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U ovom radu je objašnjen i predstavljen dvodimenzionalni grafički pogon napravljen s grafičkim sučeljem Vulkan koji je onda korišten za potrebe prikaza simulacije fluida koristeći kombinaciju Eulerovog i Lagrangeovog pristupa implementacijom FLIP algoritma. Detaljno je objašnjena implementaciju grafičkog pogona, simulacija fluida i njezina interaktivnost. Rezultati simulacije i njezinih performansi su detaljno analizirani pod utjecajem raznih parametara i opisana su moguća proširenja grafičkoga pogona i simulacije. This paper explains and presents a two-dimensional graphics engine made using the Vulkan graphics interface, which was then used for displaying a fluid simulation using a combination of Eulerian and Lagrangian approaches through the implementation of the FLIP algorithm. The implementation of the graphics engine, fluid simulation, and its interactivity are explained in detail. The simulation results and their performance are thoroughly analyzed under the influence of various parameters, and potential expansions of the graphics engine and simulation are described.

BACKGROUND & AIMS: This study aimed to assess the accuracy of functional luminal imaging probe (FLIP) Panometry to detect esophagogastric junction (EGJ) obstruction assigned by high-resolution manometry (HRM) and the Chicago Classification version 4.0 (CCv4.0). METHODS: 687 adult patients that completed FLIP and HRM for primary esophageal motility evaluation and 35 asymptomatic volunteers (“controls”) were included. EGJ opening was evaluated with 16-cm FLIP during sedated endoscopy via EGJ-distensibility index (DI) and maximum EGJ diameter. HRM was classified according to CCv4.0 and focused on studies with a conclusive disorder of EGJ outflow (i.e. achalasia subtypes I, II, or III; or EGJ outflow obstruction with abnormal timed barium esophagram) or normal EGJ outflow. RESULTS: All 35 controls had EGJ-DI >3.0mm(2)/mmHg and maximum EGJ diameter >16mm. Per HRM and CCv4.0, 245 patients had a conclusive disorder of EGJ outflow and 314 patients had normal EGJ outflow. Among the 241 patients with reduced EGJ opening (REO: EGJ-DI

BACKGROUND & AIMS: Functional luminal imaging probe (FLIP) Panometry can evaluate esophageal motility in response to sustained esophageal distension at the time of sedated endoscopy. This study aimed to describe a classification of esophageal motility using FLIP Panometry and evaluate it against high-resolution manometry (HRM) and Chicago Classification v4.0 (CCv4.0). METHODS: 539 adult patients that completed FLIP and HRM with a conclusive CCv4.0 diagnosis were included in the primary analysis. 35 asymptomatic volunteers (“controls”) and 148 patients with an inconclusive CCv4.0 diagnosis or systemic sclerosis were also described. Esophagogastric junction (EGJ) opening and the contractile response to distension (i.e. secondary peristalsis) were evaluated with 16-cm FLIP performed during sedated endoscopy and analyzed using a customize software program. HRM was classified according to CCv4.0. RESULTS: In the primary analysis, 156 patients (29%) had normal motility on FLIP Panometry, defined by normal EGJ opening (NEO) and a normal or borderline contractile response; 95% of these patients had normal motility or ineffective esophageal motility on HRM. 202 patients (37%) had obstruction with weak contractile response, defined as reduced EGJ opening and absent contractile response or impaired/disordered contractile response, on FLIP Panometry; 92% of these patients had a disorder of EGJ outflow per CCv4.0. CONCLUSIONS: Classifying esophageal motility in response to sustained distension with FLIP Panometry parallels the swallow-associated motility evaluation provided with HRM and CCv4.0. Thus, FLIP Panometry provides a well-tolerated method that can complement, or in some cases be an alternative to HRM, for evaluating esophageal motility disorders.

The Functional Luminal Imaging Probe (FLIP) has emerged as a valuable adjunctive tool in the evaluation of esophageal diseases. Using volumetric distension, FLIP can assess secondary peristalsis and detect esophageal abnormalities that may not be evaluated by high-resolution manometry (HRM). In certain clinical settings, FLIP may allow for deferral of HRM. In therapy for esophageal diseases, FLIP has demonstrated value for its real-time interpretation, which can be used intra-procedurally to tailor therapy and to predict post-therapy outcomes. The future of FLIP looks promising as surgeons and gastroenterologists place increasing emphasis on non-manometric data to diagnose esophageal motility disorders.

Introduction BCL-2 inhibition has transformed the therapeutic landscape of acute myeloid leukemia (AML). However, 10–50% of newly diagnosed patients with AML may not respond to venetoclax and HMA or LDAC, and 3–15% patients may not respond to venetoclax with intensive or non-intensive chemotherapy.1–6 In addition, up to 40% of responding patients may relapse with low rates of response of 20% to salvage therapy and poor overall survival of 2 months after relapse.7 Clinical and biological factors associated with primary and acquired resistance to venetoclax include secondary AML, monocytic differentiation, complex cytogenetics, mutations in TP53, BAX, dependence on other anti-apoptotic proteins, altered metabolism of nicotinamide, fatty acids, and oxidative phosphortylation.3,8–14

Flip-GET has been developed with the objective of optimizing engineering practicals. The innovative element of this methodology is the use of serious games, as a complement to the flipped classroom method, in the teaching-learning process of engineering studies. This methodology uses serious games to take advantage of the capacity of motivation that video games have for the current generation of students, who have been involved with digital content, software, and electronic devices. This methodology has been evaluated using the method of case studies and by an experimental evaluation carried out in different stages, each of which has been developed during an academic course. In the experimental evaluation of the methodology, the control group carried out the practicals dividing the students into subgroups, without using the Flip-GET methodology, while the experimental group performed them with the methodology.

Pesquisa sem auxílio de agências de fomento O uso de aeronaves para aplicação de pesticidas está se tornando cada vez mais comum nos dias atuais, principalmente os multirrotores, em razão de sua precisão e versatilidade. A aplicação de defensı́vel agrı́cola pode ser representada por uma massa lı́quida que compõe grande parte do peso da aeronave, ocasionando uma variação do centro de massa durante o voo, assim como o efeito de sloshing . Estruturas poderem ser inseridas no recipiente para mitigar esses efeitos, no entanto, um controle robusto permite tanto a economia de espaço quanto a diminuição de custo. Assim, um dos objetivos deste trabalho foi o desenvolvimento de um modelo unificado de simulação de transporte de lı́quido em conjunto com a dinâmica de uma aeronave multirrotor através de códigos abertos de fácil uso, para possibilitar o desenvolvimento e testes de diferentes leis de controle. Além disso, utilizou-se tilts na aeronave multirrotor para investigar o seu efeito no transporte de carga lı́quida, juntamente com a influência do sloshing na dinâmica de voo da aeronave pela interação fluı́do estrutura. Este trabalho possui dois experimentos de simulação, sendo um, a comparação com resultados experimentais para uma caixa com fluı́do submetida a acelerações externas e o outro a comparação de controle da aeronave com e sem a caixa de fluı́do. No primeiro, foi observado que apesar de algumas simplificações feitas no modelo, os valores de pressão na parede se assemelham aos existentes experimentais. Para o segundo, foi realizado um experimento com entrada step, e nele foi possı́vel observar a influência do sloshing , na dinâmica de vôo da aeronave. The use of aircraft for pesticide application is becoming increasingly common nowadays, especi- ally with multirotors, due to their precision and versatility. The application of defensibles can be represented by a liquid mass that makes up a large part of the aircraft weight, making the center of mass change during flight, as well as creating the sloshing effect. Structures can be inserted into the container to mitigate these effects. However, robust controls can save space and money. Thus, one of the objectives of this work was the development of a unified model of simulation of liquid transport together with the dynamics of a multirotor aircraft through easy-to-use open codes, to enable the development and testing of different control laws. In addition, tilts were used in the multirotor aircraft to investigate their effect on liquid cargo transport, along with the influence of sloshing on the aircraft’s flight dynamics by fluid-structure interaction. This work has two simulation experiments: one being a comparison with experimental results for a box with fluid subjected to external accelerations and the other a comparison of aircraft control with and without the fluid box. In the first one, it was observed that despite some simplifications made in the model, the pressure values on the wall had similar behaviour to the existing experimental ones. For the second, an experiment with step input was carried out, and it was possible to observe the influence of sloshing on the aircraft’s flight dynamics as well as the tilts. Dissertação (Mestrado)

In education, keeping students engaged has being a challenge in learning. Meanwhile, one of the ideas introduced by educationists in ensuring better student engagement in learning is flipped learning approach so as to enable students to carefully read their learning materials prior to the conventional class. The researchers proposed a gamified flipped learning to improve student’s performance in the Federal Polytechnic Mubi. In achieving the objective, the study examined some of the factors in flipped and gamification that have influence on students’ performance. A well-structured questionnaire was designed and used to elicit data. One Hundred and Fifty (150) copies of the questionnaire were distributed to some selected HND students from five different departments of the school to get their responses on how gamified flipped learning has impacted on their performance. The outcome of the investigation showed that reward, competition, collaboration, satisfaction and confidence have great influence on student’s performance in flipped classroom. This was recommended for use in schools by the researchers.

Regulation of anti-apoptotic protein FLICE-like inhibitory protein (FLIP) and X-linked inhibitor of apoptosis protein (XIAP) remains a crucial step in the cell fate determination and thus targeting these anti-apoptotic proteins could be a viable strategy for the treatment of cancer. However the regulation of FLIP and XIAP is not very well established till date. Here we have shown that ROS decreased XIAP and FLIP by activation of ubiquitin-proteasomal pathway in imatinib resistant K562 cells. Activation of the components of MAPK pathway, ERK and JNK, played a crucial role in XIAP and FLIP degradation because ectopic expression or knock down of ERK and JNK changed the pattern of ROS mediated down-regulation of these two proteins. We have also found that JNK and ERK differentially regulates FLIP and XIAP, respectively. Moreover, our data suggests that activated ERK decreased Akt phosphorylation and thus its binding to and stabilization of XIAP. On the other hand, JNK activation increased E3 ubiquitin ligase ITCH expression and its binding to FLIP which leads to its degradation. Thus, we have, for the first time elucidated that ROS mediated ERK-Akt crosstalk regulates XIAP. We have also shown for the first time that ROS regulates ITCH expression which controls FLIP degradation.

Vitamin E plays an exemplary role in living organisms. α-Tocopherol is the most superior and active form of naturally occurring vitamin E that meets the requirements of human beings as it possesses the α-tocopherol transfer protein (α-TTP). α-Tocopherol deficiency can lead to severe anemia, certain cancers, several neurodegenerative and cardiovascular diseases, and most importantly male infertility. As a result of the depletion of its natural sources, researchers have tried to employ metabolic engineering to enhance α-tocopherol production to meet the human consumption demand. However, the metabolic engineering approach relies on the metabolic flux of a metabolite in its biosynthetic pathway. Analysis of the metabolic flux of a metabolite needs a method that can monitor the α-tocopherol level in living cells. This study was undertaken to construct a FRET (fluorescence resonance energy transfer)-based nanosensor for monitoring the α-tocopherol flux in prokaryotic and eukaryotic living cells. The human α-TTP was sandwiched between a pair of FRET fluorophores to construct the nanosensor, which was denoted as FLIP-α (the fluorescence indicator for α-tocopherol). FLIP-α showed excellence in monitoring the α-tocopherol flux with high specificity. The sensor was examined for its pH stability for physiological applications, where it shows no pH hindrance to its activity. The calculated affinity of this nanosensor was 100 μM. It monitored the real-time flux of α-tocopherol in bacterial and yeast cells, proving its biocompatibility in monitoring the α-tocopherol dynamics in living cells. Being noninvasive, FLIP-α provides high temporal and spatial resolutions, which holds an indispensable significance in bioimaging metabolic pathways that are highly compartmentalized.

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.

Trp-cage is an artificial 20-residue protein and forms a hydrophobic core at its central cage upon folding. In the present study, the folding of Trp-cage was addressed by parallel cascade selection...

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.

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. Literature surrounding use of FLIP has demonstrated its clinical utility as a diagnostic tool and as a device to guide and measure response to therapy. FLIP can assess and guide treatments for esophageal disease states including gastroesophageal reflux disease, achalasia, and eosinophilic esophagitis. FLIP may become the initial test for patients with undifferentiated dysphagia at their index endoscopy. This article summarizes use of FLIP in assessing sphincter function, wall stiffness, and motility to guide treatments.

Human γδ T lymphocytes were reported to display anti-tumor effects against multiple cancers, including hepatocellular carcinoma (HCC). Aberrant expression of microRNAs (miRNAs) leads to a low response to immunotherapy. Thirty-five HCC tumor tissues and their adjacent healthy tissues were collected from patients with primary HCC who underwent tumor resection in the Third People’s Hospital of Hainan Province, China. The purity of the resulting γδ T cells was identified by anti-γδ-T cell receptor-phycoerythrin (anti-γδ-TCR-PE) and anti-CD3-fluorescein isothiocyanate (anti-CD3-FITC) antibodies on flow cytometry. Human HCC cell lines HepG2 and PLC were cultured. We observed that ex vivo, expanded human γδ T cells were able to induce cell lysis of HCC. Furthermore, as miR-382 was observed to be downregulated in HCC tissues and cell lines, we found that overexpression of miR-382 increased the sensitivity of HCC cells to γδ T cells. We proved that mRNA of cellular FADD-like interleukin-1β-converting enzyme-inhibitory protein (c-FLIP) was the target of miR-382. Inhibition of c-FLIP by miR-382 significantly promotes the cell lysis of HCC through strengthening the activation caspase 8 induced by γδ T cell treatment. In conclusion, overexpression of miR-382 promotes HCC cell lysis induced by γδ T cells through inhibiting the expression of c-FLIP., Graphical Abstract, We provide strong evidence that miR-382 increases sensitivity of hepatocellular carcinoma cells to γδ T cell-mediated cytotoxicity. Moreover, we explore that miR-382-promoted cell lysis is dependent on the c-FLIP/caspase-8 pathway. This study is of significance for new strategy of hepatocellular carcinoma treatment.

Vitamin C (VitC) is a potent antioxidant and contributes as an apoptosis inhibitor by preventing death receptor-triggered caspase 8 activity. Fas ligand (FasL) induces the apoptotic cell death via activation of Fas signaling, which is dependent on the expression level of anti-apoptotic molecule c-FLIP (FADD-like IL-1beta-converting enzyme-inhibitory proteins). The present study addressed the effects of VitC on survival of dendritic cells (DCs), a regulator of innate and adaptive immunity. To this end, mouse bone marrow cells were isolated and cultured to attain bone marrow-derived DCs (BMDCs). The cells were treated with FasL in the presence or absence of VitC. Real time RT-PCR, Western blotting and FACS analysis were performed to determine different hallmarks of DC apoptosis. As a result, FasL treatment resulted in activation of caspase 8 and stimulation of cell membrane scrambling, the effects were supressed when VitC was present in the cell culture or the cells were transfected with FLIP siRNA. In conclusion, VitC prevented FasL-triggered DC apoptosis mediated through the expression of c-FLIP.

Pancreatic cancer is usually advanced and drug resistant at diagnosis. A potential therapeutic approach outlined here uses nanoparticle (NP)-based drug carriers, which have unique properties that enhance intra-tumor drug exposure and reduce systemic toxicity of encapsulated drugs. Here we report that patients whose pancreatic cancers express elevated levels of Death Receptor 5 (DR5) and its downstream regulators/effectors FLIP, Caspase-8, and FADD had particularly poor prognoses. To take advantage of elevated expression of this pathway, we designed drug-loaded NPs with a surface-conjugated αDR5 antibody (AMG 655). Binding and clustering of the DR5 is a prerequisite for efficient apoptosis initiation, and the αDR5-NPs were indeed found to activate apoptosis in multiple pancreatic cancer models, whereas the free antibody did not. The extent of apoptosis induced by αDR5-NPs was enhanced by down-regulating FLIP, a key modulator of death receptor- mediated activation of caspase-8. Moreover, the DNA topoisomerase-1 inhibitor camptothecin (CPT) down-regulated FLIP in pancreatic cancer models and enhanced apoptosis induced by αDR5-NPs. CPT-loaded αDR5-NPs significantly increased apoptosis and decreased cell viability in vitro in a caspase-8- and FADD-dependent manner consistent with their expected mechanism-of-action. Importantly, CPT-loaded αDR5-NPs markedly reduced tumor growth rates in vivo in established pan- creatic tumor models, inducing regressions in one model. These proof-of-concept studies indicate that αDR5-NPs loaded with agents that downregulate or inhibit FLIP are promising candidate agents for the treatment of pancreatic cancer.

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.

p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.

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

SUMMARY: We report a previously undescribed pattern of brain metastases in patients with epidermal growth factor receptor–mutated non-small-cell lung cancer treated with tyrosine kinase inhibitors and radiation therapy. These highly distinct lesions appear to spread focally within the leptomeninges, with invasion along the perivascular spaces (FLIP). The survival of patients with FLIP was significantly better compared with patients with classic leptomeningeal disease (median survival, 21 versus 3 months; P = .003). It is unclear whether this pattern of growth is unique to epidermal growth factor receptor–mutated non-small-cell lung cancer.

Pevonedistat (MLN4924), a selective inhibitor of the NEDD8-activating enzyme E1 regulatory subunit (NAE1), has demonstrated significant therapeutic potential in several malignancies. Although multiple mechanisms-of-action have been identified, how MLN4924 induces cell death and its potential as a combinatorial agent with standard-of-care (SoC) chemotherapy in colorectal cancer (CRC) remains largely undefined. In an effort to understand MLN4924-induced cell death in CRC, we identified p53 as an important mediator of the apoptotic response to MLN4924. We also identified roles for the extrinsic (TRAIL-R2/caspase-8) and intrinsic (BAX/BAK) apoptotic pathways in mediating the apoptotic effects of MLN4924 in CRC cells, as well as a role for BID, which modulates a cross-talk between these pathways. Depletion of the anti-apoptotic protein FLIP, which we identify as a novel mediator of resistance to MLN4924, enhanced apoptosis in a p53-, TRAIL-R2/DR5-, and caspase-8-dependent manner. Notably, TRAIL-R2 was involved in potentiating the apoptotic response to MLN4924 in the absence of FLIP, in a ligand-independent manner. Moreoever, when paired with SoC chemotherapies, MLN4924 demonstrated synergy with the irinotecan metabolite SN38. The cell death induced by MLN4924/SN38 combination was dependent on activation of mitochondria through BAX/BAK, but in a p53-independent manner, an important observation given the high frequency of TP53 mutation(s) in advanced CRC. These results uncover mechanisms of cell death induced by MLN4924 and suggest that this second-generation proteostasis-disrupting agent may have its most widespread activity in CRC, in combination with irinotecan-containing treatment regimens.

We evaluated soft-error tolerance by neutrons and heavy ions on four types of flip flops (FFs) called D-type flip flop (DFF), guard-gate FF (GGFF), feedback recovery FF (FRFF), and dual FRFF (DFRFF) in a 65-nm thin buried oxide (BOX) fully depleted silicon on insulator (FDSOI). FRFF has a guard-gate structure only in the master latch. GGFF and DFRFF have the guard-gate structure in both master and slave latches. The guard-gate structure resolves a single-event transient (SET) pulse by delaying it through the guard gate. FRFF and DFRFF have smaller area and shorter delay overheads than GGFF. We revealed that the guard-gate structure has high soft-error tolerance by low-linear energy transfer (LET) heavy ions, but the larger-LET ions over 40 MeV-cm2/mg cause upset even in the guard-gate structures. We revealed that longer delay in the guard-gate can resolve these issues by circuit simulations.

Fulfillment of dual edge flip-flops gets freshly develops into the goal of countless exploration to sustain expressive accomplishment of digital schemes while compressing power expenditure. Powerful low-power flip-flops acquire absolute basic district elements Gross sudden width of histrionic organizes successive circumferences / circuits. Conclude individually and remarkable testing as long as their vulnerability, Q-Delay, Rise Time Path, Fall Time Path and Average Power Consumption. While Power reveals smart effective count regarding the latest electrifying circuit transistors, uncertainly we survive balancing, including scheming comic numbers such as transistors that suspense each number of flip-flops. Analysis / inquiry on static / stable circuits is performed by Dual Data Rate (DDR) using PTM CMOS-16 nm technology alongside 5MHZ frequencies, including their victory procedure. Sensational Dual Data Rate (DDR) Flip-Flop uses 30% less capacity / power, including 14% lower C-Q delay. This paper's proposed architecture is to analyze logic size, area, and power consumption using tanner tool.

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.

Possessing structural homology with their active enzyme counterparts but lacking catalytic activity, pseudoenzymes have been identified for all major enzyme groups. Caspases are a family of cysteine‐dependent aspartate‐directed proteases that play essential roles in regulating cell death and inflammation. Here, we discuss the only human pseudo‐caspase, FLIP(L), a paralog of the apoptosis‐initiating caspases, caspase‐8 and caspase‐10. FLIP(L) has been shown to play a key role in regulating the processing and activity of caspase‐8, thereby modulating apoptotic signaling mediated by death receptors (such as TRAIL‐R1/R2), TNF receptor‐1 (TNFR1), and Toll‐like receptors. In this review, these canonical roles of FLIP(L) are discussed. Additionally, a range of nonclassical pseudoenzyme roles are described, in which FLIP(L) functions independently of caspase‐8. These nonclassical pseudoenzyme functions enable FLIP(L) to play key roles in the regulation of a wide range of biological processes beyond its canonical roles as a modulator of cell death., FLIP(L) is to date the only true pseudo‐caspase identified in the human proteome. As a classical pseudoenzyme, it functions as a regulator of its active homologs, the apoptosis‐initiating caspases, caspase‐8 and caspase‐10. Additionally, FLIP(L) functions independently of these caspases. These nonclassical pseudoenzyme functions enable FLIP(L) to play key roles in the regulation of a wide range of biological processes beyond its canonical roles as a modulator of cell death.

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.

Objectives: Inflammation is an important predisposing and progressive factor in chronic kidney disease (CKD). Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is associated with many fundamental cellular processes, but in chronic inflammatory pathologies remains unclear. Methods: An in vitro peripheral inflammation model was established using lipopolysaccharide (LPS)-stimulated mouse RAW264.7 macrophages, followed by inflammasome activation by ATP treatment. Knockdown of hnRNPK by sihnRNPK and FLICE-like inhibitory protein (FLIP) by siFLIP transfection were achieved in Raw264.7 macrophages. ELISA was used to determine the expression of IL-1β, IL-18 and TNF-α. Real time PCR was applied to detect the mRNA levels of hnRNPK, NOD-like receptors family pyrin domain-containing 3 (NLRP3), FLIP, Caspase-1, IL-1β and IL-18. Western blot and immunofluorescence were performed to detect relevant protein expressions. Co-immunoprecipitation (Co-IP) was used to assess the interaction of hnRNPK with FLIP. Results: Results showed that LPS plus ATP activated NLRP3 inflammasome, which evidenced by the up-regulation of TNF-α, IL-1β and IL-18. Notably, hnRNPK and FLIP were significantly up-regulated in activated NLRP3 inflammasome of macrophages. HnRNPK or FLIP knockdown significantly suppressed the activation of NLRP3 inflammasome, as reflected by down-regulation of Caspase-1, IL-1β and IL-18. Importantly, hnRNPK could directly bind to FLIP in activated NLRP3 inflammasome. Discussion: Our findings suggest that hnRNPK could promote the activation of NLRP3 inflammasome by directly binding FLIP, which might provide potential new therapeutic targets for CKD., GRAPHICAL ABSTRACT

With the increase of the action time and dosing concentration, the proliferation of HepG2 cell was inhibited and its vitality gradually decreased. FCM showed that, with the increase of PN action time, the MMP gradually decreased; calcium ions flowed inwards; the cell cycle was arrested in phase G1; the cell apoptosis rate, especially late apoptosis and necrotic cells, increased. The shear expression of apoptosis-related proteins caspase3 and caspase9 was up-regulated; the shear expression of AIF, MIF and PARP1 proteins associated with Caspase-independent apoptosis, i.e. Parthanatos apoptosis, showed time-dependent up-regulation; the long and short expressions of anti-apoptosis protein FLIP showed different degrees of decrease; the ex-pression of autophagy-related proteins LC3A/B and becin-1 was up-regulated; the expression of P62 protein was down-regulated; the expression of cycle- related proteins P53, P27 and P21 increased significantly; the expression of CyclinD1 and CyclinE1 decreased. FCM was used to detect the increase of ROS with the action time of PN; and its generation level showed an increasing trend; after the combination with ROS scavenger NAC, there was no significant difference in cell viability with the control group. There was no significant difference in the expression level of relevant cell death protein with DMSO control group. There was no difference in intracellular ROS generation level with the control group. Conclusion: PN induces the ROS generation in HepG2 cell, blocks its cycle and causes apoptosis and au-tophagy to play an anti-tumor effect.

Precise information flow from the hippocampus (HP) to prefrontal cortex (PFC) emerges during early development and accounts for cognitive processing throughout life. On flip side, this flow is selectively impaired in mental illness. In mouse models of psychiatric risk mediated by gene-environment interaction (GE), the prefrontal-hippocampal coupling is disrupted already shortly after birth. While this impairment relates to local miswiring in PFC and HP, it might be also because of abnormal connectivity between the two brain areas. Here, we test this hypothesis by combining in vivo electrophysiology and optogenetics with in-depth tracing of projections and monitor the morphology and function of hippocampal afferents in the PFC of control and GE mice of either sex throughout development. We show that projections from the hippocampal CA1 area preferentially target layer 5/6 pyramidal neurons and interneurons, and to a lesser extent layer 2/3 neurons of prelimbic cortex (PL), a subdivision of PFC. In neonatal GE mice, sparser axonal projections from CA1 pyramidal neurons with decreased release probability reach the PL. Their ability to entrain layer 5/6 oscillatory activity and firing is decreased. These structural and functional deficits of hippocampal-prelimbic connectivity persist, yet are less prominent in prejuvenile GE mice. Thus, besides local dysfunction of HP and PL, weaker connectivity between the two brain areas is present in GE mice throughout development. SIGNIFICANCE STATEMENT Poor cognitive performance in mental disorders comes along with prefrontal-hippocampal dysfunction. Recent data from mice that model the psychiatric risk mediated by gene-environment (GE) interaction identified the origin of deficits during early development, when the local circuits in both areas are compromised. Here, we show that sparser and less efficient connectivity as well as cellular dysfunction are the substrate of the weaker excitatory drive from hippocampus (HP) to prefrontal cortex (PFC) as well as of poorer oscillatory coupling between the two brain areas in these mice. While the structural and functional connectivity deficits persist during the entire development, their magnitude decreases with age. The results add experimental evidence for the developmental miswiring hypothesis of psychiatric disorders., This work was supported by grants from the European Research Council (ERC-2015-CoG 681577 to I.L.H.-O.), German Research Foundation (Ha4466/11-1, SPP 1665, and SFB 936 B5 to I.L.H.-O; 368482240/GRK2416, 302153259, and 378028035 to M.S.), Horizon 2020 DEEPER 101016787, and Landesforschungsförderung Hamburg (LFF73 and LFF76 to I.L. H.-O.). We thank Dr. Joseph Gogos for providing the DISC1 mice. We also thank A. Marquardt and A. Dahlmann for excellent technical assistance.

The uncontrolled proliferation of malignant cells in growing tumors results in the generation of different stressors in the tumor microenvironment, such as nutrient shortage, hypoxia and acidosis, among others, that disrupt endoplasmic reticulum (ER) homeostasis and may lead to ER stress. As a response to ER stress, both normal and tumor cells launch a set of signaling pathways known as the unfolded protein response (UPR) to restore ER proteostasis and maintain cell viability and function. However, under sustained ER stress, an apoptotic cell death process can be induced and this has been the subject of different review articles, although the role of the TRAIL-R2/DR5-activated extrinsic pathway of apoptosis has not yet been thoroughly summarized. In this Review, we provide an updated overview of the molecular mechanisms regulating cell fate decisions in tumor cells undergoing ER stress and discuss the role of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 2 (TRAIL-R2/DR5) in the final outcome of UPR signaling. Particularly, we focus on the mechanisms controlling cellular FLICE-like inhibitory protein (FLIP) levels in tumor cells undergoing ER stress, which may represent a potential target for therapeutic intervention in cancer., This work was supported by grants from Ministerio de Economía y Competitividad (SAF2015-64383-P), Ministerio de Ciencia, Innovación y Universidades (PGC2018-093960-B-I00), Junta de Andalucía (PY20-00754), CIBERONC ISCIII CB16/12/00421 and the European Community through the regional development funding program (FEDER) to A.L.-R.

Тема выпускной квалификационной работы: «Оценка и оптимизация кузовной рамы колесного ÑÐ½ÐµÐ³Ð¾Ð±Ð¾Ð»Ð¾Ñ‚Ð¾Ñ Ð¾Ð´Ð°Â». Целью работы является оценка Ð²Ð¾Ð·Ð½Ð¸ÐºÐ°ÑŽÑ‰Ð¸Ñ Ð´ÐµÑ„Ð¾Ñ€Ð¼Ð°Ñ†Ð¸Ð¹ и напряжений в Ð¸ÑÑ Ð¾Ð´Ð½Ð¾Ð¹ пространственной раме Ð²ÐµÐ·Ð´ÐµÑ Ð¾Ð´Ð° и определение путей возможной модификации данной рамы для повышения Ð¼ÐµÑ Ð°Ð½Ð¸Ñ‡ÐµÑÐºÐ¸Ñ ÑÐ²Ð¾Ð¹ÑÑ‚Ð², а также оценка Ð¿Ñ€ÐµÐ´Ð»Ð¾Ð¶ÐµÐ½Ð½Ñ‹Ñ ÑƒÐ»ÑƒÑ‡ÑˆÐµÐ½Ð¸Ð¹. В исследовательской части представлены результаты численного моделирования кручения, изгиба и переворота рассматриваемого Ð²ÐµÐ·Ð´ÐµÑ Ð¾Ð´Ð°. В результате численного расчета Ð¸ÑÑ Ð¾Ð´Ð½Ð¾Ð¹ конструкции были определены: предельный момент до начала Ð¿Ð»Ð°ÑÑ‚Ð¸Ñ‡ÐµÑÐºÐ¸Ñ Ð´ÐµÑ„Ð¾Ñ€Ð¼Ð°Ñ†Ð¸Ð¹ и жесткость рамы на кручение, перемещения в раме при изгибе во время вывешивания колес, которые не превышают 1% на всю длину рамы и напряжения, лежащие в упругой зоне. Деформации и напряжения при перевороте машины по ÑÑ ÐµÐ¼Ðµ из ГОСТ 34065 превышают предельные значения, Ð½ÐµÐ¾Ð±Ñ Ð¾Ð´Ð¸Ð¼Ð¾Ðµ усилие на нагружающей плите не достигнуто. В соответствии с полученными результатами было выдвинуто 6 Ð²Ð¾Ð·Ð¼Ð¾Ð¶Ð½Ñ‹Ñ Ð¿ÑƒÑ‚ÐµÐ¹ улучшения конструкции, из ÐºÐ¾Ñ‚Ð¾Ñ€Ñ‹Ñ Ñ Ð¾Ñ€Ð¾ÑˆÐ¸Ðµ результаты показали: добавление поперечной стойки, уголков в Ð¼ÐµÑÑ‚Ð°Ñ ÑÐ²Ð°Ñ€ÐºÐ¸ и замена стали на алюминий с заменой сечений. Другие улучшения также имеют Ñ Ð¾Ñ€Ð¾ÑˆÐ¸Ðµ результаты по целевым параметрам, но имеют больше рисков по увеличение локальной жесткости или критичному снижению Ð¾Ð±Ñ‰Ð¸Ñ Ð¼ÐµÑ Ð°Ð½Ð¸Ñ‡ÐµÑÐºÐ¸Ñ ÑÐ²Ð¾Ð¹ÑÑ‚Ð² рамы. Работа выполнена на базе продукции компании ООО «Русак», которое предоставило Ð¸ÑÑ Ð¾Ð´Ð½ÑƒÑŽ конструкцию пространственной рамы. По итогам работы сделанные выводы и сформулированные рекомендации переданы в конструкторский отдел компании на оценку и дальнейший анализ., The subject of the graduate qualification work is "Evaluation and optimization of the body frame of the snow and swamp vehicle". The purpose of the work is to evaluate the resulting deformations and stresses in the original spatial frame of the all-terrain vehicle and to determine the ways of possible modification of this frame to improve the mechanical properties, as well as to evaluate the proposed improvements. The research part presents the results of numerical simulation of torsion, bending and overturning of the considered all-terrain vehicle. As a result of the numerical calculation of the initial structure, the following were determined: the limiting moment before the onset of plastic deformations and the torsional rigidity of the frame, the displacements in the frame during bending during the hanging of the wheels, which do not exceed 1% for the entire length of the frame, and the stresses lying in the elastic zone. Deformations and stresses during the overturn of the machine according to the scheme from GOST 34065 exceed the limit values, the required force on the loading plate is not reached. In accordance with the results obtained, 6 possible ways to improve the design were put forward, of which good results were shown: adding a transverse rack, corners at the welding points and replacing steel with aluminum with changing sections. Other improvements also have good results on target parameters but have more risks of increasing local stiffness or critically reducing the overall mechanical properties of the frame. The work was performed based on the products of Rusak LLC, which provided the initial design of the space frame. Based on the results of the work, the conclusions drawn, and the formulated recommendations were submitted to the design department of the company for evaluation and further analysis.

En Colombia cada vez es más difícil optar por vivienda propia por los altos precios asociados debido a la inflación y los incrementos en las tasas de interés para préstamos de vivienda. Así mismo, las viviendas “en la mayoría de los casos” son entregadas en obra gris (en especial las VIS y VIP) y los compradores deben invertir altos costos en remodelación, por lo que muchas veces optan por vivir en estas condiciones. Entendiendo esto se creó la compañía “Social Flip”, la cual orienta su modelo de negocio en el flipping Social, es decir a la compra, remodelación, venta, financiación y entrega de viviendas estética-terminadas a familias biparentales de la ciudad de Pasto de estratos medio bajo y bajo. Nuestra estrategia se fundamenta en realizar ventas de viviendas remodeladas a los precios más bajos de mercado y financiarlas sin tasa de interés, enfocando esfuerzos a realizar alianzas estratégicas con diferentes actores (entidades financieras, proveedores), promover la participación de las familias en la mano de obra simple para disminuir los costos y por ende el precio de venta. In Colombia it is increasingly difficult to choose your own home due to the high prices associated with inflation and increases in interest rates for home loans. Likewise, homes "in most cases" are delivered in gray works (especially VIS and VIP) and buyers must invest high costs in remodeling, so they often choose to live in these conditions. Understanding this, the company "Social Flip" was created, which focuses its business model on Social flipping, that is, the purchase, remodeling, sale, financing and delivery of aesthetically-finished homes to two-parent families in the city of Pasto de lower middle and lower strata. Our strategy is based on making sales of remodeled homes at the lowest market prices and financing them without interest rates, focusing efforts on making strategic alliances with different actors (financial entities, suppliers), promoting the participation of families in the hands of Simple work to reduce costs and therefore the sale price. Especialización Especialista en Innovación y Desarrollo de Negocios

The GRIA3 gene is located in the X chromosome and encodes for one of the subunits (iGluR3) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), an excitatory synaptic transmission receptor present in most parts of the brain. iGluR3 dysfunction has been associated with both abnormal memory for-mation and learning. It has been observed in patients with different neurological and cognitive disorders, including epilepsy. Three different de novo missense variants of GRIA3 have recently been reported in patients with Developmental and Epileptic Encephalopathy (DEE).We report on a female pediatric patient with DEE whose clinical picture mimicked structural epilepsy. We give a detailed description of our patient's most important electro-clinical features. Genetic analysis revealed that the patient carried a de novo missense variant in GRIA3 (c.2359G>A; p.Glu787Lys). The p.Glu787Lys variant had previously been reported in a male pediatric patient. Additionally, we studied iGluR3 expression in the patient and control fibroblasts. We found significantly lower iGluR3 expression in the patient's fibroblasts than in controls and different responses to glutamate treatment.In summary, our report expands knowledge of GRIA3 variants affecting boys and girls, describes functional studies of these variants, and provides an extensive review of the literature concerning GRIA3 genetic variants.

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.

Replication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires origin–recognition complex (ORC), Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with single-molecule Förster resonance energy transfer (FRET), we investigated interactions between ORC and Mcm2-7 during helicase loading. In the large majority of events, we observed a single ORC molecule recruiting both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between first- and second-helicase recruitment, a rapid change in interactions between ORC and the first Mcm2-7 occurs. Within seconds, ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7–ORC–DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly coordinated series of events through which a single ORC molecule can load two oppositely oriented helicases.

TNF stimulation generates pro-survival signals through activation of NF-κB that restrict the build-in death signaling triggered by TNF. The competition between TNF-induced survival and death signals ultimately determines the fate of a cell. Here, we report the identification of Bclaf1 as a novel component of the anti-apoptotic program of TNF. Bclaf1 depletion in multiple cells sensitizes cells to TNF-induced apoptosis but not to necroptosis. Bclaf1 exerts its anti-apoptotic function by promoting the transcription of CFLAR, a caspase 8 antagonist, downstream of NF-κB activation. Bclaf1 binds to the p50 subunit of NF-κB, which is required for Bclaf1 to stimulate CFLAR transcription. Finally, in Bclaf1 siRNA administered mice, TNF-induced small intestine injury is much more severe than in control mice with aggravated signs of apoptosis and pyroptosis. These results suggest Bclaf1 is a key regulator in TNF-induced apoptosis, both in vitro and in vivo.

The biochemical state of neurons, and of cells in general, is regulated by extracellular factors, including neurotransmitters, neuromodulators, and growth hormones. Interactions of an animal with its environment trigger neuromodulator release and engage biochemical transduction cascades to modulate synapse and cell function. Although these processes are thought to enact behavioral adaption to changing environments, when and where in the brain they are induced has been mysterious because of the challenge of monitoring biochemical state in real time in defined neurons in behaving animals. Here, we describe a method allowing measurement of activity of protein kinase A (PKA), an important intracellular effector for neuromodulators, in freely moving mice. To monitor PKA activity in vivo, we use a genetically targeted sensor (FLIM-AKAR) and fluorescence lifetime photometry (FLiP). This article describes how to set up a FLiP system and obtain robust recordings of net PKA phosphorylation state in vivo. The methods should be generally useful to monitor other pathways for which fluorescence lifetime reporters exist. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Building a FLiP system Basic Protocol 2: FLIM-AKAR viral injection and fiber implantation for FLiP measurement Basic Protocol 3: Performing measurements using FLiP.