[Circulatory failure after bupropion overdose]. / Allt fler allvarliga förgiftningar med preparatet bupropion.

A case of massive overdose of sustained release bupropion tablets is described. The patient presented with GCS 3, tachycardic and in vasoplegic shock. ECHO and EKG were initially normal. The hemodynamic situation was stabilised with vasopressors, but 18 h after presentation the patient deteriorated with wide complex arrhythmias rapidly progressing to cardiac arrest. The patient was put on VA-ECMO after 35 minutes of CPR. Circulation could be stabilized and ECMO was discontinued after 36 h. The patient was extubated on day 6 and made a complete recovery on discharge two weeks after presentation. At 34h, with ongoing ECMO, 236 tablets (with visible print identifying them as bupropion) were evacuated from the patient's stomach by gastroscopy. The tablets were analysed by NMR (nuclear magnetic resonance) but no longer contained any active substance. Blood levels of bupropion and hydroxybupropion at 36h were 790 and 1300 µg/l. The case illustrates a worrying surge in serious bupropion poisonings as noted by the Swedish Poisons Information Centre during the last 5 years.

Knockout validation of LAMP2A antibodies for immunostaining in human cancer cells.

LAMP2A is the rate-limiting factor of chaperone-mediated autophagy (CMA), a unique selective protein degradative pathway. To date LAMP2A antibodies are not knockout (KO)-validated in human cells. We have recently generated human isoform-specific LAMP2A KO cells, and here we assessed the specificity of select commercial LAMP2A antibodies on wild-type and LAMP2A KO human cancer cells. While all tested antibodies were suitable for immunoblotting, the anti-LAMP2A antibody (ab18528) is likely to exhibit an off-target reactivity in immunostaining approaches using human cancer cells, and alternative antibodies, which seem more appropriate, are available.

The nutritional supplement taurine activates p53-dependent and independent tumor suppressor mechanisms in various cellular models of ovarian cancer.

Loss of treatment-induced ovarian carcinoma (OC) growth suppression poses a major clinical challenge because it leads to disease recurrence. Therefore, there is a compelling need for well- -tolerated approaches that can support tumor growth-suppression after therapy is stopped. We have profiled ascites as OC tumor microenvironments to search for potential non-toxic soluble components that would activate tumor suppressor pathways in OC cells. Our investigations revealed that low levels of taurine, a non-proteogenic sulfonic amino acid, were present within OC ascites. Taurine supplementation, beyond levels found in ascites, induced growth suppression without causing cytotoxicity in various OC cells, including chemotherapy-resistant cell clones and patient-derived organoids representing primary or chemotherapy recovered disease. Inhibition of proliferation by taurine was linked to increased mutant or wild-type p53 proteins binding to DNA, induction of p21, and independently of p53, TIGAR expression. Taurine-induced activation of p21 and TIGAR was associated with suppression of cell-cycle progression, glycolysis, and mitochondrial respiration. Expression of p21 or TIGAR in OC cells mimicked taurine-induced growth suppression. Our studies support the potential therapeutic value of taurine supplementation in OC.

A Method to Identify Potential Prognostic Markers Across Distinct Tumor Types.

The identification of novel biomarkers in cancer patients often requires both survival and gene expression analyses. The Kaplan-Meier survival analysis is one of the most common methods to assess the fraction of subjects living for a certain amount of time.Here, we describe a method for researchers to identify potential prognostic markers across distinct tumor types. We utilize The Cancer Genome Atlas (TCGA) as this is one of the most extensive and successful cancer genomics programs to date that includes expression data and clinical follow-up information for up to 33 distinct tumor types. Nevertheless, the method described here can also be applied to any open-source dataset where the RNA expression and clinical outcome are provided.We provide detailed practical instructions and advices for investigators to be able to successfully identify prognostic markers in cancer patients.

A Method for Coexpression Analysis.

Gene coexpression network analysis is a commonly used approach in bioinformatics and biomedical research to construct coexpression networks and detect coexpressed genes. This type of analysis has proven valuable for gene function prediction and for disease biomarker discovery.Here, we introduce and guide researchers through a method of differential coexpression analysis focusing on key autophagy and metabolic genes. We utilized the open-source Cancer Cell Line Encyclopedia (CCLE ) project as this is one of the most comprehensive genomic and transcriptomic resources including more than 1000 cell lines of distinct origins. However, the coexpression analysis method described here can also be applied to any open-source dataset where the RNA expression are provided.We here provide detailed comprehensive practical instructions for investigators to successfully identify novel coexpression signatures.

Extraction of Metabolites from Cancer Cells.

Cancer cells possess an elevated demand for nutrients and metabolites due to their uncontrolled proliferation and need to survive in unfavorable conditions. Autophagy is a conservative degradation pathway that counters lack of nutrients and provides organelle and protein quality control, beyond maintenance of cellular metabolism.Mass spectrometry-based metabolomics is a powerful tool to study the metabolome of a cell. Such analysis requires proper sample preparation including the extraction of metabolites. Here, we provide a protocol for the extraction of metabolites from adherent cancer cells suitable for global metabolome profiling by mass spectrometry.

Regional left ventricular systolic dysfunction associated with critical illness: incidence and effect on outcome.

AIMS: Left ventricular (LV) dysfunction can be triggered by non-cardiac disease, such as sepsis, hypoxia, major haemorrhage, or severe stress (Takotsubo syndrome), but its clinical importance is not established. In this study, we evaluate the incidence and impact on mortality of LV dysfunction associated with critical illness. METHODS AND RESULTS: In this single-centre, observational study, consecutive patients underwent an echocardiographic examination within 24 h of intensive care unit (ICU) admission. LV systolic dysfunction was defined as an ejection fraction (EF) < 50% and/or regional wall motion abnormalities (RWMA). A cardiologist assessed patients with LV dysfunction for the presence of an acute or chronic cardiac disease, and coronary angiography was performed in high-risk patients. Of the 411 patients included, 100 patients (24%) had LV dysfunction and in 52 (13%) of these patients, LV dysfunction was not attributed to a cardiac disease. Patients with LV dysfunction and non-cardiac disease had higher mortality risk score (Simplified Acute Physiologic Score 3 score), heart rate, noradrenaline doses, and lactate levels as well as decreased EF, stroke volume, and cardiac output compared with patients with normal LV function. Diagnoses most commonly associated with LV dysfunction and non-cardiac disease were sepsis, respiratory insufficiency, major haemorrhage, and neurological disorders. RWMA (n = 40) with or without low EF was more common than global hypokinesia (n = 12) and was reversible in the majority of cases. Twelve patients had a circumferential pattern of RWMA in concordance with Takotsubo syndrome. Crude 30 day mortality was higher in patients with LV dysfunction and non-cardiac disease compared with patients with normal LV function (33% vs. 18%, P = 0.023), but not after risk adjustment (primary outcome) {odds ratio [OR] 1.56 [confidence interval (CI) 0.75-3.39], P = 0.225}. At 90 days, crude mortality was 44% and 22% (P = 0.002), respectively, in these groups. This difference was also significant after risk adjustment [OR 2.40 (CI 1.18-4.88), P = 0.016]. CONCLUSIONS: Left ventricular systolic dysfunction is commonly triggered by critical illness, is frequently seen as regional hypokinesia, and is linked to an increased risk of death. The prognostic importance of LV dysfunction in critical illness might be underestimated.

Quantitative proteomic analysis of temporal lysosomal proteome and the impact of the KFERQ-like motif and LAMP2A in lysosomal targeting.

Autophagic pathways are regulated mechanisms that play important roles in lysosome-mediated cellular degradation. Yet, the contribution of different autophagic pathways in lysosomal targeting, and characterization of the extent and specificity in their degradome remains largely uncharacterized. By undertaking a multiplex quantitative mass spectrometry approach, we have previously analyzed the lysosomal proteome during chaperone-mediated autophagy (CMA)-stimulated conditions in cancer cells. Here, we have extended our multiplex quantitative mass spectrometry and bioinformatics analysis on the proteome from isolated lysosomes to gain a comprehensive view of the temporal enriched lysosomal content upon non-macroautophagy-activated conditions. In parallel, we describe the functional dependency of LAMP2A on, and to what degree the presence of KFERQ-like motifs in proteins influences, their lysosomal targeting. These findings establish a framework for a better understanding of the degradome mediated by autophagic pathways beyond macroautophagy, and present characterization of the impact of LAMP2A in lysosomal targeting in cancer cells.Abbreviations: CMA: chaperone-mediated autophagy; ER: endoplasmic reticulum; EIF4A1: eukaryotic translation initiation factor 4A1; eMI: endosomal microautophagy; FC: fold change; GO: gene ontology; ISR: integrated stress response; LAMP2A: lysosomal associated membrane protein 2A; MA: macroautophagy; MI: microautophagy; MS: mass spectrometry; PCA: principal component analysis; TAX1BP1: Tax1 binding protein 1.

The deubiquitinase JOSD2 is a positive regulator of glucose metabolism.

Cancer cells undergo complex metabolic alterations. The mechanisms underlying the tuning of cancer metabolism are under active investigation. Here, we identify the uncharacterized deubiquitinase JOSD2 as a positive regulator of cancer cell proliferation by displaying comprehensive effects on glucose catabolism. We found that JOSD2 directly controls a metabolic enzyme complex that includes Aldolase A, Phosphofructokinase-1 and Phosphoglycerate dehydrogenase, in vitro and in vivo. Further, JOSD2 expression, but not a catalytically inactive mutant, deubiquitinates and stabilizes the enzyme complex, thereby enhancing their activities and the glycolytic rate. This represents a selective JOSD2 feature that is not shared among other Machado-Joseph disease DUBs or observed in nontransformed cells. JOSD2 deficiency displays cytostatic effects and reduces glycolysis in a broad spectrum of tumor cells of distinct origin and its expression correlates with poor prognosis in non-small cell lung cancer. Overall, our study provides evidence for a previously unknown biological mechanism in which JOSD2 integrates glucose and serine metabolism with potential therapeutic implications.

Systematic analysis reveals a functional role for STAMBPL1 in the epithelial-mesenchymal transition process across multiple carcinomas.

BACKGROUND: Deubiquitinating enzymes (DUBs) are linked to cancer progression and dissemination, yet less is known about their regulation and impact on epithelial-mesenchymal transition (EMT). METHODS: An integrative translational approach combining systematic computational analyses of The Cancer Genome Atlas cancer cohorts with CRISPR genetics, biochemistry and immunohistochemistry methodologies to identify and assess the role of human DUBs in EMT. RESULTS: We identify a previously undiscovered biological function of STAM-binding protein like 1 (STAMBPL1) deubiquitinase in the EMT process in lung and breast carcinomas. We show that STAMBPL1 expression can be regulated by mutant p53 and that its catalytic activity is required to affect the transcription factor SNAI1. Accordingly, genetic depletion and CRISPR-mediated gene knockout of STAMBPL1 leads to marked recovery of epithelial markers, SNAI1 destabilisation and impaired migratory capacity of cancer cells. Reversely, STAMBPL1 expression reprogrammes cells towards a mesenchymal phenotype. A significant STAMBPL1-SNAI1 co-signature was observed across multiple tumour types. Importantly, STAMBPL1 is highly expressed in metastatic tissues compared to matched primary tumour of the same lung cancer patient and its expression predicts poor prognosis. CONCLUSIONS: Our study provides a novel concept of oncogenic regulation of a DUB and presents a new role and predictive value of STAMBPL1 in the EMT process across multiple carcinomas.

Left ventricular dysfunction in potential heart donors and its influence on recipient outcomes.

OBJECTIVES: New onset of left ventricular (LV) dysfunction in organ donors is frequent and considered as a contraindication for utilization of the heart. However, such dysfunction might be caused by sympathetic stress and could be transient (Takotsubo syndrome). In this study, we assessed the incidence, pattern, and predictors of LV dysfunction in potential heart donors and evaluated its influence on recipient outcomes. METHODS: Donor records of consecutive organ donors in western Sweden between 2006 and 2016 were reviewed. Recipients of transplanted donor hearts were identified in the Scandiatransplant database. RESULTS: Of 641 potential heart donors who underwent echocardiographic assessment, LV dysfunction (ejection fraction <50% and/or regional hypokinesia) was found in 155 donors (24%). Regional hypokinesia was seen in 113 donors of whom 46 had a Takotsubo-like circumferential hypokinetic pattern. Independent donor variables associated with LV dysfunction were a younger age, cardiac arrest as a contributing factor to death, need for inotropic support, and a shorter time from admission to declaration of brain death. A total of 338 (54%) donor hearts were transplanted, of which 45 (14%) had LV dysfunction. LV dysfunction was a major determinant of not transplanting the heart (P < .001). After transplantation, LV function normalized in the recipients. Neither short-term outcomes nor the composite end point of death or retransplantation over time differed between recipients of donor hearts with versus without LV dysfunction (P = .587). CONCLUSIONS: LV dysfunction is common among potential heart donors. These hearts were safely transplanted in this study. The use of these hearts might significantly increase transplantation rates.

Mutant p53 as a Regulator and Target of Autophagy.

One of the most notoriously altered genes in human cancer is the tumor-suppressor TP53, which is mutated with high frequency in more cancers than any other tumor suppressor gene. Beyond the loss of wild-type p53 functions, mutations in the TP53 gene often lead to the expression of full-length proteins with new malignant properties. Among the defined oncogenic functions of mutant p53 is its effect on cell metabolism and autophagy. Due to the importance of autophagy as a stress adaptive response, it is frequently dysfunctional in human cancers. However, the role of p53 is enigmatic in autophagy regulation. While the complex action of the wild-type p53 on autophagy has extensively been described in literature, in this review, we focus on the conceivable role of distinct mutant p53 proteins in regulating different autophagic pathways and further discuss the available evidence suggesting a possible autophagy stimulatory role of mutant p53. Moreover, we describe the involvement of different autophagic pathways in targeting and degrading mutant p53 proteins, exploring the potential strategies of targeting mutant p53 in cancer by autophagy.

Targetome analysis of chaperone-mediated autophagy in cancer cells.

Chaperone-mediated autophagy (CMA) is a lysosomal degradation pathway of select soluble proteins. Nearly one-third of the soluble proteins are predicted to be recognized by this pathway, yet only a minor fraction of this proteome has been identified as CMA substrates in cancer cells. Here, we undertook a quantitative multiplex mass spectrometry approach to study the proteome of isolated lysosomes in cancer cells during CMA-activated conditions. By integrating bioinformatics analyses, we identified and categorized proteins of multiple cellular pathways that were specifically targeted by CMA. Beyond verifying metabolic pathways, we show that multiple components involved in select biological processes, including cellular translation, was specifically targeted for degradation by CMA. In particular, several proteins of the translation initiation complex were identified as bona fide CMA substrates in multiple cancer cell lines of distinct origin and we show that CMA suppresses cellular translation. We further show that the identified CMA substrates display high expression in multiple primary cancers compared to their normal counterparts. Combined, these findings uncover cellular processes affected by CMA and reveal a new role for CMA in the control of translation in cancer cells. Abbreviations: 6-AN: 6-aminonicotinamide; ACTB: actin beta; AR7: atypical retinoid 7; CHX: cycloheximide; CMA: chaperone-mediated autophagy; CQ: chloroquine; CTS: cathepsins; DDX3X: DEAD-box helicase 3 X-linked; EEF2: eukaryotic translation elongation factor 2; EIF4A1: eukaryotic translation initiation factor 4A1; EIF4H: eukaryotic translation initiation factor 4H; GEO: Gene Expression Omnibus; GO: Gene Ontology; GSEA: gene set enrichment analysis; HK2: hexokinase 2; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; LAMP: lysosomal-associated membrane protein; LDHA: lactate dehydrogenase A; NES: normalized enrichment score; NFKBIA: NFKB inhibitor alpha; PCA: principle component analysis; PQ: paraquat; S.D.: standard deviation; SUnSET: surface sensing of translation; TMT: tandem mass tags; TOMM40/TOM40: translocase of outer mitochondrial membrane 40.

miR-100-5p confers resistance to ALK tyrosine kinase inhibitors Crizotinib and Lorlatinib in EML4-ALK positive NSCLC.

Lung cancer causes the highest number of cancer-related deaths worldwide. Resistance to therapy is a major clinical issue contributing to the poor prognosis of lung cancer. In recent years, targeted therapy has become a concept where subgroups of non-small cell lung cancer (NSCLC) with genetically altered receptor tyrosine kinases are targeted by tyrosine kinase inhibitors (TKIs). One such subgroup harbors a gene fusion of echinoderm microtubule-associated protein-like 4 (EML4) with anaplastic lymphoma kinase (ALK). Although most NSCLC patients with EML4-ALK fusions initially respond to ALK TKI-therapy they eventually develop resistance. While ALK kinase domain mutations contribute to ALK TKI-refractoriness, they are only present in a fraction of all ALK TKI-resistant tumors. In this study we sought to explore a possible involvement of microRNAs (miRNAs) in conferring resistance to ALK TKIs in ALK TKI-refractory NSCLC cell lines. We subjected our ALK TKI-refractory cancer cells along with parental cancer cells to systematic miRNA expression arrays. Furthermore, ALK TKI-refractory cancer cells were exposed to a synthetic miRNA inhibitory Locked Nucleic Acid (LNA)-library in the presence of ALK TKIs Crizotinib or Lorlatinib. The outcome of the combined approaches uncovered miR-100-5p to confer resistance to Crizotinib and Lorlatinib in EML4-ALK NSCLC cells and to be a potential therapeutic target in drug resistance.

Impact of Acute Cardiac Complications After Subarachnoid Hemorrhage on Long-Term Mortality and Cardiovascular Events.

BACKGROUND: Cardiac complications frequently occur after subarachnoid hemorrhage (SAH) and are associated with an increased risk of neurological complications and poor outcomes. The aim of this study was to evaluate the impact of acute cardiac complications after SAH on long-term mortality and cardiovascular events. METHODS: All patients admitted to our Neuro intensive care unit with verified SAH from January 2010 to April 2015, and electrocardiogram, echocardiogram, and troponin T or NTproBNP data obtained within 72 h of admission were included in the study. Mortality data were obtained from the Swedish population register. Data regarding cause of death and hospitalization for cardiovascular events were obtained from the Swedish Board of Health and Welfare. RESULTS: A total of 455 patients were included in the study analysis. There were 102 deaths during the study period. Cardiac troponin release (HR 1.08, CI 1.02-1.15 per 100 ng/l, p = 0.019), NTproBNP (HR 1.05, CI 1.01-1.09 per 1000 ng/l, p = 0.018), and ST-T abnormalities (HR 1.53, CI 1.02-2.29, p = 0.040) were independently associated with an increased risk of death. However, these associations were significant only during the first 3 months after the hemorrhage. Cardiac events were observed in 25 patients, and cerebrovascular events were observed in 62 patients during the study period. ST-T abnormalities were independently associated with an increased risk of cardiac events (HR 5.52, CI 2.07-14.7, p < 0.001), and stress cardiomyopathy was independently associated with an increased risk of cerebrovascular events (HR 3.65, CI 1.55-8.58, p = 0.003). CONCLUSION: Cardiac complications after SAH are associated with an increased risk of short-term death. Patients with electrocardiogram abnormalities and stress cardiomyopathy need appropriate follow-up for the identification of cardiac disease or risk factors for cardiovascular disease.

Characterization of a fully integrated heterogeneous silicon/III-V colliding pulse mode-locked laser with on-chip feedback.

A fully integrated heterogeneous silicon/III-V colliding pulse mode-locked laser with tunable on-chip optical feedback operating in the O-band is extensively investigated. The 19-GHz colliding pulsed laser operates in a wide mode-locking regime with good mode locking quality. By precisely controlling the strength and phase of the on-chip optical feedback signal, the laser exhibits clear periodic pulse shortening effects. The RF 3 dB linewidth was reduced by a factor of 4.7 down to 6 kHz, as compared to the free running state.

USP10 regulates the stability of the EMT-transcription factor Slug/SNAI2.

Epithelial-to-mesenchymal transition (EMT) is a fundamental mechanism governing the switch of cells from an epithelial to a motile mesenchymal-like state. This transdifferentiation is regulated by key transcription factors, including Slug. The stability and function of Slug can be regulated by multiple mechanisms, including ubiquitin-mediated post-translational modifications. Here, by using a genome wide siRNA screen for human deubiquitinating enzymes (DUBs), we identified USP10 as a deubiquitinase for Slug in cancer cells. USP10 interacts with Slug and mediates its degradation by the proteasome. Importantly, USP10 is concomitantly highly expressed with Slug in cancer biopsies. Genetic knockdown of USP10 leads to suppressed Slug levels with a decreased expression of the mesenchymal marker Vimentin. Further, it reduces the migratory capacity of cancer cells. Reversely, overexpression of USP10 elevates the level of both Slug and Vimentin. Our study identifies USP10 as a regulator of the EMT-transcription factor Slug and cell migration.

An optical-frequency synthesizer using integrated photonics.

Optical-frequency synthesizers, which generate frequency-stable light from a single microwave-frequency reference, are revolutionizing ultrafast science and metrology, but their size, power requirement and cost need to be reduced if they are to be more widely used. Integrated-photonics microchips can be used in high-coherence applications, such as data transmission 1 , highly optimized physical sensors 2 and harnessing quantum states 3 , to lower cost and increase efficiency and portability. Here we describe a method for synthesizing the absolute frequency of a lightwave signal, using integrated photonics to create a phase-coherent microwave-to-optical link. We use a heterogeneously integrated III-V/silicon tunable laser, which is guided by nonlinear frequency combs fabricated on separate silicon chips and pumped by off-chip lasers. The laser frequency output of our optical-frequency synthesizer can be programmed by a microwave clock across 4 terahertz near 1,550 nanometres (the telecommunications C-band) with 1 hertz resolution. Our measurements verify that the output of the synthesizer is exceptionally stable across this region (synthesis error of 7.7 × 10-15 or below). Any application of an optical-frequency source could benefit from the high-precision optical synthesis presented here. Leveraging high-volume semiconductor processing built around advanced materials could allow such low-cost, low-power and compact integrated-photonics devices to be widely used.

Resistant to Targeted Therapy - Aim for Metabolic Liabilities.

The advent of targeted therapies generated much optimism when discovered. Targeted therapies, are however associated with rapid acquisition of resistance. In a recent study by Dong et al. (Theranostics 2018; 8(7):1808-1823. doi:10.7150/thno.23177) it was shown that lung tumors resistant to the EGFR-inhibitor (Erlotinib), reprogram their metabolism and acquire a pro-survival dependency on Phosphoglycerate Dehydrogenase (PHGDH) that can be targeted to eliminate resistant tumors.