with intraoperative FLIP and 82 without FLIP). Video recordings were available for 85% of the FLIP cohort. Review of these operative recordings revealed that 65% of patients who underwent FLIP had additional myotomy performed following the initial postmyotomy FLIP measurement. At 12 months after POEM, the FLIP cohort had significantly more clinical successes (defined as ES ≤ 3) than patients in whom FLIP was not used (93% vs. 81%, p

The functional luminal imaging probe (FLIP) can be used to measure the esophagogastric junction distensibility index (DI) during myotomy for achalasia and increased DI has been shown to predict superior clinical outcomes. The objective of this study was to determine if the intraoperative DI and the changes produced by per oral endoscopic myotomy (POEM) differed between achalasia subtypes. FLIP measurements were performed during POEM for achalasia at a single institution. DI (defined as the minimum cross-sectional area (CSA) at the EGJ divided by distensive pressure) was measured at three time points: after induction of anesthesia, after submucosal tunneling, and after myotomy. Measurements were reported at the 40 mL fill volume for the 8 cm FLIP (EF-325) and at the 60 mL fill volume for the 16 cm FLIP (EF-322). Measurements were compared using chi-square and Kruskal–Wallis tests. 142 patients had intraoperative FLIP performed during POEM for achalasia between 2012 and 2019 (30 type I, 68 type II, 27 type III, and 17 variant). Patients with type I achalasia had a significantly higher induction DI (median 1.7 mm2/mmHg) than type II (0.8 mm2/mmHg), type III (0.9 mm2/mmHg), and variants (1.1 mm2/mmHg; p

TRAIL-R2 (DR5) is a clinically-relevant therapeutic target and a key target for immune effector cells. Herein, we identify a novel interaction between TRAIL-R2 and the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2). We find that SCFSkp2 can interact with both TRAIL-R2’s pre-ligand association complex (PLAC) and ligand-activated death-inducing signalling complex (DISC). Moreover, Cullin-1 interacts with TRAIL-R2 in its active NEDDylated form. Inhibiting Cullin-1’s DISC recruitment using the NEDDylation inhibitor MLN4924 (Pevonedistat) or siRNA increased apoptosis induction in response to TRAIL. This correlated with enhanced levels of the caspase-8 regulator FLIP at the TRAIL-R2 DISC, particularly the long splice form, FLIP(L). We subsequently found that FLIP(L) (but not FLIP(S), caspase-8, nor the other core DISC component FADD) interacts with Cullin-1 and Skp2. Importantly, this interaction is enhanced when FLIP(L) is in its DISC-associated, C-terminally truncated p43-form. Prevention of FLIP(L) processing to its p43-form stabilises the protein, suggesting that by enhancing its interaction with SCFSkp2, cleavage to the p43-form is a critical step in FLIP(L) turnover. In support of this, we found that silencing any of the components of the SCFSkp2 complex inhibits FLIP ubiquitination, while overexpressing Cullin-1/Skp2 enhances its ubiquitination in a NEDDylation-dependent manner. DISC recruitment of TRAF2, previously identified as an E3 ligase for caspase-8 at the DISC, was also enhanced when Cullin-1’s recruitment was inhibited, although its interaction with Cullin-1 was found to be mediated indirectly via FLIP(L). Notably, the interaction of p43-FLIP(L) with Cullin-1 disrupts its ability to interact with FADD, caspase-8 and TRAF2. Collectively, our results suggest that processing of FLIP(L) to p43-FLIP(L) at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP(L)’s interactions with other DISC components and promoting its ubiquitination and degradation, thereby modulating TRAIL-R2-mediated apoptosis.

Ring flips of phenylalanine and tyrosine are a hallmark of protein dynamics. They report on transient breathing motions of proteins. In addition, flip rates also depend on stabilizing interactions in the ground state, like aromatic stacking or cation–π interaction. So far, experimental studies of ring flips have almost exclusively been performed on aromatic rings without stabilizing interactions. Here we investigate ring flip dynamics of Phe and Tyr in the aromatic cluster in GB1. We found that all four residues of the cluster, Y3, F30, Y45 and F52, display slow ring flips. Interestingly, F52, the central residue of the cluster, which makes aromatic contacts with all three others, is flipping significantly faster, while the other rings are flipping with the same rates within margin of error. Determined activation enthalpies and activation volumes of these processes are in the same range of other reported ring flips of single aromatic rings. There is no correlation of the number of aromatic stacking interactions to the activation enthalpy, and no correlation of the ring’s extent of burying to the activation volume. Because of these findings, we speculate that F52 is undergoing concerted ring flips with each of the other rings. Electronic supplementary material The online version of this article (10.1007/s10858-020-00303-3) contains supplementary material, which is available to authorized users.

A flip is a minimal move between two triangulations of a polytope. The set of triangulations of a polytope was shown by Santos to not always be connected by flips, and it is an interesting problem to find large classes of polytopes for which it is. One such class which has received considerable attention is the product of two simplices. Santos proved that the set of triangulations of a product of two simplices is connected by flips when one of the simplices is a triangle. However, the author showed that it is not connected when one of the simplices is four-dimensional and the other has very large dimension. In this paper we show that it is connected when one of the simplices is a tetrahedron, thereby extending Santos’s result as far as possible.

c-FLIP (cellular FLICE-like inhibitory protein) protein is mostly known as an apoptosis modulator. However, increasing data underline that c-FLIP plays multiple roles in cellular homoeostasis, influencing differently the same pathways depending on its expression level and isoform predominance. Few and controversial data are available regarding c-FLIP function in autophagy. Here we show that autophagic flux is less effective in c-FLIP−/− than in WT MEFs (mouse embryonic fibroblasts). Indeed, we show that the absence of c-FLIP compromises the expression levels of pivotal factors in the generation of autophagosomes. In line with the role of c-FLIP as a scaffold protein, we found that c-FLIPL interacts with Beclin-1 (BECN1: coiled-coil, moesin-like BCL2-interacting protein), which is required for autophagosome nucleation. By a combination of bioinformatics tools and biochemistry assays, we demonstrate that c-FLIPL interaction with Beclin-1 is important to prevent Beclin-1 ubiquitination and degradation through the proteasomal pathway. Taken together, our data describe a novel molecular mechanism through which c-FLIPL positively regulates autophagy, by enhancing Beclin-1 protein stability.

The assembly of the death-inducing signaling complex (DISC) and death effector domain (DED) filaments at CD95/Fas initiates extrinsic apoptosis. Procaspase-8 activation at the DED filaments is controlled by short and long c-FLIP isoforms. Despite apparent progress in understanding the assembly of CD95-activated platforms and DED filaments, the detailed molecular mechanism of c-FLIP action remains elusive. Here, we further addressed the mechanisms of c-FLIP action at the DISC using biochemical assays, quantitative mass spectrometry, and structural modeling. Our data strongly indicate that c-FLIP can bind to both FADD and procaspase-8 at the DED filament. Moreover, the constructed in silico model shows that c-FLIP proteins can lead to the formation of the DISCs comprising short DED filaments as well as serve as bridging motifs for building a cooperative DISC network, in which adjacent CD95 DISCs are connected by DED filaments. This network is based on selective interactions of FADD with both c-FLIP and procaspase-8. Hence, c-FLIP proteins at the DISC control initiation, elongation, and composition of DED filaments, playing the role of control checkpoints. These findings provide new insights into DISC and DED filament regulation and open innovative possibilities for targeting the extrinsic apoptosis pathway.

The functional luminal imaging probe (FLIP) uses high-resolution planimetry to provide a three-dimensional image of the esophageal lumen by measuring diameter, volume, and pressure changes. The goal of this review is to summarize the most recent advances in applying the (FLIP) to esophageal disorders. The FLIP has been studied in esophageal disease states including gastroesophageal reflux disease (GERD), achalasia, and eosinophilic esophagitis. It has also been used in the investigation of dysphagia. The FLIP is a valuable tool for the diagnosis of esophageal diseases as well as guiding treatments and predicting treatment response. As further research is done, the FLIP may become the initial test for the patient with undifferentiated dysphagia at the time of their index endoscopy.

Aurora kinase B (AKB), a Ser/Thr kinase that plays a crucial role in mitosis, is overexpressed in several cancers. Clinical inhibitors targeting AKB bind to the active DFG “in” conformation of the kinase. It would be beneficial, however, to understand if AKB is susceptible to type II kinase inhibitors that bind to the inactive, DFG “out” conformation, since type II inhibitors achieve higher kinome selectivity and higher potency in vivo. The DFG “out” conformation of AKB is not yet experimentally determined which makes the design of type II inhibitors exceedingly difficult. An alternate approach is to simulate the DFG “out” conformation from the experimentally determined DFG “in” conformation using atomistic molecular dynamics (MD) simulation. In this work, we employed metadynamics (MTD) approach to simulate the DFG “out” conformation of AKB by choosing the appropriate collective variables. We examined structural changes during the DFG-flip and determined the interactions crucial to stabilize the kinase in active and inactive states. Interestingly, the MTD approach also identified a unique transition state (DFG “up”), which can be targeted by small molecule inhibitors. Structural insights about these conformations is essential for structure-guided design of next-generation AKB inhibitors. This work also emphasizes the usefulness of MTD simulations in predicting macromolecular conformational changes at reduced computational costs.

We give an example of a three-dimensional zonotope whose set of tight zonotopal tilings is not connected by flips. Using this, we show that the set of triangulations of $\Delta^4 \times \Delta^n$ is not connected by flips for large $n$. Our proof makes use of a non-explicit probabilistic construction., Comment: 28 pages

Flip the switch: BTG2–PRMT1 protein complexes antagonize pre-B-cell proliferation to promote B-cell development

We show that computing canonical representations for circular-arc (CA) graphs reduces to computing certain subsets of vertices called flip sets. For a broad class of CA graphs, which we call uniform, it suffices to compute a CA representation to find such flip sets. As a consequence canonical representations for uniform CA graphs can be obtained in polynomial-time. We then investigate what kind of CA graphs pose a challenge to this approach. This leads us to introduce the notion of restricted CA matrices and show that the canonical representation problem for CA graphs is logspace-reducible to that of restricted CA matrices. As a byproduct, we obtain the result that CA graphs without induced 4-cycles can be canonized in logspace., 24 pages

The functional luminal imaging probe (FLIP) provides a novel method to evaluate esophageal physiology and function in both health and disease. FLIP enables assessment of a previously underexplored component of esophageal function, distensibility, by utilizing high-resolution impedance planimetry to evaluate the relationship of the esophageal luminal geometry with pressure during controlled volumetric distension. Recent studies have described the potential utility of FLIP to evaluate the severity and effectiveness of therapy for esophageal diseases including achalasia, gastroesophageal reflux disease, and eosinophilic esophagitis. This review summarizes these emerging clinical applications of FLIP in the evaluation and management of esophageal diseases.

We lack an understanding of how the full range of genetic variants that occur in individuals can interact. To address this shortcoming, here we combine diverse mutations between genes in a model regulatory network, the galactose (GAL) switch of budding yeast. The effects of thousands of pairs of mutations fall into a limited number of phenotypic classes. While these effects are mostly predictable using simple rules that capture the ‘stereotypical’ genetic interactions of the network, some double mutants have unexpected outcomes including constituting alternative functional switches. Each of these ‘harmonious’ genetic combinations exhibits altered dependency on other regulatory genes. These cases illustrate how both pairwise and higher epistasis determines gene essentiality and how combinations of mutations rewire regulatory networks. Together, our results provide an overview of how broad spectra of mutations interact, how these interactions can be predicted, and how diverse genetic solutions can achieve ‘wild-type’ phenotypic behavior., Studying how genetic variants in different genes interact and their combinatorial output is experimentally and analytically challenging. Here, the authors quantify the effects of more than 5000 mutation pairs in the yeast GAL regulatory system, finding that many combinations can be predicted with statistical models.

FLICE-like inhibitory protein (FLIP) is a critical regulator of death receptor-mediated apoptosis. Here, we found ubiquitin-specific peptidase 8 (USP8) to be a novel deubiquitylase of the long isoform of FLIP (FLIPL). USP8 directly deubiquitylates and stabilizes FLIPL, but not the short isoform. USP8 depletion induces FLIPL destabilization, promoting anti-Fas-, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- and tumor necrosis factor alpha-induced extrinsic apoptosis by facilitating death-inducing signaling complex or TNFR1 complex II formation, which results in the activation of caspase-8 and caspase-3. USP8 mRNA levels are elevated in melanoma and cervical cancers, and the protein levels of USP8 and FLIPL are positively correlated in these cancer cell lines. Xenograft analyses using ME-180 cervical cancer cells showed that USP8 depletion attenuated tumor growth upon TRAIL injection. Taken together, our data indicate that USP8 functions as a novel deubiquitylase of FLIPL and inhibits extrinsic apoptosis by stabilizing FLIPL.

The CD95/Fas/APO-1 death-inducing signaling complex (DISC), comprising CD95, FADD, procaspase-8, procaspase-10, and c-FLIP, has a key role in apoptosis induction. Recently, it was demonstrated that procaspase-8 activation is driven by death effector domain (DED) chains at the DISC. Here, we analyzed the molecular architecture of the chains and the role of the short DED proteins in regulating procaspase-8 activation in the chain model. We demonstrate that the DED chains are largely composed of procaspase-8 cleavage products and, in particular, of its prodomain. The DED chain also comprises c-FLIP and procaspase-10 that are present in 10 times lower amounts compared with procaspase-8. We show that short c-FLIP isoforms can inhibit CD95-induced cell death upon overexpression, likely by forming inactive heterodimers with procaspase-8. Furthermore, we have addressed mechanisms of the termination of chain elongation using experimental and mathematical modeling approaches. We show that neither c-FLIP nor procaspase-8 prodomain terminates the DED chain, but rather the dissociation/association rates of procaspase-8 define the stability of the chain and thereby its length. In addition, we provide evidence that procaspase-8 prodomain generated at the DISC constitutes a negative feedback loop in procaspase-8 activation. Overall, these findings provide new insights into caspase-8 activation in DED chains and apoptosis initiation.

Chemotherapy remains the core of anticancer treatment. However, despite the tremendous strides made in the development of targeted anticancer therapies, emergence of resistance to chemotherapeutic drugs is still a major obstacle in the successful management of resistant tumors. Therefore, profound investigation into the in-depth molecular mechanisms of drug resistance is essential and may hopefully translate into effective therapies that can flip the switch from drug resistance to susceptibility. To develop novel-targeted therapy holds promise for conquering chemotherapy resistance, one of the major hurdles in current colon cancer treatment. Previous studies indicate that CD147 is involved in the progression of chemotherapy resistance in breast cancer and ovarian cancer cells and its expression is negative regulated by miR-492 in muscles cells. In the present study, we found that lower level of miR-492 is accompanied with increased expression of CD147 in Oxaliplatin-resistant colon cancer cell line LS174T/L-OHP as compared with its parental cell line LS174T. Exogenous expression of miR-492 in LS174T/L-OHP could sensitize its reaction on the treatment of Oxaliplatin, which is coincided with its directly reducing the expression of CD147. Furthermore, we found that knockdown of CD147 in LS174T/L-OHP could also sensitize its reaction of the treatment with Oxaliplatin. Besides, intratumoral delivering of miR-492 could also restore Oxaliplatin treatment response in Oxaliplatin-resistant xenografts in vivo. These findings provide direct evidences that the miR-492/CD147 axis might play an essential role in the Oxaliplatin resistance of colon cancer cells, suggesting that the miR-492/CD147 signaling cohort could be served as a novel therapeutic target for the treatment of chemotherapy resistant in colon cancer.

In this study, we developed an image analysis algorithm for quantification of two potential apoptotic biomarkers in non-small-cell lung cancer (NSCLC): FLIP and procaspase-8. Immunohistochemical expression of FLIP and procaspase-8 in 184 NSCLC tumors were assessed. Individual patient cores were segmented and classified as tumor and stroma using the Definiens Tissue Studio. Subsequently, chromogenic expression of each biomarker was measured separately in the nucleus and cytoplasm and reported as a quantitative histological score. The software package pROC was applied to define biomarker thresholds. Cox proportional hazards analysis was applied to generate hazard ratios (HRs) and associated 95% CI for survival. High cytoplasmic expression of tumoral (but not stromal) FLIP was associated with a 2.5-fold increased risk of death in lung adenocarcinoma patients, even when adjusted for known confounders (HR 2.47, 95% CI 1.14–5.35). Neither nuclear nor cytoplasmic tumoral procaspase-8 expression was associated with overall survival in lung adenocarcinoma patients; however, there was a significant trend (P for trend=0.03) for patients with adenocarcinomas with both high cytoplasmic FLIP and high cytoplasmic procaspase-8 to have a multiplicative increased risk of death. Notably, high stromal nuclear procaspase-8 expression was associated with a reduced risk of death in lung adenocarcinoma patients (adjusted HR 0.31, 95% CI 0.15–0.66). On further examination, the cells with high nuclear procaspase-8 were found to be of lymphoid origin, suggesting that the better prognosis of patients with tumors with high stromal nuclear procaspase-8 is related to immune infiltration, a known favorable prognostic factor. No significant associations were detected in analysis of lung squamous cell carcinoma patients. Our results suggest that cytoplasmic expression of FLIP in the tumor and nuclear expression of procaspase-8 in the stroma are prognostically relevant in non-small-cell adenocarcinomas but not in squamous cell carcinomas of the lung.

To present a case of unintentional tracheal extubation in a prone positioned patient with a known difficult airway.This case report describes the unintended tracheal extubation of an achondroplastic dwarf with kyphosis undergoing spinal fusion and instrumentation. The patient had a history of obstructive sleep apnea and a difficult airway requiring fibreoptic-guided tracheal intubation through an air-Q™ supraglottic airway device. Abrupt head movement during a wake-up test to evaluate lost motor-evoked potential signals resulted in dislodgement of the tracheal tube. Airway obstruction was evidenced by rapid oxygen desaturation and the absence of end-tidal capnography waveforms despite apparent chest excursions. An air-Q was used for successfully rescuing the airway and quickly re-establishing oxygenation and ventilation, which eliminated the need for emergent supine positioning for airway management. The air-Q was then used as a conduit for fibreoptic-guided tracheal intubation while the patient remained in the prone position.This case highlights some of the safety advantages of supraglottic airway devices for airway rescue and subsequent tracheal intubation even with the patient in the prone position. The use of an air-Q may have the advantages of not requiring an intubation introducer technique and allowing for direct tracheal intubation with an appropriately sized cuffed tracheal tube.

Successful translation of findings derived from preclinical studies into effective therapies is critical in biomedical research. Lack of robustness and reproducibility of the preclinical data, due to insufficient number of repeats, inadequate cell-based and mouse models contribute to the poor success rate. Antibodies are widely used in preclinical research, notably to determine the expression of potential therapeutic targets in tissues of interest, including tumors, but also to identify disease and/or treatment response biomarkers. We sought to determine whether the current antibody characterization standards in preclinical research are sufficient to ensure reliability of the data found in peer-reviewed publications. To address this issue, we used detection of the protein c-FLIP, a major factor of resistance to apoptosis, as a proof of concept. Accurate detection of endogenous c-FLIP levels in the preclinical settings is imperative since it is considered as a potential theranostic biomarker. Several sources of c-FLIP antibodies validated by their manufacturer and recommended for western blotting were therefore rigorously tested. We found a wide divergence in immune recognition properties. While these antibodies have been used in many publications, our results show that several of them failed to detect endogenous c-FLIP protein by Western blotting. Our results suggest that antibody validation standards are inadequate, and that systematic use of genetic knockdowns and/or knockouts to establish proof of specificity is critical, even for antibodies previously used in the scientific literature. Because antibodies are fundamental tools in both preclinical and clinical research, ensuring their specificity is crucial.

Background The immunoprecipitation (IP) assay is a valuable molecular biology tool applied across a breadth of fields. The standard assay couples IP to immunoblotting (IP/IB), a procedure severely limited as it is not easily scaled for high-throughput analysis. Results Here we describe and characterize a new methodology for fast and reliable evaluation of an immunoprecipitation reaction. FLIP (FLuorescence IP) relies on the expression of the target protein as a chromophore-tagged protein and couples IP with the measurement of fluorescent signal coating agarose beads. We show here that FLIP displays similar sensitivity to the standard IP/IB procedure but is amenable to high-throughput analysis. We applied FLIP to the screening of mouse monoclonal antibodies of unknown behavior in IP procedures. The parallel analysis of the considered antibodies using FLIP and IP/western shows good correlation between the two procedures. We also show application of FLIP using unpurified antibodies (hybridoma supernatant) and we developed a publicly available tool for the easy analysis and quantification of FLIP signals. Conclusions Altogether, our characterizations of this new methodology show that FLIP is an appealing and reliable tool for any application of high-throughput IP. Electronic supplementary material The online version of this article (doi:10.1186/s12575-016-0046-x) contains supplementary material, which is available to authorized users.

The human β2-adrenergic receptor (β2AR) belongs to the G protein-coupled receptor (GPCR) family and due to its central role in bronchodilation, is an important drug target. The inter-individual variability in β2AR has been implicated in disease susceptibility and differential drug response. In this work, we identified nine potentially deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) using a consensus approach. The deleterious nsSNPs were found to cluster near the ligand binding site and towards the G-protein binding site. To assess their molecular level effects, we built structural models of these receptors and performed atomistic molecular dynamics simulations. Most notably, in the Phe290Ser variant we observed the rotameric flip of Trp2866.48, a putative activation switch that has not been reported in β2AR thus far. In contrast, the variant Met82Lys was found to be the most detrimental to epinephrine binding. Additionally, a few of the nsSNPs were seen to cause perturbations to the lipid bilayer, while a few lead to differences at the G-protein coupling site. We are thus able to classify the variants as ranging from activating to damaging, prioritising them for experimental studies.

We study ¿ip graphs of triangulations whose maximum vertex degree is bounded by a constant k. In particular, we consider triangulations of sets of n points in convex position in the plane and prove that their ¿ip graph is connected if and only if k > 6; the diameter of the ¿ip graph is O(n2). We also show that, for general point sets, ¿ip graphs of pointed pseudo-triangulations can be disconnected for k = 9, and ¿ip graphs of triangulations can be disconnected for any k. Additionally, we consider a relaxed version of the original problem. We allow the violation of the degree bound k by a small constant. Any two triangulations with maximum degree at most k of a convex point set are connected in the ¿ip graph by a path of length O(n log n), where every intermediate triangulation has maximum degree at most k + 4. Keywords: Flip graphs - Triangulations - Rotation distance - Connectivity - Degree bounds

This paper presents a novel, semi-transparent structure to implement single-edge and dual-edge triggered flip-flops. These two novel flip-flops can achieve high speed and low power due to their short data paths and fewer redundant transitions. Simulation results show that the proposed semi-transparent flip-flop and dual-edge triggered semi-transparent flip-flop perform best compared with conventional flip-flops. STFF reduces the power-delay-product 33.1% compared with Antonio’s sense amplifier based flip-flop. DSTFF improves the PDP 9.1% and 47.8% as compared with dual-edge triggered sense amplifier based flip-flop and high speed dual-edge triggered modified hybrid latch flip-flop respectively.

A memory unit is a sequential logic which is an essential part of a computational machine. Realization of fast optical memory units is very challenging. In this communication the authors propose a method of implementation of different frequency encoded flip flops JK, D and T exploiting semiconductor optical amplifier based polarization rotation, filtering and frequency conversion. The main advantage of the frequency encoding technique is that it is free from intensity loss dependent problem and provides ultra fast speed of operation.

FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-specific inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as efficient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as 'FLIP inhibitors'.

Binding of TNF to TNF receptor-1 can give a pro-survival signal through activation of p65/RelA NF-κB, but also signals cell death. To determine the roles of FLICE-inhibitory protein (FLIP) and caspase-8 in TNF-induced activation of NF-κB and apoptosis, we used mouse embryonic fibroblasts derived from FLIP and caspase-8 gene-deleted mice, and treated them with TNF and a smac-mimetic compound that causes degradation of cellular inhibitor of apoptosis proteins (cIAPs). In cells treated with smac mimetic, TNF and Fas Ligand caused wild-type and FLIP(-/-) MEFs to die, whereas caspase-8(-/-) MEFs survived, indicating that caspase-8 is necessary for death of MEFs triggered by these ligands when IAPs are degraded. By contrast, neither caspase-8 nor FLIP was required for TNF to activate p65/RelA NF-κB, because IκB was degraded, p65 translocated to the nucleus, and an NF-κB reporter gene activated normally in caspase-8(-/-) or FLIP(-/-) MEFs. Reconstitution of FLIP(-/-) MEFs with the FLIP isoforms FLIP-L, FLIP-R, or FLIP-p43 protected these cells from dying when treated with TNF or FasL, whether or not cIAPs were depleted. These results show that in MEFs, caspase-8 is necessary for TNF- and FasL-induced death, and FLIP is needed to prevent it, but neither caspase-8 nor FLIP is required for TNF to activate NF-κB.

Gap junction communication is an essential component in the mechanosensitive response of tenocytes. However, little is known about direct mechanoregulation of gap junction turnover and permeability. The present study tests the hypothesis that mechanical loading alters gap junction communication between tenocyte within tendon fascicles. Viable tenocytes within rat tail tendon fasicles were labelled with calcein-AM and subjected to a fluorescent loss induced by photobleaching (FLIP) protocol. A designated target cell within a row of tenocytes was continuously photobleached at 100% laser power whilst recording the fluorescent intensity of neighbouring cells. A mathematical compartment model was developed to estimate the intercellular communication between tenocytes based upon the experimental FLIP data. This produced a permeability parameter, k, which quantifies the degree of functioning gap functions between cells as confirmed by the complete inhibition of FLIP by the inhibitor 18α-glycyrrhentic acid. The application of 1N static tensile load for 10 min had no effect on gap junction communication. However, when loading was increased to 1 h, there was a statistically significant reduction in gap junction permeability. This coincided with suppression of connexin 43 protein expression in loaded samples as determined by confocal immunofluorescence. However, there was an upregulation of connexin 43 mRNA. These findings demonstrate that tenocytes remodel their gap junctions in response to alterations in mechanical loading with a complex mechanosensitive mechanism of breakdown and remodelling. This is therefore the first study to show that tenocyte gap junctions are not only important in transmitting mechanically activated signals but that mechanical loading directly regulates gap junction permeability.

Previous studies have suggested that the caspase 8 inhibitor FLIP is a promising anti-cancer therapeutic target. In this study, we characterised a novel FLIP-targeted antisense phosphorothioate oligonucleotide (AS PTO). FLIP AS and control PTOs were assessed in vitro in transient transfection experiments and in vivo using xenograft models in Balb/c nude mice. FLIP expression was assessed by QPCR and Western. Apoptosis induction was determined by flow cytometry and Western. Of 5 sequences generated, one potently down-regulated FLIP. AS PTO-mediated down-regulation of FLIP resulted in caspase 8 activation and apoptosis induction in non-small cell lung (NSCLC) cells but not in normal lung cells. Similar results were observed in colorectal and prostate cancer cells. Furthermore, the FLIP AS PTO sensitized cancer cells but not normal lung cells to apoptosis induced by rTRAIL. Moreover, the FLIP AS PTO enhanced chemotherapy-induced apoptosis in NSCLC cells. Importantly, compared to a control non-targeted PTO, intra-peritoneal delivery of FLIP AS PTO inhibited the growth of NSCLC xenografts and enhanced the in vivo antitumour effects of cisplatin. We have identified a novel FLIP-targeted AS PTO that has in vitro and in vivo activity and which therefore has potential for further pre-clinical development.

Past studies have identified a number of distinct mechanisms that contribute to the resistance of melanoma cells against apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL). In this report we show that cystatin B is another endogenous inhibitor of TRAIL-induced apoptosis. Cystatin B-deficient melanoma cell lines established by shRNA knockdown displayed increased apoptosis that was associated with enhanced activation of caspase-8 induced by TRAIL. This was not related to the inhibitory effect of cystatin B on the lysosomal cysteine proteases, cathepsin B and L, as they did not have a role in TRAIL-induced apoptosis in most melanoma cell lines even when cystatin B was inhibited. Instead, sensitization of melanoma cells to TRAIL-induced apoptosis by inhibition of cystatin B appeared associated with decreased stability of FLIP(L) as the levels of FLIP(L) were reduced because of shortened half-life time in melanoma cells deficient in cystatin B. In contrast, over-expression of cystatin B increased the levels of FLIP(L), decreased the amount of the E3 ligase Itch associated with FLIP(L), and reduced FLIP(L) ubiquitination. Inhibition of Itch by siRNA restored the levels of FLIP(L) and blocked sensitization to TRAIL-induced apoptosis associated with deficiency in cystatin B. Taken together, these results indicate that cystatin B regulates Itch-mediated degradation of FLIP(L) and thereby TRAIL-induced apoptosis in melanoma cells.

Human immunodeficiency virus (HIV) infection-induced apoptosis of infected CD4 T cells as well as uninfected (bystander) CD4 T cells and other types of cells is a major factor in the pathogenesis of AIDS. Clinically, HIV-2 patients have a higher CD4 cell count at the time of an AIDS diagnosis, and generally have longer survival after development of symptoms. The mortality after an AIDS diagnosis has been reported to be more influenced by CD4 cell count than HIV type. Previous studies have shown significant variations in cytopathic effects following in vitro infection with primary isolates of HIV-1 or HIV-2 subtypes; however, the relative contributions of HIV-1 and HIV-2 infection leading to cell death remain unclear. Using a human cell line, Jurkat, we examined differences in key molecules involved in apoptotic signaling pathways during infection with either HIV-1 or HIV-2. HIV-1 infection generated more reactive oxygen species (ROS), increased the expression of a larger number of molecules involved in cell signaling such as p47, p38alpha, JNK, c-Yes, total PKC, and decreased the expression of molecules such as p38beta, ERK1/2, and XIAP relative to HIV-2 infection. HIV-1 induced a higher degree of cell death through stronger activation of both apoptotic pathways. HIV-1 infection downregulated both Bcl-X(L) and FLIP expressions at later time points postinfection, while HIV-2 infection dramatically upregulated both Bcl-X(L) and FLIP expression. We also found that the expression of Bcl-X(L) or FLIP resulted in significant inhibition of HIV replication in Jurkat cells. These findings suggest that HIV-1 infection with high levels of cytotoxicity results in a higher level of cell death through apoptosis during a short time postinfection. The longer period of infection observed with HIV-2 with a lower degree of cytotoxicity was accompanied by increased Bcl-X(L) and FLIP expression. High protein levels of Bcl-X(L) or FLIP inhibit HIV replication and may be one explanation for the clinical observation that HIV-2 infected patients generally tend to be long-term nonprogressors with high CD4 lymphocyte counts compared with HIV-1 infected persons.

Although Akt is a determinant of cisplatin (cis-diaminedichloroplatinum (CDDP)) resistance in ovarian cancer cells, which is related in part to its inhibitory action on p53 activation, precisely how Akt confers CDDP resistance is unclear. In this study, we show that CDDP induced p53-dependent Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory protein (FLIP) degradation in chemosensitive ovarian cancer cells but not their resistant counterparts. CDDP induced FLIP-p53-Itch interaction, colocalization and FLIP ubiquitination in chemosensitive but not chemoresistant ovarian cancer cells. Moreover, although activated Akt inhibited CDDP-induced FLIP degradation and apoptosis in sensitive cells, these responses were facilitated by dominant-negative Akt expression in chemoresistant cells. Inhibition of Akt function also facilitated p53-FLIP interaction and FLIP ubiquitination, which were attenuated by p53 silencing. These results suggest that Akt confers resistance, in part, by modulating CDDP-induced, p53-dependent FLIP ubiquitination. Understanding the precise etiology of chemoresistance may improve treatment for ovarian cancer.

Apoptosis is central to the interaction between pathogenic mycobacteria and host macrophages. Caspase-8-dependent apoptosis of infected macrophages, which requires activation of the mitogen-activated protein (MAP) kinase p38, lowers the spread of mycobacteria. Here we establish a link between the release of tumor necrosis factor (TNF) and mycobacteria-mediated macrophage apoptosis. TNF activated a pathway involving the kinases ASK1, p38 and c-Abl. This pathway led to phosphorylation of FLIP(S), which facilitated its interaction with the E3 ubiquitin ligase c-Cbl. This interaction triggered proteasomal degradation of FLIP(S), which promoted activation of caspase-8 and apoptosis. Our findings identify a previously unappreciated signaling pathway needed for Mycobacterium tuberculosis-triggered macrophage cell death.

We found that procaspase 8 was overexpressed in non-small-cell lung cancers (NSCLCs) compared with matched normal tissues. The caspase 8 inhibitor FLICE-inhibitory protein (FLIP) was also overexpressed in the majority of NSCLCs. Silencing FLIP induced caspase 8 activation and apoptosis in NSCLC cell lines, but not in normal lung cell lines. Apoptosis induced by FLIP silencing was mediated by the TRAIL death receptors DR4 and DR5, but was not dependent on ligation of the receptors by TRAIL. Furthermore, the apoptosis induced by FLIP silencing was dependent on the overexpression of procaspase 8 in NSCLC cells. Moreover, in NSCLC cells, but not in normal cells, FLIP silencing induced co-localization of DR5 and ceramide, and disruption of this co-localization abrogated apoptosis. FLIP silencing supra-additively increased TRAIL-induced apoptosis of NSCLC cells; however, normal lung cells were resistant to TRAIL, even when FLIP was silenced. Importantly, FLIP silencing sensitized NSCLC cells but not normal cells to chemotherapy in vitro, and silencing FLIP in vivo retarded NSCLC xenograft growth and enhanced the anti-tumour effects of cisplatin. Collectively, our results suggest that due to frequent procaspase 8 overexpression, NSCLCs may be particularly sensitive to FLIP-targeted therapies.

CCAAT/enhancer-binding protein alpha (C/EBPalpha) is mutated in 10% of acute myeloid leukemias, resulting in either a truncated protein or an altered leucine zipper (C/EBPalphaLZ) that prevents DNA binding. C/EBPalpha induces bcl-2 in cooperation with nuclear factor-kappaB (NF-kappaB) p50 to inhibit apoptosis. We now demonstrate that C/EBPalpha or a C/EBPalphaLZ oncoprotein binds the bcl-2 P2 promoter in chromatin immunoprecipitation assays and induces the promoter dependent on the integrity of a kappaB site. C/EBPalpha expressed as a transgene in B cells binds and activates the bcl-2 promoter, but not in nfkb1-/- mice lacking NF-kappaB p50. Bcl-2 is central to the intrinsic apoptotic pathway, whereas FLICE inhibitory protein (FLIP) modulates caspase-8, the initiator caspase of the extrinsic pathway. C/EBPalpha and C/EBPalphaLZ also bind the FLIP promoter and induce its expression dependent upon NF-kappaB p50. Moreover, induction of FLIP by C/EBPalpha protects splenocytes from Fas ligand-induced apoptosis, but only if p50 is present. We also demonstrate the direct interaction between bacterially produced C/EBPalpha and NF-kappaB p50, mediated by the C/EBPalpha basic region. These findings indicate that C/EBPalpha or its oncoproteins activate the bcl-2 and FLIP genes by tethering to their promoters through bound NF-kappaB p50. Targeting their interaction may favor apoptosis of transformed cells.

This study aimed to reveal the pathophysiological signalling responsible for radiation-induced sensitization of hepatocytes to TNF-alpha-mediated apoptosis. IkappaB was upregulated in irradiated hepatocytes. Administration of IkappaB antisense oligonucleotides prior to irradiation inhibited occurrence of apoptosis after TNF-alpha administration. Caspases-8, -9 and -3 activities were increased in irradiated hepatocytes and downregulation of apoptosis by IkappaB antisense oligonucleotides was mediated by suppression of caspases-9 and -3 activation but not of caspase-8 activation, suggesting that radiation-induced sensitization of hepatocytes to TNF-alpha-mediated apoptosis additionally requires changes upstream of caspase-8 activation. Herein, upregulation of FLIP may play a crucial role. Cleavage of bid, upregulation of bax, downregulation of bcl-2 and release of cytochrome c after TNF-alpha-administration depend on radiation-induced upregulation of IkappaB, thus demonstrating an apoptosis permitting effect of IkappaB.

Apoptosis is an essential process to get rid of injured or unwanted cells. In this study, we proposed a mathematical modeling for death receptor mediated apoptosis to investigate the role of c-FLIP in controlling the balance between apoptosis and survival. In order to get insight into how NF-kappa B mediated pro-survival pathway affects the outcome of our modeling, we implemented reduced models without taking such regulation into consideration. Our simulation revealed that c-FLIP could act as a pivotal death or life switch and this switch-like behavior is bistable, irreversible, and robust. We introduce a new term, probability apoptosis, to delineate the likelihood in occurrence of apoptosis events. This simulation system is plausible and may offer several valuable clinical indications for the abnormal apoptosis related disease, such as cancer.

Androgen-withdrawal-induced apoptosis (AWIA) is deregulated in androgen refractory prostate cancer. Androgens have been shown to positively regulate expression of the antiapoptotic FADD-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory protein (FLIP), and reduced FLIP expression precedes apoptosis after androgen withdrawal. Here, we show that FLIP protein expression is downregulated in castrated rats, while in LNCaP cells, androgens regulate FLIP in a manner that is dependent on phosphoinositol-3-kinase (PI3K) and Akt signaling. Specifically, treatment of LNCaP cells with LY294002, or expression of either PTEN or a non-phosphorylatable form of FOXO3a (FOXO3aTM), downregulates FLIP protein and mRNA. Conversely, treatment with androgens in the absence of PI3/Akt signaling, or following expression of FOXO3aTM, leads to increased FLIP expression. A FOXO3a binding site was identified in the FLIP promoter and shown necessary for the combined effects of androgens and FOXO3a on FLIP transcription. FOXO3a binds the androgen receptor, suggesting that the transcriptional synergy depends on an interaction between these proteins. Finally, LNCaP cells are sensitized to TRAIL-induced apoptosis by PTEN or LY294002, and rescued by androgens. FOXO3aTM also sensitizes cells to androgen-inhibited TRAIL apoptosis. Androgen rescue was diminished when either FOXO3a or FLIP was reduced by siRNA. These data support a role for FOXO3a in AWIA.

Cellular FLICE-inhibitory protein (c-FLIP) proteins are known as potent inhibitors of death receptor-mediated apoptosis by interfering with caspase-8 activation at the death-inducing signaling complex (DISC). Among the three human isoforms, c-FLIP(long), c-FLIP(short) and c-FLIP(R), the latter isoform is poorly characterized. We report here the characterization of murine c-FLIP(R) and show that it is the only short c-FLIP isoform expressed in mice. By generating several mutants, we demonstrate that both death effector domains (DEDs) are required for DISC binding and the antiapoptotic function of c-FLIP(R). Surprisingly, the C-terminal tail is important for both protein stability and DISC recruitment. Three-dimensional modeling of c-FLIP(R) revealed a substantial similarity of the overall structures and potential interaction motifs with the viral FLIP MC159. We found, however, that c-FLIP(R) uses different structural motifs for its DISC recruitment. Whereas MC159 interferes with interaction and self-oligomerization of the DISC component FADD by its extensive hydrophilic surface, a narrow hydrophobic patch of c-FLIP(R) on the surface of DED2 is crucial for DISC association. Thus, despite the presence of similar tandem DEDs, viral and cellular FLIPs inhibit apoptosis by remarkably divergent mechanisms.

Protein tyrosine kinases differ widely in their propensity to undergo rearrangements of the N-terminal Asp–Phe–Gly (DFG) motif of the activation loop, with some, including FGFR1 kinase, appearing refractory to this so-called ‘DFG flip'. Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a ‘DFG-out' state. Here we use conformationally selective inhibitors as chemical probes for interrogation of the structural and dynamic features that appear to govern the DFG flip in FGFR1. Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the αH helix, towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib. We conclude that the αC-β4 loop and ‘molecular brake' regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1., Receptor tyrosine kinases are key mediators of cell proliferation that have been implicated in several disease states for which they represent promising drug targets. Here the authors determine the thermodynamic basis for the low propensity of FGFR1 to access the DFG-Phe-out conformation required to bind type-II inhibitors.

Dendritic cells (DCs) are critical for immune homeostasis. To target DCs, we generated a mouse line with Flip deficiency in cells that express cre under the CD11c promoter (CD11c-Flip-KO). CD11c-Flip-KO mice spontaneously develop erosive, inflammatory arthritis, resembling rheumatoid arthritis, which is dramatically reduced when these mice are crossed with Rag−/− mice. The CD8α+ DC subset is significantly reduced, along with alterations in NK cells and macrophages. Autoreactive CD4+ T cells and autoantibodies specific for joint tissue are present, and arthritis severity correlates with the number of autoreactive CD4+ T cells and plasmablasts in the joint-draining lymph nodes. Reduced T regulatory cells (Tregs) inversely correlate with arthritis severity, and the transfer of Tregs ameliorates arthritis. This KO line identifies a model that will permit in depth interrogation of the pathogenesis of rheumatoid arthritis, including the role of CD8α+ DCs and other cells of the immune system., Dendritic cells are critical for initiation of immune responses and for induction of tolerance. Here the authors show that deletion of survival factor c-flip in CD11c-expressing cells subset perturbs CD8a+ dendritic cell, NK and macrophage pools, and leads to development of autoimmune arthritis.

Glutamine and glucose are often controlled at low levels in fed-batch strategies to limit ammonia and lactate accumulation and improve productivity of mammalian cell cultures. However, this risks triggering apoptosis if cells are depleted of glutamine or glucose. To examine the apoptosis cascade during glutamine or glucose limitation, the transcriptional profile of FAS, FASL, FADD, FLIP, BAX, p53 and PEG3 in CRL 1606 hybridoma culture was investigated using quantitative real-time PCR. Activities of caspases 2, 3, 8 and 9 were also analyzed. Increase in the activities of the caspases was observed with up-regulation in the expression of FAS (6-8-fold) and PEG3 (2.5-fold), suggesting that the cells experienced apoptotic cell death via both the death receptor and mitochondrial pathways.

Purpose: In this study, we have investigated the effect of trichostatin A (TSA) pretreatment on the cytotoxicity of TRAIL in TRAIL-resistant myeloma cells. Methods and Results: MM1S myeloma cells exhibited resistance to TRAIL-induced apoptosis even at high doses of TRAIL and was sensitive to low doses of TSA. Sequential treatment of myeloma cells with TSA followed by TRAIL enhanced TRAIL cytotoxicity. TSA induced the transcription of TRAIL death receptor DR5 without affecting the transcription of DR4 and the decoy receptors; DcR1 and DcR2. However, the surface expression of both DR4 and DR5 was not modulated by TSA treatment. TSA treatment repressed the transcription and downregulated the expression of the antiapoptotic Bcl-2 proteins; Bcl-2 and Bcl-XL. Surprisingly, the effect of TSA on the proapoptotic Bcl-2 proteins was mixed, the two isoforms of PUMA (α, β), Noxa, Bax were downregulated, while Bim was upregulated. Although MM1S cells showed higher expression level of FLIPS than other TRAIL-sensitive myeloma cells, the enhancing effect of TSA was not accompanied by FLIPS downregulation. Conclusion: In conclusion, TSA sensitized TRAIL-resistant myeloma cells by downregulating the antiapoptotic Bcl-2 protein without altering FLIPS expression.

Patients with malignant gliomas have a poor prognosis and new treatment paradigms are needed against this disease. TRAIL/Apo2L selectively induces apoptosis in malignant cells sparing normal cells and is hence of interest as a potential therapeutic agent against gliomas. To determine the factors that modulate sensitivity to TRAIL, we examined the differences in TRAIL-activated signaling pathways in glioma cells with variable sensitivities to the agent. Apoptosis in response to TRAIL was unrelated to DR5 expression or endogenous p53 status in a panel of 8 glioma cell lines. TRAIL activated the extrinsic (cleavage of caspase-8, caspase-3 and PARP) and mitochondrial apoptotic pathways and reduced FLIP levels. It also induced caspase-dependent JNK activation, which did not influence TRAIL-induced apoptosis. Because the pro-survival PI3K/Akt pathway is highly relevant to gliomas, we assessed whether Akt could protect against TRAIL-induced apoptosis. Pretreatment with SH-6, a novel Akt inhibitor, enhanced TRAIL-induced apoptosis, suggesting a protective role for Akt. Conversely, TRAIL induced caspase-dependent cleavage of Akt neutralizing its anti-apoptotic effects. These results demonstrate that TRAIL-induced apoptosis in gliomas involves both activation of death pathways and downregulation of survival pathways. Additional studies are warranted to determine the therapeutic potential of TRAIL against gliomas. Apoptosis