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Supplementary Table 1. Antibodies used in the study Figure S1. AICAR-induced downregulation of IEGs mRNA expression in ALL cells. Figure S2. The mechanism of AICAR-induced IEGs downregulation is AMPK-independent. Figure S3. The PKC inhibitor GF109203X suppresses IEGs expression in ALL cells. Figure S4. PKD inhibition downregulates IEGs transcription in ALL cells. Figure S5. The PKD inhibitor CRT synergizes with regorafenib and dexamethasone in ALL cells.

PDF file - 1.3MB, Dose-response of 2-DG and AIX in CCRF-CEM

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-d-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with d-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG–treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DG's cytotoxicity in ALL. 2-DG–treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival. Mol Cancer Res; 10(7); 969–78. ©2012 AACR.

Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death in children and adolescents, and cure rates for relapsed/refractory ALL remain dismal, highlighting the need for novel targeted therapies. To identify genome-wide metabolic-stress regulated genes, we used RNA-sequencing in ALL cells treated with AICAR, an AMPK activator. RNA-sequencing identified the immediate early genes (IEGs) as a subset of genes downregulated by AICAR. We show that AICAR-induced IEGs downregulation was blocked by an adenosine uptake inhibitor indicating AICAR was responsible for IEGs reprogramming. Using pharmacologic and genetic models we established this mechanism was AMPK-independent. Further investigations using kinase assays, PKD/PKC inhibitors and rescue experiments, demonstrated that AICAR directly inhibited PKD kinase activity and identified PKD as responsible for IEGs downregulation. Mechanistically, PKD inhibition suppressed phosphorylation and nuclear export of class IIa HDACs, which lowered histone H3 acetylation and decreased NFκB(p65) recruitment to IEGs promoters. Finally, PKD inhibition induced apoptosis via DUSP1/DUSP6 downregulation eliciting a DNA damage response. More importantly, ALL patient cells exhibited the same PKD-HDACs-IEGs–mediated mechanism. As proof of principle of the therapeutic potential of targeting PKD, we established the in vivo relevance of our findings using an NSG ALL mouse model. In conclusion, we identified a previously unreported PKD-dependent survival mechanism in response to AICAR-induced cellular stress in ALL through regulation of DUSPs and IEGs' expression.Implications:PKD mediates early transcriptional responses in ALL cells as an adaptive survival mechanism to overcome cellular stress.

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PDF file - 707K, Salubrinal does not protect ALL from apoptosis induced by the inhibitors of N-linked glycosylation

PDF file - 240K, Effects of D-mannose on ALL cytotoxicity induced by equimolar concentrations of 2-DG and 2-FDG

PDF file - 415K, Inhibition of mTOR with CCI-779 does not protect ALL cells from 2-DG-cytotoxicity

PDF file - 385K, The AMP analogue AICAr does not significantly affect 2-DG-induced cytotoxicity in ALL cell lines

Background: During the COVID-19 pandemic, the CoMix study, a longitudinal behavioral survey, was designed to monitor social contacts and public awareness in multiple countries, including Belgium. As a longitudinal survey, it is vulnerable to participants’ “survey fatigue”, which may impact inferences. Methods: A negative binomial generalized additive model for location, scale, and shape (NBI GAMLSS) was adopted to estimate the number of contacts reported between age groups and to deal with under-reporting due to fatigue within the study. The dropout process was analyzed with first-order auto-regressive logistic regression to identify factors that influence dropout. Using the so-called next generation principle, we calculated the effect of under-reporting due to fatigue on estimating the reproduction number. Results: Fewer contacts were reported as people participated longer in the survey, which suggests under-reporting due to survey fatigue. Participant dropout is significantly affected by household size and age categories, but not significantly affected by the number of contacts reported in any of the two latest waves. This indicates covariate-dependent missing completely at random (MCAR) in the dropout pattern, when missing at random (MAR) is the alternative. However, we cannot rule out more complex mechanisms such as missing not at random (MNAR). Moreover, under-reporting due to fatigue is found to be consistent over time and implies a 15-30% reduction in both the number of contacts and the reproduction number (R0) ratio between correcting and not correcting for under-reporting. Lastly, we found that correcting for fatigue did not change the pattern of relative incidence between age groups also when considering age-specific heterogeneity in susceptibility and infectivity. Conclusions: CoMix data highlights the variability of contact patterns across age groups and time, revealing the mechanisms governing the spread/transmission of COVID-19/airborne diseases in the population. Although such longitudinal contact surveys are prone to bias, participant fatigue, and drop-out, we showed that these factors can be identified and corrected using NBI GAMLSS. This information can be used to improve the design of similar, future surveys.

Background: National immunisation programmes globally are at risk of suspension due to the severe health system constraints and physical distancing measures in place to mitigate the ongoing COVID-19 pandemic. We aimed to compare the health benefits of sustaining routine childhood immunisation in Africa with the risk of acquiring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection through visiting routine vaccination service delivery points. Methods: We considered a high-impact scenario and a low-impact scenario to approximate the child deaths that could be caused by immunisation coverage reductions during COVID-19 outbreaks. In the high-impact scenario, we used previously reported country-specific child mortality impact estimates of childhood immunisation for diphtheria, tetanus, pertussis, hepatitis B, Haemophilus influenzae type b, Streptococcus pneumoniae, rotavirus, measles, meningitis A, rubella, and yellow fever to approximate the future deaths averted before 5 years of age by routine childhood vaccination during a 6-month COVID-19 risk period without catch-up campaigns. In the low-impact scenario, we approximated the health benefits of sustaining routine childhood immunisation on only the child deaths averted from measles outbreaks during the COVID-19 risk period. We assumed that contact-reducing interventions flattened the outbreak curve during the COVID-19 risk period, that 60% of the population will have been infected by the end of that period, that children can be infected by either vaccinators or during transport, and that upon child infection the whole household will be infected. Country-specific household age structure estimates and age-dependent infection-fatality rates were applied to calculate the number of deaths attributable to the vaccination clinic visits. We present benefit–risk ratios for routine childhood immunisation, with 95% uncertainty intervals (UIs) from a probabilistic sensitivity analysis. Findings: In the high-impact scenario, for every one excess COVID-19 death attributable to SARS-CoV-2 infections acquired during routine vaccination clinic visits, 84 (95% UI 14–267) deaths in children could be prevented by sustaining routine childhood immunisation in Africa. The benefit–risk ratio for the vaccinated children is 85 000 (4900–546 000), for their siblings (60 years) is 96 (14–307). In the low-impact scenario that approximates the health benefits to only the child deaths averted from measles outbreaks, the benefit–risk ratio to the households of vaccinated children is 3 (0–10); if the risk to only the vaccinated children is considered, the benefit–risk ratio is 3000 (182–21 000). Interpretation: The deaths prevented by sustaining routine childhood immunisation in Africa outweigh the excess risk of COVID-19 deaths associated with vaccination clinic visits, especially for the vaccinated children. Routine childhood immunisation should be sustained in Africa as much as possible, while considering other factors such as logistical constraints, staff shortages, and reallocation of resources during the COVID-19 pandemic. Funding: Gavi, the Vaccine Alliance; Bill & Melinda Gates Foundation.

Endozepines, known as the endogenous ligands of benzodiazepine-binding sites, include the diazepam binding inhibitor (DBI) and its processing products, the triakontatetraneuropeptide (TTN) and the octadecaneuropeptide (ODN). Despite indisputable evidences of the binding of ODN on GABAAR-BZ-binding sites, their action on this receptor lacks compelling electrophysiological observations, some studies reporting that ODN acts as a negative allosteric modulator (NAM) of GABAAR while others suggest the opposite (positive allosteric modulation, PAM effect). All these studies were carried out in vitro with various neuronal cell types. To further elucidate the role of ODN on neuronal excitability, we tested its effect in vivo in the cortex of the anesthetized mouse. Spontaneous neuronal spikes were recorded by the mean of an extracellular pipette in the vicinity of which ODN was micro-infused, either at high dose (10-5M) or low dose (10-11M). ODN at high dose induced a significant increase of neuronal spiking. This effect could be antagonized by the GABAAR-BZ-binding sites blocker flumazenil. In sharp contrast, at low concentration, ODN reduced neuronal spiking in a magnitude similar to GABA itself. Interestingly, this decrease of neuronal activity by low dose of ODN was not flumazenil dependent suggesting that this effect is mediated by another receptor. Finally, we show that astrocytes in culture, known to be stimulated by picomolar dose of ODN via a GPCR, increased their export of GABA when stimulated by low dose of ODN. Our results confirm the versatility of ODN in the control of GABA transmission, but suggest that its PAM-like effect is, at least in part, mediated via an astrocytic non-GABAAR ODN receptor.

Resume Introduction Chez le sujet âge, la prise en charge de l’anxiete, des troubles du sommeil, des troubles dyspeptiques et de la constipation est presque systematiquement dependante de therapies medicamenteuses. Cela induit une iatrogenie importante. Materiel et methodes Les ordonnances de 104 patients institutionnalises et souffrant de dependance cognitive et/ou fonctionnelle ont ete reevaluees lors d’entretiens medicopharmaceutiques afin de s’assurer de la pertinence des prescriptions des anxiolytiques et hypnotiques, des inhibiteurs de la pompe a protons et pansements gastriques et des laxatifs. Le rapport benefice-risque de chaque prescription a ete evalue en vue d’optimiser la prise en charge par la reduction de posologie ou la deprescription de ces medicaments. L’objectif principal de ce travail est de demontrer la tolerance clinique des patients pour les modifications therapeutiques. L’objectif secondaire de la reevaluation est de reduire la consommation globale de ces medicaments et donc les depenses associees. Resultats Dans la population etudiee, l’efficacite a long terme de la deprescription de ces medicaments (a l’exception des laxatifs) a semble evidente. Cependant l’economie generee par la seule reduction des consommations a ete derisoire.

Bacteriophage (“phage”) are both predators and evolutionary drivers for bacteria, notably contributing to the spread of antimicrobial resistance (AMR) genes by generalised transduction. Our current understanding of the dual nature of this relationship is limited. We used an interdisciplinary approach to quantify how these interacting dynamics can lead to the evolution of multi-drug resistant bacteria. We co-cultured two strains of Methicillin-resistantStaphylococcus aureus, each harbouring a different antibiotic resistance gene, with 80α generalized transducing phage. After a growth phase of 8h, bacteria and phage surprisingly coexisted at a stable equilibrium in our culture, the level of which was dependent on the starting concentration of phage. We detected double-resistant bacteria as early as 7h, indicating that transduction of AMR genes had occurred. We developed multiple mathematical models of the bacteria and phage relationship, and found that phage-bacteria dynamics were best captured by a model in which the phage burst size decreases as the bacteria population reaches stationary phase, and where phage predation is frequency-dependent. We estimated that one in every 108new phage generated was a transducing phage carrying an AMR gene, and that double-resistant bacteria were always predominantly generated by transduction rather than by growth. Our results suggest a shift in how we understand and model phage-bacteria dynamics. Although rates of generalised transduction could be interpreted as too rare to be significant, they are sufficient to consistently lead to the evolution of multi-drug resistant bacteria. Currently, the potential of phage to contribute to the growing burden of AMR is likely underestimated.

As several countries gradually release social distancing measures, rapid detection of new localized COVID-19 hotspots and subsequent intervention will be key to avoiding large-scale resurgence of transmission. We introduce ASMODEE (automatic selection of models and outlier detection for epidemics), a new tool for detecting sudden changes in COVID-19 incidence. Our approach relies on automatically selecting the best (fitting or predicting) model from a range of user-defined time series models, excluding the most recent data points, to characterize the main trend in an incidence. We then derive prediction intervals and classify data points outside this interval as outliers, which provides an objective criterion for identifying departures from previous trends. We also provide a method for selecting the optimal breakpoints, used to define how many recent data points are to be excluded from the trend fitting procedure. The analysis of simulated COVID-19 outbreaks suggests ASMODEE compares favourably with a state-of-art outbreak-detection algorithm while being simpler and more flexible. As such, our method could be of wider use for infectious disease surveillance. We illustrate ASMODEE using publicly available data of National Health Service (NHS) Pathways reporting potential COVID-19 cases in England at a fine spatial scale, showing that the method would have enabled the early detection of the flare-ups in Leicester and Blackburn with Darwen, two to three weeks before their respective lockdown. ASMODEE is implemented in the free R package trendbreaker . This article is part of the theme issue ‘Modelling that shaped the early COVID-19 pandemic response in the UK’.

Before the coronavirus 2019 (COVID-19) pandemic began, antimicrobial resistance (AMR) was among the top priorities for global public health. Already a complex challenge, AMR now needs to be addressed in a changing healthcare landscape. Here, we analyse how changes due to COVID-19 in terms of antimicrobial usage, infection prevention, and health systems affect the emergence, transmission, and burden of AMR. Increased hand hygiene, decreased international travel, and decreased elective hospital procedures may reduce AMR pathogen selection and spread in the short term. However, the opposite effects may be seen if antibiotics are more widely used as standard healthcare pathways break down. Over 6 months into the COVID-19 pandemic, the dynamics of AMR remain uncertain. We call for the AMR community to keep a global perspective while designing finely tuned surveillance and research to continue to improve our preparedness and response to these intersecting public health challenges.

Background Assessing the societal perspective in economic evaluations of new interventions requires estimates of indirect non-medical costs caused by the disease. Different methods exist for measuring the labor input function as a surrogate for these costs. They rarely specify the effect of health on labor and who gains and who loses money. Social accounting matrix (SAM) is an established framework that evaluates public policies with multiple perspectives that could help. Objectives We evaluated the use of a modified SAM to assess money flows between different economic agents resulting in economic transactions following policy changes of medical interventions. Methods We compared conventional methods of measuring indirect non-medical costs related to rotavirus vaccination in the Netherlands with a modified SAM framework. To compare the outcome of each method, we calculated returns on investment (ROI) as the net amount of money per euro invested in the vaccine. One-way and probabilistic sensitivity analyses were carried out for each method, focusing on critical variables with the largest impact on indirect cost estimates. Results The ROI was higher for the modified SAM (1.33) than for the conventional methods assessing income calculations (range - 0.178 to 1.22). Probabilistic sensitivity analyses showed wide distributions in the ROI estimates, with variation in the variable impact on the indirect cost results per method selected. Conclusions In contrast to conventional methods, the SAM approach provides detailed and comprehensive assessments of the impact of new interventions on the indirect non-medical costs and the financial interactions between agents, disclosing useful information for different stakeholders.

Introduction: Contact tracing has the potential to control outbreaks without the need for stringent physical distancing policies, e.g. civil lockdowns. Unlike forward contact tracing, backward contact tracing identifies the source of newly detected cases. This approach is particularly valuable when there is high individual-level variation in the number of secondary transmissions (overdispersion). Methods: By using a simple branching process model, we explored the potential of combining backward contact tracing with more conventional forward contact tracing for control of COVID-19. We estimated the typical size of clusters that can be reached by backward tracing and simulated the incremental effectiveness of combining backward tracing with conventional forward tracing. Results: Across ranges of parameter values consistent with dynamics of SARS-CoV-2, backward tracing is expected to identify a primary case generating 3-10 times more infections than a randomly chosen case, typically increasing the proportion of subsequent cases averted by a factor of 2-3. The estimated number of cases averted by backward tracing became greater with a higher degree of overdispersion. Conclusion: Backward contact tracing can be an effective tool for outbreak control, especially in the presence of overdispersion as is observed with SARS-CoV-2.

Introduction: Contact tracing has the potential to control outbreaks without the need for stringent physical distancing policies, e.g. civil lockdowns. Unlike forward contact tracing, backward contact tracing identifies the source of newly detected cases. This approach is particularly valuable when there is high individual-level variation in the number of secondary transmissions (overdispersion). Methods: By using a simple branching process model, we explored the potential of combining backward contact tracing with more conventional forward contact tracing for control of COVID-19. We estimated the typical size of clusters that can be reached by backward tracing and simulated the incremental effectiveness of combining backward tracing with conventional forward tracing. Results: Across ranges of parameter values consistent with dynamics of SARS-CoV-2, backward tracing is expected to identify a primary case generating 3-10 times more infections than a randomly chosen case, typically increasing the proportion of subsequent cases averted by a factor of 2-3. The estimated number of cases averted by backward tracing became greater with a higher degree of overdispersion. Conclusion: Backward contact tracing can be an effective tool for outbreak control, especially in the presence of overdispersion as is observed with SARS-CoV-2.

Introduction: Contact tracing has the potential to control outbreaks without the need for stringent physical distancing policies, e.g. civil lockdowns. Unlike forward contact tracing, backward contact tracing identifies the source of newly detected cases. This approach is particularly valuable when there is high individual-level variation in the number of secondary transmissions (overdispersion). Methods: By using a simple branching process model, we explored the potential of combining backward contact tracing with more conventional forward contact tracing for control of COVID-19. We estimated the typical size of clusters that can be reached by backward tracing and simulated the incremental effectiveness of combining backward tracing with conventional forward tracing. Results: Across ranges of parameter values consistent with dynamics of SARS-CoV-2, backward tracing is expected to identify a primary case generating 3-10 times more infections than average, typically increasing the proportion of subsequent cases averted by a factor of 2-3. The estimated number of cases averted by backward tracing became greater with a higher degree of overdispersion. Conclusion: Backward contact tracing can be an effective tool for outbreak control, especially in the presence of overdispersion as was observed with SARS-CoV-2.

Background: The isolation of symptomatic cases and tracing of contacts has been used as an early COVID-19 containment measure in many countries, with additional physical distancing measures also introduced as outbreaks have grown. To maintain control of infection while also reducing disruption to populations, there is a need to understand what combination of measures—including novel digital tracing approaches and less intensive physical distancing—might be required to reduce transmission. We aimed to estimate the reduction in transmission under different control measures across settings and how many contacts would be quarantined per day in different strategies for a given level of symptomatic case incidence. Methods: For this mathematical modelling study, we used a model of individual-level transmission stratified by setting (household, work, school, or other) based on BBC Pandemic data from 40 162 UK participants. We simulated the effect of a range of different testing, isolation, tracing, and physical distancing scenarios. Under optimistic but plausible assumptions, we estimated reduction in the effective reproduction number and the number of contacts that would be newly quarantined each day under different strategies. Results: We estimated that combined isolation and tracing strategies would reduce transmission more than mass testing or self-isolation alone: mean transmission reduction of 2% for mass random testing of 5% of the population each week, 29% for self-isolation alone of symptomatic cases within the household, 35% for self-isolation alone outside the household, 37% for self-isolation plus household quarantine, 64% for self-isolation and household quarantine with the addition of manual contact tracing of all contacts, 57% with the addition of manual tracing of acquaintances only, and 47% with the addition of app-based tracing only. If limits were placed on gatherings outside of home, school, or work, then manual contact tracing of acquaintances alone could have an effect on transmission reduction similar to that of detailed contact tracing. In a scenario where 1000 new symptomatic cases that met the definition to trigger contact tracing occurred per day, we estimated that, in most contact tracing strategies, 15 000–41 000 contacts would be newly quarantined each day. Interpretation: Consistent with previous modelling studies and country-specific COVID-19 responses to date, our analysis estimated that a high proportion of cases would need to self-isolate and a high proportion of their contacts to be successfully traced to ensure an effective reproduction number lower than 1 in the absence of other measures. If combined with moderate physical distancing measures, self-isolation and contact tracing would be more likely to achieve control of severe acute respiratory syndrome coronavirus 2 transmission. Funding: Wellcome Trust, UK Engineering and Physical Sciences Research Council, European Commission, Royal Society, Medical Research Council.

As several countries gradually release social distancing measures, rapid detection of new localised COVID-19 hotspots and subsequent intervention will be key to avoiding large-scale resurgence of transmission. We introduce ASMODEE (Automatic Selection of Models and Outlier Detection for Epidemics), a new tool for detecting sudden changes in COVID-19 incidence. Our approach relies on automatically selecting the best (fitting or predicting) model from a range of user-defined time series models, excluding the most recent data points, to characterise the main trend in an incidence. We then derive prediction intervals and classify data points outside this interval as outliers, which provides an objective criterion for identifying departures from previous trends. We also provide a method for selecting the optimal breakpoints, used to define how many recent data points are to be excluded from the trend fitting procedure. The analysis of simulated COVID-19 outbreaks suggest ASMODEE compares favourably with a state-of-art outbreak-detection algorithm while being simpler and more flexible. We illustrate our method using publicly available data of NHS Pathways reporting potential COVID-19 cases in England at a fine spatial scale, for which we provide a template automated analysis pipeline. ASMODEE is implemented in the free R package trendbreaker.

MINERvA reports inclusive charged-current cross sections for muon neutrinos on hydrocarbon in the NuMI beamline. We measured the double-differential cross section in terms of the longitudinal and transverse muon momenta, as well as the single-differential cross sections in those variables. The data used in this analysis correspond to an exposure of $3.34 \times 10^{20}$ protons on target with a peak neutrino energy of approximately 3.5 GeV. Measurements are compared to the GENIE, NuWro and GiBUU neutrino cross-section predictions, as well as a version of GENIE modified to produce better agreement with prior exclusive MINERvA measurements. None of the models or variants were able to successfully reproduce the data across the entire phase space, which includes areas dominated by each interaction channel.

Background: Concern about the health impact of novel coronavirus SARS-CoV-2 has resulted in widespread enforced reductions in people’s movement (“lockdowns”). However, there are increasing concerns about the severe economic and wider societal consequences of these measures. Some countries have begun to lift some of the rules on physical distancing in a stepwise manner, with differences in what these “exit strategies” entail and their timeframes. The aim of this work was to inform such exit strategies by exploring the types of indoor and outdoor settings where transmission of SARS-CoV-2 has been reported to occur and result in clusters of cases. Identifying potential settings that result in transmission clusters allows these to be kept under close surveillance and/or to remain closed as part of strategies that aim to avoid a resurgence in transmission following the lifting of lockdown measures. Methods: We performed a systematic review of available literature and media reports to find settings reported in peer reviewed articles and media with these characteristics. These sources are curated and made available in an editable online database. Results: We found many examples of SARS-CoV-2 clusters linked to a wide range of mostly indoor settings. Few reports came from schools, many from households, and an increasing number were reported in hospitals and elderly care settings across Europe. Conclusions: We identified possible places that are linked to clusters of COVID-19 cases and could be closely monitored and/or remain closed in the first instance following the progressive removal of lockdown restrictions. However, in part due to the limits in surveillance capacities in many settings, the gathering of information such as cluster sizes and attack rates is limited in several ways: inherent recall bias, biased media reporting and missing data.

Survival rates for relapsed/refractory acute lymphoblastic leukemia (ALL), the most common cancer in children and adolescents, remain dismal. We and others have reported that ALL cells are vulnerable to conditions inducing energy/ER-stress mediated by AMP-activated protein kinase (AMPK) activation. AMPK has been reported to interact with chromatin-associated proteins (e.g., histone H2B) in MEF cells to epigenetically regulate gene expression in response to environmental/cellular stress (Bungard et al. Science, 2010; 329:1201). To identify genome-wide genes regulated by direct association of AMPK to chromatin in response to energy/metabolic stress, we first constructed Bp-ALL (NALM6, REH) and T-ALL (CCRF-CEM, KE-37) stable cell lines expressing HA-AMPKα1 or HA-AMPKα2. Next, using HA and RNA pol II antibodies, we performed ChIP-seq assays in CCRF-CEM/HA-AMPKα2 (CN2) grown in glucose-free RPMI for 24 h. ChIP-seq differentially identified 171 candidate genes in CN2 treated with no-glucose vs. 431 genes in untreated controls. Data analysis using the Encode and ChEA database identified the TATA-Box Binding Protein Associated Factor, CCAAT Enhancer Binding Protein Delta (CEBPD), the negative elongation factor complex member E (NELFE), and the Promyelocytic leukemia protein (PML) among highly ranked transcription factors (TFs) may associated with AMPKα2 on chromatin. To correlate the level of gene mRNA expression and recruitment of AMPKα2 to chromatin gene loci regulated in response to energy/metabolic stress, we performed RNA-seq assays in CN2 cells treated with or without glucose deprivation for 24 h. RNA-seq data analysis indicated that of the 3497 genes altered by AMPK activation, two thirds were downregulated whereas the remaining were upregulated. Kyoto Encyclopedia of Genes and Genomes gene set and BioPlanet 2019 gene set analysis identified metabolic pathways, DNA replication/metabolism, and cell cycle as the main biological processes altered in CN2 cells in response to metabolic stress. Among downregulated genes in response to metabolic stress, we uncovered a cluster of histone genes. To confirm and validate our data, we used RT-qPCR and ChIP-qPCR assays on selected histone gene candidates (H1-2/ HIST1H1C, H1-3/HIST1H1D, H4C4/HIST1H4D) which exhibited both decreased recruitment of HA-AMPKα2 to chromatin and mRNA downregulation in response to metabolic stress. Further ChIP-qPCR assays using an AMPKα2 antibody confirmed these data in KASUMI-2 cells (Bp-ALL). Similar data were also observed in CN2 cells treated with AICAR, another AMPK activator. Additional experiments were conducted using the allosteric AMPK activators compound 991 and PF-06409577. Using Co-IP experiments, we uncovered that AMPKα2 interacted with putative members of an AMPK/chromatin-associated transcription factor complex which included the TATA-Box Binding Protein Associated Factor (TAF), integrator (INT), and RNA polymerase II. To investigate the role of AMPK kinase activity on AMPKα2-associated chromatin regulated gene targets, we determined the effect of genetic constructs encoding a constitutively active (CA) form of AMPKα2 on histone gene mRNA expression in CCRF-CEM and MEF AMPKα1/AMPKα2 double knockout (DKO) cells, and found that in both cell types the expression of the full-length CA-AMPKα2 (T172D) lead to decreased mRNA expression of the histone genes examined, suggesting AMPK kinase activity is required to regulate histone genes in response to energy/metabolic stress. In conclusion, our data show that in response to metabolic stress, AMPK binds directly to a multi-protein complex on chromatin to reprogram gene expression to promote cellular adaptation/survival in ALL. Further elucidation of AMPK's interactions with members of the putative AMPK/chromatin-associated transcription complex may lead to unique opportunities for epigenetic-based therapeutic interventions and combination strategies to exploit synthetic lethality in relapse/refractory ALL and other hematological malignancies. Disclosures No relevant conflicts of interest to declare.

We measure neutrino charged current quasielastic-like scattering on hydrocarbon at high statistics using the wide-band NuMI beam with neutrino energy peaked at 6 GeV. The double-differential cross section is reported in terms of muon longitudinal and transverse momentum. Cross-section contours versus lepton momentum components are approximately described by a conventional generator-based simulation, however discrepancies are observed for transverse momenta above 0.5 GeV/c for longitudinal momentum ranges 3 to 5 GeV/c and 9 to 20 GeV/c. The single differential cross section versus momentum transfer squared ($d\sigma/dQ_{QE}^2$) is measured over a four-decade range of $Q^2$ that extends to $10~GeV^2$. The cross section turn-over and fall-off in the $Q^2$ range 0.3 to $10~GeV^2$ is not fully reproduced by generator predictions that rely on dipole form factors. Our measurement probes the axial-vector content of the hadronic current and complements the electromagnetic form factor data obtained using electron-nucleon elastic scattering. These results help oscillation experiments because they probe the importance of various correlations and final-state interaction effects within the nucleus, which have different effects on the visible energy in detectors., Comment: 10 pages, 7 figures

PURPOSE Actionable oncogenic fusion FGFR3-TACC3 (F3T3) is found in 3% of gliomas. Our goal was to characterize the clinical, radiological and molecular features of patients with F3T3-positive glioma. PATIENTS AND METHODS Overall, we screened 1112 gliomas by RT-PCR (861 WHO grade IV, 140 grade III and 111 grade II) and identified 50 F3T3-positive cases. We performed a radiological and radiomic case control study. RESULTS F3T3 fusion was exclusively found in IDH wild-type gliomas. F3T3 was mutually exclusive with the EGFR amplification (0/38 versus 143/336 of F3T3-negative cases, P=0.01), whereas it was associated with CDK4 amplification (7/33 versus 22/321 of F3T3-negative cases, P=0.04) and MDM2 amplification (6/33 versus 15/354 of F3T3-negative cases, P=0.03). F3T3-positive gliomas showed a longer overall survival than F3T3-negative gliomas (median OS 40.1 and 20.0 months, P=0.02), particularly in the grade IV subgroup (40.1 versus 19.0 months, P=0.006). Multivariate analysis showed that F3T3 fusion is an independent predictor of favorable outcome for glioblastoma patients. F3T-positive gliomas were associated with involvement in non-eloquent areas (r=0.31 P=0.007), cortico-subcortical regions and insula involvement (adjusted p CONCLUSION Gliomas harboring F3T3 gene fusions show specific molecular features, distinct radiological and radiomic features and a less aggressive clinical evolution.

Antimicrobial resistance (AMR) is one of the greatest public health challenges we are currently facing. To develop effective interventions against this, it is essential to understand the processes behind the spread of AMR. These are partly dependent on the dynamics of horizontal transfer of resistance genes between bacteria, which can occur by conjugation (direct contact), transformation (uptake from the environment) or transduction (mediated by bacteriophages). Mathematical modelling is a powerful tool to investigate the dynamics of AMR; however, the extent of its use to study the horizontal transfer of AMR genes is currently unclear. In this systematic review, we searched for mathematical modelling studies that focused on horizontal transfer of AMR genes. We compared their aims and methods using a list of predetermined criteria and used our results to assess the current state of this research field. Of the 43 studies we identified, most focused on the transfer of single genes by conjugation inEscherichia coliin culture and its impact on the bacterial evolutionary dynamics. Our findings highlight the existence of an important research gap in the dynamics of transformation and transduction and the overall public health implications of horizontal transfer of AMR genes. To further develop this field and improve our ability to control AMR, it is essential that we clarify the structural complexity required to study the dynamics of horizontal gene transfer, which will require cooperation between microbiologists and modellers.

Unregulated growth and replication as well as an abnormal microenvironment, leads to elevated levels of stress which is a common trait of cancer. By inducing both energy and endoplasmic reticulum (ER) stress, 2-Deoxy-glucose (2-DG) is particularly well-suited to take advantage of the therapeutic window that heightened stress in tumors provides. Under hypoxia, blocking glycolysis with 2-DG leads to significant lowering of ATP resulting in energy stress and cell death in numerous carcinoma cell types. In contrast, under normoxia, 2-DG at a low-concentration is not toxic in most carcinomas tested, but induces growth inhibition, which is primarily due to ER stress. Here we find a synergistic toxic effect in several tumor cell lines in vitro combining 2-DG with fenofibrate (FF), a drug that has been safely used for over 40 years to lower cholesterol in patients. This combination induces much greater energy stress than either agent alone, as measured by ATP reduction, increased p-AMPK and downregulation of mtor. Inhibition of mtor results in blockage of GRP78 a critical component of the unfolded protein response which we speculate leads to greater ER stress as observed by increased p-eif2 alpha. Moreover, to avoid an insulin response and adsorption by the liver, 2-DG is delivered by slow-release pump yielding significant anti-tumor control when combined with FF. Our results provide promise for developing this combination clinically and others that combine 2-DG with agents that act synergistically to selectively increase energy and ER stress to a level that is toxic to numerous tumor cell types., NA

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer related death in children, and cure rates for relapsed/refractory ALL remain dismal, highlighting the need for novel targeted therapies. We previously uncovered that ALL are vulnerable to metabolic/energy stress and ER-stress via AMP-activated protein kinase (AMPK) activation. In order to identify genome wide metabolic stress and AMPK transcriptionally regulated genes, we used RNA-Seq and compared mRNA profiles in ALL cells treated with the adenosine analog AICAR, an activator of AMPK. RNA-Seq data indicated that high dose AICAR (15 mM/45 min) induced a robust downregulation of a cluster of genes known as the immediate early genes (IEGs), which are critical for cell survival, proliferation and adaptation. AICAR-induced downregulation on IEG expression was dose- and time-dependent, and observed in other cell types (HEK293T, Hela, MEF), indicating this mechanism is conserved in mammalian cells. Using MEF AMPKα2 and AMPKα1/α2 knockout cell lines, we found that these alterations were AMPK-independent. Characterization of AICAR’s mechanism of action identified the protein kinase D1 (PKD1) as responsible for these effects. PKD1 is a Ser/Thr protein kinase involved in many cellular processes important for cancer development and progression, including proliferation, survival, apoptosis, invasion, cell adhesion and angiogenesis. We uncovered that high dose AICAR significantly inhibited PKD1 activation (Ser-910) at the plasma membrane which prevented its nuclear translocation. When PKD1 activity was pharmacologically inhibited by CRT0066101 or downregulated by shRNA, we observed similar IEGs’ downregulation in ALL cells. Conversely, the effect of AICAR on IEGs’ expression was abrogated by PMA, a direct activator of PKD1. In addition, when PKD1 was overexpressed in HEK293T cells, AICAR-induced IEG’s downregulation was partially restored. Using a kinase assay, we found that AICAR, but not ZMP, directly inhibited PKD1 kinase activity. Further, we determined that AICAR suppressed phosphorylation and nuclear export of PKD1-targeted histone deacetylases HDAC4/5, which led to decreased histone H3 acetylation at the IEGs’ promoter region. Finally, ChIP-qPCR indicated that AICAR-induced PKD1 inhibition prevented NF-κB recruitment to IEGs’ promoters. Inhibition of PKD1 activity led to decreased cell proliferation and promoted apoptosis in ALL cells. To confirm the in vivo relevance of our data, single agent and combination experiments using our NSG ALL mouse model are underway. Taken together, we have identified a novel AMPK-independent mechanism leading to AICAR’s inhibition of PKD1-mediated ALL survival. Consequently, co-targeting PDK1 and other pro-survival stress response pathways in ALL cells offers novel strategies to overcome therapeutic resistance. Citation Format: Anna Shvab, Guangyan Sun, Bin Li, Felipe Beckedorff, Guy J. Leclerc, Ramin Shiekhattar, Julio C. Barredo. AICAR inhibits protein kinase D1 activity leading to epigenetic downregulation of immediate early genes via the NF-kB pathway in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1289.

Le chylothorax apres oesogastrectomie polaire superieure est une complication rare (3,8 %) mais potentiellement mortelle. Nous rapportons le cas d’un chylothorax traite par lymphangiographie apres echec des traitements medico-chirurgicaux. Il s’agit d’une patiente de 58 ans presentant une recidive d’un carcinome epidermoide du bas œsophage ayant beneficie d’une oesogastrectomie polaire superieure par double voie avec ligature elective du canal thoracique. On constatait en post-operatoire immediat l’apparition d’un chylothorax estime a 2 litres/24H qui persistait apres mise a jeun et nutrition parenterale. Une reprise chirurgicale au 4e jour ne permettait pas de retrouver le canal thoracique. Un traitement medical par somatostatine etait ensuite entrepris mais sans succes. Au 14e jour, une lymphangiographie au Lipiodol etait realisee, remontant progressivement jusqu’a L2 sans opacifier la citerne de Pecquet (ne permettant pas l’embolisation du canal thoracique). Cependant, on observait un tarissement rapide de la fistule chyleuse autorisant le retrait des drains au 17e jour. Il n’existe pas de consensus dans le traitement du chylothorax compliquant une oesogastrectomie polaire superieure. L’embolisation percutanee du canal thoracique est une option therapeutique decrite. Cependant une lymphangiographie diagnostique meme incomplete peut s’averer efficace.

SIRT1 and AMP-activated protein kinase (AMPK) share common activators, actions and target molecules. Previous studies have suggested that a putative SIRT1-AMPK regulatory network could act as the prime initial sensor for calorie restriction-induced adaptations in skeletal muscle-the major site of insulin-stimulated glucose disposal. Our study aimed to investigate whether a feedback loop exists between AMPK and SIRT1 in skeletal muscle and how this may be involved glucose tolerance.To investigate this, we used skeletal muscle-specific AMPKα1/2 knockout mice (AMPKα1/2(-/-)) fed ad libitum (AL) or a 30% calorie restricted (CR) diet and L6 rat myoblasts incubated with SIRT1 inhibitor (EX527).CR-AMPKα1/2(-/-) displayed impaired glucose tolerance (*p0.05), in association with down-regulated SIRT1 and PGC-1α expression (300% vs. CR-WT, (±±)p0.01). Moreover, AMPK activity was decreased following SIRT1 inhibition in L6 cells (~0.5-fold vs. control, *p0.05).This study demonstrates that skeletal muscle-specific AMPK deficiency impairs the beneficial effects of CR on glucose tolerance and that these effects may be dependent on reduced SIRT1 levels.

Background AMP-activated protein kinase (AMPK) is an evolutionarily conserved sensor of cellular energy status that contributes to restoration of energy homeostasis by slowing down ATP-consuming pathways and activating ATP-producing pathways. Unexpectedly, in different systems, AMPK is also required for proper cell division. In the current study, we evaluated the potential effect of the AMPK catalytic subunit, AMPKα1, on hepatocyte proliferation. Methods Hepatocyte proliferation was determined in AMPKα1 knockout and wild-type mice in vivo after two thirds partial hepatectomy, and in vitro in primary hepatocyte cultures. The activities of metabolic and cell cycle-related signaling pathways were measured. Results After partial hepatectomy, hepatocytes proliferated rapidly, correlating with increased AMPK phosphorylation. Deletion of AMPKα1 delayed liver regeneration by impacting on G1/S transition phase. The proliferative defect of AMPKα1-deficient hepatocytes was cell autonomous, and independent of energy balance. The priming phase, lipid droplet accumulation, protein anabolic responses and growth factor activation after partial hepatectomy occurred normally in the absence of AMPKα1 activity. By contrast, mRNA and protein expression of cyclin A2, a key driver of S phase progression, were compromised in the absence of AMPK activity. Importantly, AMPKα1 controlled cyclin A2 transcription mainly through the ATF/CREB element. Conclusions Our study highlights a novel role for AMPKα1 as a positive regulator of hepatocyte division occurring independently of energy balance.

Das erhohte Risiko von Anasthesisten fur einen Substanzmissbrauch u. a. von Opioiden und Propofol ist bekannt. Es gibt jedoch nur wenige Selbstbeschreibungen suchtkranker Arzte mit i.v.-Analgetika- und Anasthetikamissbrauch, speziell auch von Propofol, die aber dazu betragen konnten, die Dynamik in der Entwicklung der Abhangigkeit besser zu verstehen. Der hier vorgestellte Fallbericht enthalt ein Interview mit einer ehemals suchtkranken Anasthesistin, die nach langjahrigem Tilidin- und Naloxonmissbrauch zu i.v.-Fentanyl- und spater auch Propofolinjektionen wechselte. Hierunter kam es zu mehreren lebensbedrohlichen Zwischenfallen. Erst nach stationarer Behandlung und nach einigen Ruckschlagen gelang die berufliche Rehabilitation auserhalb der Anasthesie. Exemplarisch zeigt dieser Fall in Ubereinstimmung mit dem aktuellen Schrifttum, dass einerseits Warnsymptome bei abhangigen Arzten in Kliniken leicht missachtet werden, aber auch dass eine professionelle Therapie unter laufender Uberwachung die Moglichkeit zur Rehabilitation eroffnet. Der Fall unterstreicht zudem die Notwendigkeit, die Abgabe von Propofol zu kontrollieren, um dieses lebensbedrohliche „Berufsrisiko“ von Anasthesisten zu reduzieren.

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children, and cure rates for relapsed/refractory ALL in children and adults remain dismal, highlighting the need for novel targeted therapies capable of overcoming resistance in relapsed/refractory disease. We previously uncovered that ALL cells are vulnerable to metabolic/energy stress and endoplasmic reticulum (ER)-stress via AMP-activated protein kinase (AMPK) activation leading to unfolded protein response (UPR)-mediated apoptosis. In order to identify genome-wide metabolic-stress and AMPK-transcriptionally regulated genes in ALL cells undergoing metabolic/energy stress, we used RNA-Seq and compared mRNA transcript profiles in ALL cells treated with acadesine (adenosine analog 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside or AICAR), known to activate AMPK. RNA-Seq data indicated that acadesine treatment (15 mM/45 min) induced a robust and rapid alteration in gene expression in ALL cells. The most significant acadesine-induced gene signature represented a cluster of genes known as immediate early genes (IEGs), which are fundamental in critical biological pathways for cell survival/proliferation/adaptation. We interpreted these changes as a compensatory pro-survival mechanism in ALL cells undergoing energy/metabolic stress. Among the acadesine-induced downregulated IEGs, we selected DUSP1, JUNB and NFKBIA for further characterization. Downregulation of these IEGs was confirmed using RT-qPCR. We found that the effect of acadesine-induced downregulation on IEGs expression was dose- and time-dependent, and these effects were observed in other cell types (HeLa, HEK293T, mouse embryonic fibroblasts(MEF)), indicating this mechanism of acadesine-induced downregulation of IEGs expression is conserved in mammalian cells. Interestingly, when we used lower doses of acadesine (the half-maximal inhibition concentration for IEGs (IC50)), the IEGs mRNA levels returned to baseline after 3 hours of exposure, suggesting the effect of acadesine on these IEGs was transient at IC50 dose. Using NALM6 AMPKα1 knockdown and MEF AMPKα1/α2 knockout cell lines, we uncovered that high-dose/short-time exposure to acadesine led to changes in IEGs expression that were independent of AMPK. Consistent with these findings, ALL cells co-treated with acadesine plus adenosine kinase inhibitors (ABT702 or 5-Iodotubercidin), which prevent its conversion to ZMP, exhibited the same gene expression signature. Characterization of acadesine's mechanism of action identified protein kinase D1 (PKD1) as responsible for acadesine-induced downregulation of IEGs. PKD1 is a serine/threonine protein kinase involved in many cellular processes important to cancer development and progression, including proliferation, survival, apoptosis, motility, cell adhesion and angiogenesis. Acadesine induced strong inhibition of PKD1 activity which resulted in PKD1 accumulation in the cytoplasm and prevented its nuclear translocation. When ALL cells were treated with protein kinase D (PKD) inhibitors (CRT0066101, GF109203X), we observed a similar rapid, robust and transient downregulation of IEGs, suggesting acadesine interacts with the PKD1 pathway. Conversely, the effect of acadesine on IEGs expression was abrogated by phorbol 12-myristate 13-acetate (PMA), a direct activator of PKD. Further, we determined that acadesine suppresses PKD1-regulated class II Histone deacetylase (HDAC4/5) phosphorylation and nuclear export, which led to decreased histone H3 acetylation levels at the IEG's promoter region. Finally, ChIP-qPCR experiments uncovered that the acadesine/PKD1 axis regulates the recruitment of nuclear factor-κB (NF-κB) to the promoter region of selected IEGs. Consequently, we have identified a novel, AMPK-independent transcription regulation mechanism of acadesine thorugh PKD1 in ALL cells, and co-targeting PDK1 and other pro-survival stress response pathways in ALL cells vulnerable to energy/metabolic stress offers potential novel strategies to overcome therapeutic resistance. Disclosures No relevant conflicts of interest to declare.