Macro- and micro-scale culture environment differentially regulate the effects of crowding on macrophage function.

Macrophages hold vital roles in immune defense, wound healing, and tissue homeostasis, and have the exquisite ability to sense and respond to dynamically changing cues in their microenvironment. Much of our understanding of their behavior has been derived from studies performed using in vitro culture systems, in which the cell environment can be precisely controlled. Recent advances in miniaturized culture platforms also offer the ability to recapitulate some features of the in vivo environment and analyze cellular responses at the single-cell level. Since macrophages are sensitive to their surrounding environments, the specific conditions in both macro- and micro-scale cultures likely contribute to observed responses. In this study, we investigate how the presence of neighboring cells influence macrophage activation following proinflammatory stimulation in both bulk and micro-scale culture. We found that in bulk cultures, higher seeding density negatively regulated the average TNF-α secretion from individual macrophages in response to inflammatory agonists, and this effect was partially caused by the reduced cell-to-media volume ratio. In contrast, studies conducted using microwells to isolate single cells and groups of cells revealed that increasing numbers of cells positively influences their inflammatory activation, suggesting that the absolute cell numbers in the system may be important. In addition, a single inflammatory cell enhanced the inflammatory state of a small group of cells. Overall, this work helps to better understand how variations of macroscopic and microscopic culture environments influence studies in macrophage biology and provides insight into how the presence of neighboring cells and the soluble environment influences macrophage activation.

Testing the social motivation theory of autism: the role of co-occurring anxiety.

BACKGROUND: The Social Motivation Theory proposes that social reward processing differences underlie autism. However, low social motivation has also been linked to higher anxiety. Given the co-occurrence between autism and anxiety, it is possible that anxiety drives the association between social motivation and autistic characteristics. This study tests the mechanisms underlying the association between social motivation and autistic traits. METHODS: Participants were 165 adolescents (71 male), aged 10-16 years, from the Mapping profiles of cognition, motivation and attention in childhood (C-MAPS) study, enriched for autistic traits (70 participants with an autism diagnosis, 37 male). Participants completed a battery of online experimental tasks, including a Choose-a-Movie social motivation task and social cognition measures (theory of mind; emotion recognition), alongside parent-reported child anxiety and autistic traits. RESULTS: Higher social motivation was significantly associated with lower autistic traits (ß = -.26, p < .001). Controlling for social cognition did not change the association between social motivation and autistic traits. Controlling for anxiety did significantly reduce the strength of the association (unstandardized coefficient change: p = .003), although social motivation remained associated with autistic traits (ß = -.16, p = .004). Post hoc analyses demonstrated differential sex-effects: The association between social motivation and autistic traits was significant only in the females (ß = -.38, p < .001), as was the attenuation by anxiety (unstandardized coefficient change: p < .001). CONCLUSIONS: The association between social motivation and autistic traits could be partially attributed to co-occurring anxiety. Sex-specific effects found in females may be due to environmental factors such as increased social demands in adolescent female relationships. Results are consistent with self-report by autistic individuals who do not identify as having reduced social motivation.

Social threat processing in adults and children: Faster orienting to, but shorter dwell time on, angry faces during visual search.

Attention to emotional signals conveyed by others is critical for gleaning information about potential social partners and the larger social context. Children appear to detect social threats (e.g., angry faces) faster than non-threatening social signals (e.g., neutral faces). However, methods that rely on behavioral responses alone are limited in identifying different attentional processes involved in threat detection or responding. To address this question, we used a visual search paradigm to assess behavioral (i.e., reaction time to select a target image) and attentional (i.e., eye-tracking fixations, saccadic shifts, and dwell time) responses in children (ages 7-10 years old, N = 42) and adults (ages 18-23 years old, N = 46). In doing so, we compared behavioral responding and attentional detection and engagement with threatening (i.e., angry and fearful faces) and non-threatening (i.e., happy faces) social signals. Overall, children and adults were faster to detect social threats (i.e., angry faces), but spent a smaller proportion of time dwelling on them and had slower behavioral responses. Findings underscore the importance of combining different measures to parse differences between processing versus responding to social signals across development. RESEARCH HIGHLIGHTS: Children and adults are slower to select angry faces when measured by time to mouse-click but faster to detect angry faces when measured by time to first eye fixation. The use of eye-tracking addresses some limitations of prior visual search tasks with children that rely on behavioral responses alone. Results suggest shorter time to first fixation, but subsequently, shorter duration of dwell on social threat in children and adults.

Automated production of gene-modified chimeric antigen receptor T cells using the Cocoon Platform.

Autologous cell-based therapeutics have gained increasing attention in recent years because of their efficacy at treating diseases with limited therapeutic options. Chimeric antigen receptor (CAR) T-cell therapy has demonstrated clinical success in hematologic oncology indications, providing critically ill patients with a potentially curative therapy. Although engineered cell therapies such as CAR T cells provide new options for patients with unmet needs, the high cost and complexity of manufacturing may hinder clinical and commercial translation. The Cocoon Platform (Lonza, Basel, Switzerland) addresses many challenges, such as high labor demand, process consistency, contamination risks and scalability, by enabling efficient, functionally closed and automated production, whether at clinical or commercial scale. This platform is customizable and easy to use and requires minimal operator interaction, thereby decreasing process variability. We present two processes that demonstrate the Cocoon Platform's capabilities. We employed different T-cell activation methods-OKT3 and CD3/CD28 Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA)-to generate final cellular products that meet the critical quality attributes of a clinical autologous CAR T-cell product. This study demonstrates a manufacturing solution for addressing challenges with manual methods of production and facilitating the scale-up of autologous cell therapy.

Associations between emotion recognition and autistic and callous-unemotional traits: differential effects of cueing to the eyes.

BACKGROUND: Although autism and callous-unemotional (CU) traits are distinct conditions, both are associated with difficulties in emotion recognition. However, it is unknown whether the emotion recognition difficulties characteristic of autism and CU traits are driven by comparable underpinning mechanisms. METHODS: We tested whether cueing to the eyes improved emotion recognition in relation to autistic and CU traits in a heterogeneous sample of children enhanced for social, emotional and behavioural difficulties. Participants were 171 (n = 75 male) children aged 10-16 years with and without a diagnosis of autism (n = 99 autistic), who completed assessments of emotion recognition with and without cueing to the eyes. Parents completed the assessment of autistic and CU traits. RESULTS: Associations between autistic and CU traits and emotion recognition accuracy were dependent upon gaze cueing. CU traits were associated with an overall decrease in emotion recognition in the uncued condition, but better fear recognition when cued to the eyes. Conversely, autistic traits were associated with decreased emotion recognition in the cued condition only, and no interactions between autistic traits and emotion were found. CONCLUSIONS: The differential effect of cueing to the eyes in autistic and CU traits suggests different mechanisms underpin emotion recognition abilities. Results suggest interventions designed to promote looking to the eyes may be beneficial for children with CU traits, but not for children with autistic characteristics. Future developmental studies of autism and CU characteristics are required to better understand how different pathways lead to overlapping socio-cognitive profiles.

Safety and efficacy of short-course intravenous antibiotics after complicated appendicitis in selected patients.

BACKGROUND: After surgery for complicated appendicitis (CA), common practice is to treat all patients with a standardised long-course of intravenous antibiotics (IVAB) to reduce the risk of postoperative surgical infections (PSI). The aim of the current study was to evaluate the safety and efficacy of a short-course IVAB after CA in selected patients. METHODS: The Department's prospectively collected database identified CA patients treated between2015 and 2019. Baseline and treatment characteristics and postoperative outcomes were analysed. The cut-off between short- and long-course IVAB was 2 days. Outcomes of interest were PSI and 30-day unplanned readmission. RESULTS: In total, 226 patients had CA: Ninety-nine CA (43.8%) received short-course IVAB and 127 (56.2%) received long-course. PSI occurred in 6% and 10% of the short-course and long-course patients, respectively (p = 0.34). Length of IVAB after a PSI was comparable to that of patients without PSI (median 3 and 2 days of IVAB respectively; p = 0.28). 30-day unplanned readmission rates were 7% and 6%, respectively (p = 0.99). Length of IVAB for readmitted patients was similar to those who were not readmitted (median 3 days of IVAB in both; p = 0.91). Multivariable analysis showed that the intraoperative findings of the appendix (p = 0.04) was a prognostic predictor for PSI. ASA score (p = 0.02) and surgical approach (p = 0.05) were prognostic predictors for 30-day unplanned readmission. CONCLUSIONS: This study shows that when patients respond well, a short-course IVAB can safely be applied after CA without increasing risk of PSI or 30-day unplanned readmission.

Surface Inactivation of Highly Mutated SARS-CoV-2 Variants of Concern: Alpha, Delta, and Omicron.

Continued SARS-CoV-2 transmission among the human population has meant the evolution of the virus to produce variants of increased infectiousness and virulence, coined variants of concern (VOCs). The last wave of pandemic infections was driven predominantly by the delta VOC, but because of continued transmission and adaptive mutations, the more highly transmissible omicron variant emerged and is now dominant. However, due to waning immunity and emergence of new variants, vaccines alone cannot control the pandemic. The application of an antiviral coating to high-touch surfaces and physical barriers such as masks are an effective means to inactivate the virus and their spread. Here, we demonstrate an environmentally friendly water-borne polymer coating that can completely inactivate SARS-CoV-2 independent of the infectious variant. The polymer was designed to target the highly glycosylated spike protein on the virion surface and inactivate the virion by disruption of the viral membrane through a nano-mechanical process. Our findings show that, even with low amounts of coating on the surface (1 g/m2), inactivation of alpha, delta, and omicron VOCs and degradation of their viral genome were complete. Furthermore, our data shows that the polymer induces little to no skin sensitization in mice and is non-toxic upon oral ingestion in rats. We anticipate that our transparent polymer coating can be applied to face masks and many other surfaces to capture and inactivate the virus, aiding in the reduction of SARS-CoV-2 transmission and evolution of new variants of concern.

Endovascular Repair of Complicated Type B Aortic Intramural Haematoma: A Single Centre Long Term Experience.

OBJECTIVE: To evaluate the efficacy of thoracic endovascular aortic repair (TEVAR) in the treatment of patients with complicated type B aortic intramural haematoma (IMH). METHODS: A retrospective observational study of patients treated between January 2002 and December 2017 was performed. Complicated type B IMH was defined as persistent pain, rapid dilatation, presence of ulcer-like projections (ULPs), haemothorax, and other signs of (impending) rupture. Thirty day results and long term follow up outcomes were reported. RESULTS: Thirty-nine patients were included for analysis (mean age 68 ± 8 years, 36% male). The thirty day mortality rate was 5%, stroke rate 10%, and re-intervention rate 3%. The median follow up duration was 49 months (25th - 75th percentile: 2 - 96 months). At 10 years, estimated freedom from all cause mortality was 66 ± 9%. During follow up, nine re-interventions were performed, leading to a 10 year estimated freedom from re-intervention rate of 72 ± 8%. Estimated freedom from aortic growth at 10 years was 85 ± 9%. CONCLUSION: Complicated type B IMH can be treated effectively by TEVAR, thus preventing death from aortic rupture. However, severe early post-operative complications, most importantly stroke, are of concern. Long term outcomes are excellent, although re-interventions are not uncommon, either for progression of proximal or distal aortic disease or due to stent graft related complications.

Inhibitory control and problem solving in early childhood: Exploring the burdens and benefits of high self-control.

Low inhibitory control (IC) is sometimes associated with enhanced problem-solving amongst adults, yet for young children high IC is primarily framed as inherently better than low IC. Here, we explore associations between IC and performance on a novel problem-solving task, amongst 102 English 2- and 3-year-olds (Study 1) and 84 Swedish children, seen at 18-months and 4-years (Study 2). Generativity during problem-solving was negatively associated with IC, as measured by prohibition-compliance (Study 1, both ages, Study 2 longitudinally from 18-months). High parent-reported IC was associated with poorer overall problem-solving success, and greater perseveration (Study 1, 3-year-olds only). Benefits of high parent-reported IC on persistence could be accounted for by developmental level. No concurrent association was observed between problem-solving performance and IC as measured with a Delay-of-Gratification task (Study 2, concurrent associations at 4-years). We suggest that, for young children, high IC may confer burden on insight- and analytic-aspects of problem-solving.

Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability.

Nitric oxide (NO) regulates neuronal function and thus is critical for tuning neuronal communication. Mechanisms by which NO modulates protein function and interaction include posttranslational modifications (PTMs) such as S-nitrosylation. Importantly, cross signaling between S-nitrosylation and prenylation can have major regulatory potential. However, the exact protein targets and resulting changes in function remain elusive. Here, we interrogated the role of NO-dependent PTMs and farnesylation in synaptic transmission. We found that NO compromises synaptic function at the Drosophila neuromuscular junction (NMJ) in a cGMP-independent manner. NO suppressed release and reduced the size of available vesicle pools, which was reversed by glutathione (GSH) and occluded by genetic up-regulation of GSH-generating and de-nitrosylating glutamate-cysteine-ligase and S-nitroso-glutathione reductase activities. Enhanced nitrergic activity led to S-nitrosylation of the fusion-clamp protein complexin (cpx) and altered its membrane association and interactions with active zone (AZ) and soluble N-ethyl-maleimide-sensitive fusion protein Attachment Protein Receptor (SNARE) proteins. Furthermore, genetic and pharmacological suppression of farnesylation and a nitrosylation mimetic mutant of cpx induced identical physiological and localization phenotypes as caused by NO. Together, our data provide evidence for a novel physiological nitrergic molecular switch involving S-nitrosylation, which reversibly suppresses farnesylation and thereby enhances the net-clamping function of cpx. These data illustrate a new mechanistic signaling pathway by which regulation of farnesylation can fine-tune synaptic release.

Saccade dysmetria indicates attenuated visual exploration in autism spectrum disorder.

BACKGROUND: Visual exploration in autism spectrum disorder (ASD) is characterized by attenuated social attention. The underlying oculomotor function during visual exploration is understudied, whereas oculomotor function during restricted viewing suggested saccade dysmetria in ASD by altered pontocerebellar motor modulation. METHODS: Oculomotor function was recorded using remote eye tracking in 142 ASD participants and 142 matched neurotypical controls during free viewing of naturalistic videos with and without human content. The sample was heterogenous concerning age (6-30 years), cognitive ability (60-140 IQ), and male/female ratio (3:1). Oculomotor function was defined as saccade, fixation, and pupil-dilation features that were compared between groups in linear mixed models. Oculomotor function was investigated as ASD classifier and features were correlated with clinical measures. RESULTS: We observed decreased saccade duration (∆M = -0.50, CI [-0.21, -0.78]) and amplitude (∆M = -0.42, CI [-0.12, -0.72]), which was independent of human video content. We observed null findings concerning fixation and pupil-dilation features (POWER = .81). Oculomotor function is a valid ASD classifier comparable to social attention concerning discriminative power. Within ASD, saccade features correlated with measures of restricted and repetitive behavior. CONCLUSIONS: We conclude saccade dysmetria as ASD oculomotor phenotype relevant to visual exploration. Decreased saccade amplitude and duration indicate spatially clustered fixations that attenuate visual exploration and emphasize endogenous over exogenous attention. We propose altered pontocerebellar motor modulation as underlying mechanism that contributes to atypical (oculo-)motor coordination and attention function in ASD.

Adaptation of sperm whales to open-boat whalers: rapid social learning on a large scale?

Animals can mitigate human threats, but how do they do this, and how fast can they adapt? Hunting sperm whales was a major nineteenth century industry. Analysis of data from digitized logbooks of American whalers in the North Pacific found that the rate at which whalers succeeded in harpooning ('striking') sighted whales fell by about 58% over the first few years of exploitation in a region. This decline cannot be explained by the earliest whalers being more competent, as their strike rates outside the North Pacific, where whaling had a longer history, were not elevated. The initial killing of particularly vulnerable individuals would not have produced the observed rapid decline in strike rate. It appears that whales swiftly learned effective defensive behaviour. Sperm whales live in kin-based social units. Our models show that social learning, in which naive social units, when confronted by whalers, learned defensive measures from grouped social units with experience, could lead to the documented rapid decline in strike rate. This rapid, large-scale adoption of new behaviour enlarges our concept of the spatio-temporal dynamics of non-human culture.

RBM3 mediates structural plasticity and protective effects of cooling in neurodegeneration.

In the healthy adult brain synapses are continuously remodelled through a process of elimination and formation known as structural plasticity. Reduction in synapse number is a consistent early feature of neurodegenerative diseases, suggesting deficient compensatory mechanisms. Although much is known about toxic processes leading to synaptic dysfunction and loss in these disorders, how synaptic regeneration is affected is unknown. In hibernating mammals, cooling induces loss of synaptic contacts, which are reformed on rewarming, a form of structural plasticity. We have found that similar changes occur in artificially cooled laboratory rodents. Cooling and hibernation also induce a number of cold-shock proteins in the brain, including the RNA binding protein, RBM3 (ref. 6). The relationship of such proteins to structural plasticity is unknown. Here we show that synapse regeneration is impaired in mouse models of neurodegenerative disease, in association with the failure to induce RBM3. In both prion-infected and 5XFAD (Alzheimer-type) mice, the capacity to regenerate synapses after cooling declined in parallel with the loss of induction of RBM3. Enhanced expression of RBM3 in the hippocampus prevented this deficit and restored the capacity for synapse reassembly after cooling. RBM3 overexpression, achieved either by boosting endogenous levels through hypothermia before the loss of the RBM3 response or by lentiviral delivery, resulted in sustained synaptic protection in 5XFAD mice and throughout the course of prion disease, preventing behavioural deficits and neuronal loss and significantly prolonging survival. In contrast, knockdown of RBM3 exacerbated synapse loss in both models and accelerated disease and prevented the neuroprotective effects of cooling. Thus, deficient synapse regeneration, mediated at least in part by failure of the RBM3 stress response, contributes to synapse loss throughout the course of neurodegenerative disease. The data support enhancing cold-shock pathways as potential protective therapies in neurodegenerative disorders.

Emotion Recognition Performance in Children with Callous Unemotional Traits is Modulated by Co-occurring Autistic Traits.

OBJECTIVE: Atypical emotion recognition (ER) is characteristic of children with high callous unemotional (CU) traits. The current study aims to 1) replicate studies showing ER difficulties for static faces in relation to high CU-traits; 2) test whether ER difficulties remain when more naturalistic dynamic stimuli are used; 3) test whether ER performance for dynamic stimuli is moderated by eye-gaze direction and 4) assess the impact of co-occurring autistic traits on the association between CU and ER. METHODS: Participants were 292 (152 male) 7-year-olds from the Wirral Child Health and Development Study (WCHADS). Children completed a static and dynamic ER eye-tracking task, and accuracy, reaction time and attention to the eyes were recorded. RESULTS: Higher parent-reported CU-traits were significantly associated with reduced ER for static expressions, with lower accuracy for angry and happy faces. No association was found for dynamic expressions. However, parent-reported autistic traits were associated with ER difficulties for both static and dynamic expressions, and after controlling for autistic traits, the association between CU-traits and ER for static expressions became non-significant. CU-traits and looking to the eyes were not associated in either paradigm. CONCLUSION: The finding that CU-traits and ER are associated for static but not naturalistic dynamic expressions may be because motion cues in the dynamic stimuli draw attention to emotion-relevant features such as eyes and mouth. Further, results suggest that ER difficulties in CU-traits may be due, in part, to co-occurring autistic traits. Future developmental studies are required to tease apart pathways toward the apparently overlapping cognitive phenotype.

International Multicenter Experience of Isolated Limb Infusion for In-Transit Melanoma Metastases in Octogenarian and Nonagenarian Patients.

BACKGROUND: Isolated limb infusion (ILI) is used to treat in-transit melanoma metastases confined to an extremity. However, little is known about its safety and efficacy in octogenarians and nonagenarians (ON). PATIENTS AND METHODS: ON patients (≥ 80 years) who underwent a first ILI for American Joint Committee on Cancer seventh edition stage IIIB/IIIC melanoma between 1992 and 2018 at nine international centers were included and compared with younger patients (< 80 years). A cytotoxic drug combination of melphalan and actinomycin-D was used. RESULTS: Of the 687 patients undergoing a first ILI, 160 were ON patients (median age 84 years; range 80-100 years). Compared with the younger cohort (n = 527; median age 67 years; range 29-79 years), ON patients were more frequently female (70.0% vs. 56.9%; p = 0.003), had more stage IIIB disease (63.8 vs. 53.3%; p = 0.02), and underwent more upper limb ILIs (16.9% vs. 9.5%; p = 0.009). ON patients experienced similar Wieberdink limb toxicity grades III/IV (25.0% vs. 29.2%; p = 0.45). No toxicity-related limb amputations were performed. Overall response for ON patients was 67.3%, versus 64.6% for younger patients (p = 0.53). Median in-field progression-free survival was 9 months for both groups (p = 0.88). Median distant progression-free survival was 36 versus 23 months (p = 0.16), overall survival was 29 versus 40 months (p < 0.0001), and melanoma-specific survival was 46 versus 78 months (p = 0.0007) for ON patients compared with younger patients, respectively. CONCLUSIONS: ILI in ON patients is safe and effective with similar response and regional control rates compared with younger patients. However, overall and melanoma-specific survival are shorter.

Tropical Rainforest Restoration Plantations Are Slow to Restore the Soil Biological and Organic Carbon Characteristics of Old Growth Rainforest.

Widespread and continuing losses of tropical old-growth forests imperil global biodiversity and alter global carbon (C) cycling. Soil organic carbon (SOC) typically declines with land use change from old-growth forest, but the underlying mechanisms are poorly understood. Ecological restoration plantations offer an established means of restoring aboveground biomass, structure and diversity of forests, but their capacity to recover the soil microbial community and SOC is unknown due to limited empirical data and consensus on the mechanisms of SOC formation. Here, we examine soil microbial community response and SOC in tropical rainforest restoration plantings, comparing them with the original old-growth forest and the previous land use (pasture). Two decades post-reforestation, we found a statistically significant but small increase in SOC in the fast-turnover particulate C fraction. Although the δ13C signature of the more stable humic organic C (HOC) fraction indicated a significant compositional turnover in reforested soils, from C4 pasture-derived C to C3 forest-derived C, this did not translate to HOC gains compared with the pasture baseline. Matched old-growth rainforest soils had significantly higher concentrations of HOC than pasture and reforested soils, and soil microbial enzyme efficiency and the ratio of gram-positive to gram-negative bacteria followed the same pattern. Restoration plantings had unique soil microbial composition and function, distinct from baseline pasture but not converging on target old growth rainforest within the examined timeframe. Our results suggest that tropical reforestation efforts could benefit from management interventions beyond re-establishing tree cover to realize the ambition of early recovery of soil microbial communities and stable SOC.

Optimizing the Availability of Enhanced Prehospital Care Team Resources.

OBJECTIVE: After recent developments within the North West Air Ambulance (NWAA), we undertook a service evaluation to determine if resource use could be improved. We sought to answer the following questions: (1) At what time of day do major trauma incidents occur in the North West of England? 2) Do current NWAA operating hours meet the needs of these major trauma patients? 3) Where do major trauma incidents occur in the North West of England? and 4) Are current NWAA resources optimally located to meet the needs of these major trauma patients? METHODS: We reviewed records from the Trauma Audit and Research Network database for the North West of England (the counties of Cheshire, Merseyside, Greater Manchester, Cumbria, and Lancashire) between January 1, 2017, and December 31, 2017. These data were supplemented by incident records from the North West Ambulance Service National Health Service Trust. Analysis was undertaken using Excel (Microsoft, Redmond, WA) and MapInfo Pro (Pitney Bowes, Stamford, CT). A survey will be conducted to give insight into the level of cover provided by other UK helicopter emergency medical services. RESULTS: Data from 2,318 incidents were analyzed. Major trauma occurs in higher numbers at certain times of day, varies from weekday to weekend, and takes place in higher concentrations in certain locations, appearing related to population density. CONCLUSION: There is a significant difference between the current trauma care provision and the demand of major trauma incidents. The findings of this study suggest an expansion in cover provided by the NWAA may be appropriate.

Combination treatment of cancer cells with pan-Akt and pan-mTOR inhibitors: effects on cell cycle distribution, p-Akt expression level and radiolabelled-choline incorporation.

Signal transduction pathways, which regulate cell growth and survival, are up-regulated in many cancers and there is considerable interest in their pharmaceutical modulation for cancer treatment. However inhibitors of single pathway components induce feedback mechanisms that overcome the growth moderating effect of the inhibitor. Combination treatments have been proposed to provide a more complete pathway inhibition. Here the effect of dual treatment of cancer cells with a pan-Akt and a pan-mTOR inhibitor was explored. Breast (SKBr3 and MDA-MB-468) and colorectal (HCT8) cancer cells were treated with the pan-Akt inhibitor MK2206 and pan-mTOR inhibitor AZD8055. Cytotoxic effect of the two drugs were determined using the MTT assay and the Combination Index and isobolomic analysis used to determine the nature of the interaction of the two drugs. Flow cytometry and western blot were employed to demonstrate drug effects on cell cycle distribution and phosph-Aktser473 expression. Radiolabelled ([methyl-3H]) Choline uptake was measured in control and drug-treated cells to determine the modulatory effects of the drugs on choline incorporation. The two drugs acted synergistically to inhibit the growth rate of each cancer cell line. Flow cytometry demonstrated G0/G1 blockade with MK2206 and AZD8055 which was greater when cells were treated with both drugs. The incorporation of [methyl-3H] choline was found be decreased to a greater extent in cells treated with both drugs compared with cells treated with either drug alone. Conclusions Pan-mTOR and pan-Akt inhibition may be highly effective in cancer treatment and measuring changes in choline uptake could be useful in detecting efficacious drug combinations.

The GORE TAG conformable thoracic stent graft with the new ACTIVE CONTROL deployment system.

OBJECTIVE: The aim of this study was to describe single-center intraoperative details and early outcome of the new GORE TAG conformable stent graft with ACTIVE CONTROL (C-TAG ACTIVE CONTROL; W. L. Gore & Associates, Flagstaff, Ariz). METHODS: Between September 2017 and June 2018, a consecutive 30 patients underwent thoracic endovascular aortic repair with C-TAG ACTIVE CONTROL. This new system provides an intermediate deployment step at 50% and optional angulation control of the proximal part of the stent graft. Indications for thoracic endovascular aortic repair varied widely, including chronic postdissection and degenerative aneurysms, complicated acute dissections, and intramural hematomas, among others, in an elective (63.3%), urgent (13.3%), or emergent (23.3%) setting. The proximal landing zone (LZ) was LZ 2 in 23.3%, LZ 3 in 43.3%, and LZ 4 in 33.3%. Data were collected retrospectively and analyzed for technical and clinical success. RESULTS: In all patients, the stent graft was successfully introduced and deployed at the intended position, with complete exclusion of aortic disease and without intraoperative mortality (primary technical success, 100%). One emergent patient died at 2 days of ongoing septic shock (clinical success at 30 days, 96.6%). Median follow-up was 107 days (range, 33-271 days); late mortality was 3.4% (one patient died of stent graft infection at 40 days). Freedom from type I and type III endoleak at the early follow-up was 100%. No patients required other surgical or endovascular procedures for the primary treated aortic disease. CONCLUSIONS: Our reported initial experience in 30 patients with the C-TAG ACTIVE CONTROL showed excellent early results, with high deployment accuracy and conformability. The additional features of staged deployment and angulation control may be of great help in challenging aortic arch diseases, allowing precise positioning and preventing bird-beak effect.

CDP-choline accumulation in breast and colorectal cancer cells treated with a GSK-3-targeting inhibitor.

PURPOSE: Glycogen synthase kinase 3 (GSK3) is a key controlling element of many cellular processes including cell-cycle progression and recent studies suggest that GSK3 is a potential anticancer target. Changes in glucose metabolism associated with GSK3 inhibition may impact on lipid synthesis, whilst lipid metabolites can act as molecular response markers. METHODS: Here, SKBr3 breast and HCT8 colorectal cancer cells were treated with the GSK3 inhibitor SB216763, and [14C (U)] glucose and [3H] choline incorporation into lipids was determined. Cell extracts from treated cells were subject to 31P NMR spectroscopy. RESULTS: SB216763 treatment decreased choline incorporation into lipids and caused an accumulation of CDP-choline which was accompanied by decreased conversion of glucose into lipid components. CONCLUSION: SB216763 profoundly inhibits phospholipid synthesis in cancer cells which demonstrate accumulation of CDP-choline detectable by 31P NMR spectroscopy. Metabolic changes in lipid metabolism present potential response markers to drugs targeting GSK3.