Desorption Electrospray Ionization Mass Spectrometry Imaging of Powder-Treated Fingermarks on Forensic Gelatin Lifters and its Application for Separating Overlapping Fingermarks.

Fingermarks are frequently collected at crime scenes by using gelatin lifters for preservation and transport of the marks to a forensic laboratory for inspection. The gelatin lifters preserve both the imprint of the fingermark pattern necessary for identification purposes and the chemical residue of the mark potentially useful for profiling the person who left the fingermark. The fingermark patterns are traditionally recorded using photography/optical imaging, but methods for chemical analysis of fingermark residues on gelatin lifters are scarce. Here we report the first method for the chemical analysis of fingermarks on gelatin lifters using desorption electrospray ionization mass spectrometry (DESI-MS) imaging. The imaging can be done directly on the gelatin support without any sample preparation, supporting immediate operational use of the method for fingermarks collected at crime scenes. Operational use of the method is further supported by successful chemical imaging of fingermarks enhanced by traditional dusting with forensic powders and lifted off different surfaces (glass, stainless steel, painted aluminum, polystyrene, cardboard, and plastic) as well as fingermarks lifted multiple times. We also demonstrate that the present method can be used to visually separate natural overlapping powder-treated fingermarks, and the chemical composition of the fingermarks can be analyzed on the gelatin support by DESI-MS/MS. The presented method has potential for integration into the traditional workflow for fingermark analysis, and will allow more fingermarks collected at crime scenes to be evaluated both visually and chemically.

Combined in vivo metabolic effects of quetiapine and methadone in brain and blood of rats.

Changes in pharmacokinetics and endogenous metabolites may underlie additive biological effects of concomitant use of antipsychotics and opioids. In this study, we employed untargeted metabolomics analysis and targeted analysis to examine the changes in drug metabolites and endogenous metabolites in the prefrontal cortex (PFC), midbrain, and blood of rats following acute co-administration of quetiapine and methadone. Rats were divided into four groups and received cumulative increasing doses of quetiapine (QTP), methadone (MTD), quetiapine + methadone (QTP + MTD), or vehicle (control). All samples were analyzed using liquid chromatography-mass spectrometry (LC-MS). Our findings revealed increased levels of the quetiapine metabolites: Norquetiapine, O-dealkylquetiapine, 7-hydroxyquetiapine, and quetiapine sulfoxide, in the blood and brain when methadone was present. Our study also demonstrated a decrease in methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in the rat brain when quetiapine was present. Despite these findings, there were only small differences in the levels of 225-296 measured endogenous metabolites due to co-administration compared to single administrations. For example, N-methylglutamic acid, glutaric acid, p-hydroxyphenyllactic acid, and corticosterone levels were significantly decreased in the brain of rats treated with both compounds. Accumulation of serotonin in the midbrain was additionally observed in the MTD group, but not in the QTP + MTD group. In conclusion, this study in rats suggests a few but important additive metabolic effects when quetiapine and methadone are co-administered.

Non-imaging metasurface design for collimated beam shaping.

Non-imaging optical lenses can shape the light intensity from incoherent sources to a desired target intensity profile, which is important for applications in lighting, solar light concentration, and optical beam shaping. Their surface curvatures are designed to ensure optimal transfer of energy from the light source to the target. The performance of such lenses is directly linked to their asymmetric freeform surface curvature, which is challenging to manufacture. Metasurfaces can mimic any surface curvature without additional fabrication difficulty by imparting a spatially-dependent phase delay using optical antennas. As a result, metasurfaces are uniquely suited to realize non-imaging optics, but non-imaging design principles have not yet been established for metasurfaces. Here, we take an important step in connecting non-imaging optics and metasurface optics, by presenting a phase-design method for beam shaping based on the concept of optimal transport. We establish a theoretical framework that enables a collimated beam to be redistributed by a metasurface to a desired output intensity profile. The optimal transport formulation leads to metasurface phase profiles that transmit all energy from the incident beam to the output beam, resulting in an efficient beam shaping process. Through a variety of examples, we show that our approach accommodates a diverse range of different input and output intensity profiles. Last but not least, a full field simulation of a metasurface has been done to verify our phase-design framework.

Validation of an indirect ELISA assay for assessment of autoantibodies against full-length TRIM21 and its individual domains.

Anti-SSA-autoantibodies are common in patients with rheumatologic disease, especially Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis. They consist of both autoantibodies towards Ro60 and Ro52, the latter also known as TRIM21. TRIM21 is an intracellular protein consisting of four domains; PRY/SPRY, Coiled-Coil, B-box and RING. The aim of this study was to establish an indirect ELISA detecting autoantibodies towards both the full-length TRIM21 protein and its four domains. We expressed the five constructs, created, and validated indirect ELISA protocols for each target using plasma from anti-SSA positive patients and healthy controls. Our findings were validated to the clinically used standards. We measured significantly higher levels of autoantibodies towards our full-length TRIM21, and the PRY/SPRY, Coiled-Coil and RING domains in patients compared to healthy controls. No significant difference in the level of autoantibodies were detected against the B-box domain. Our setups had a signal to noise ratio in the range of 30 to 184, and an OD between 2 and 3. Readings did not decline using NaCl of 500 mM as wash, affirming the high binding affinity of the autoantibodies measured. Our protocols allow us to further study the different autoantibodies of anti-SSA positive patients. This creates the possibility to stratify our patients into subgroups regarding autoantibody profile and specific pheno- or endotype.

Assessment of XCMS Optimization Methods with Machine-Learning Performance.

The metabolomics field is under rapid development. In particular, biomarker identification and pathway analysis are growing, as untargeted metabolomics is usable for discovery research. Frequently, new processing and statistical strategies are proposed to accommodate the increasing demand for robust and standardized data. One such algorithm is XCMS, which processes raw data into integrated peaks. Multiple studies have tried to assess the effect of optimizing XCMS parameters, but it is challenging to quantify the quality of the XCMS output. In this study, we investigate the effect of two automated optimization tools (Autotuner and isotopologue parameter optimization (IPO)) using the prediction power of machine learning as a proxy for the quality of the data set. We show that optimized parameters outperform default XCMS settings and that manually chosen parameters by liquid chromatography-mass spectrometry (LC-MS) experts remain the best. Finally, the machine-learning approach of quality assessment is proposed for future evaluations of newly developed optimization methods because its performance directly measures the retained signal upon preprocessing.

Comparison of 16 Serological SARS-CoV-2 Immunoassays in 16 Clinical Laboratories.

Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to support clinical diagnosis and epidemiological investigations. Recently, assays for large-scale detection of total antibodies (Ab), immunoglobulin G (IgG), and IgM against SARS-CoV-2 antigens have been developed, but there are limited data on the diagnostic accuracy of these assays. This study was a Danish national collaboration and evaluated 15 commercial and one in-house anti-SARS-CoV-2 assays in 16 laboratories. Sensitivity was evaluated using 150 samples from individuals with asymptomatic, mild, or moderate COVID-19, nonhospitalized or hospitalized, confirmed by nucleic acid amplification tests (NAAT); samples were collected 13 to 73 days either from symptom onset or from positive NAAT (patients without symptoms). Specificity and cross-reactivity were evaluated in samples collected prior to the SARS-CoV-2 epidemic from >586 blood donors and patients with autoimmune diseases, cytomegalovirus or Epstein-Barr virus infections, and acute viral infections. A specificity of ≥99% was achieved by all total-Ab and IgG assays except one, DiaSorin Liaison XL IgG (97.2%). Sensitivities in descending order were Wantai ELISA total Ab (96.7%), CUH-NOVO in-house ELISA total Ab (96.0%), Ortho Vitros total Ab (95.3%), YHLO iFlash IgG (94.0%), Ortho Vitros IgG (93.3%), Siemens Atellica total Ab (93.2%), Roche Elecsys total Ab (92.7%), Abbott Architect IgG (90.0%), Abbott Alinity IgG (median 88.0%), DiaSorin Liaison XL IgG (median 84.6%), Siemens Vista total Ab (81.0%), Euroimmun/ELISA IgG (78.0%), and Snibe Maglumi IgG (median 78.0%). However, confidence intervals overlapped for several assays. The IgM results were variable, with the Wantai IgM ELISA showing the highest sensitivity (82.7%) and specificity (99%). The rate of seropositivity increased with time from symptom onset and symptom severity.

P53, Somatostatin receptor 2a and Chromogranin A immunostaining as prognostic markers in high grade gastroenteropancreatic neuroendocrine neoplasms.

BACKGROUND: High grade gastroenteropancreatic (GEP) neuroendocrine neoplasms (NEN) with a Ki67 proliferation index > 20%, include well-differentiated tumours grade 3 (NET G3) and poorly differentiated (PD) neuroendocrine carcinomas (NEC). Abnormal p53-expression is a feature of PD tumours, while expression of chromogranin A (CgA) and somatostatin-receptor 2a (SSTR-2a) may be a feature of well-differentiated tumours. The aim of this study was to elucidate the expression and prognostic value of these three markers in 163 GEP-NEN patients with a Ki67-index > 20%. METHOD: Clinical data, histopathology and overall survival were analysed according to Kaplan-Meier's method and Cox regression. The expression of SSTR-2a, CgA and synaptophysin was analysed in tumour specimens by immunohistochemistry, and semi-quantitatively scored as negative (< 5%), heterogeneously positive (5-30%) or strongly positive (> 30%). P53 was defined as normal when scored as heterogeneously positive (1-30%), and abnormal when negative (0%) or strongly positive (> 30%). RESULTS: In multivariate analysis, better survival was observed among patients with heterogeneously positive p53 compared to strongly positive (p < 0.001). When dichotomised, tumours with a heterogeneously positive p53 vs. negative and strongly positive p53 also showed a significantly better survival (p = 0.002). Survival was significantly worse for negative CgA compared to heterogeneously positive CgA (p = 0.02). Strongly positive SSTR-2a expression was found in 26% of the 163 included patients. Well-differentiated morphology correlated with strong expression of SSTR-2a and CgA, and heterogeneously positive p53-staining, and was more frequent in pancreatic primaries. In pancreatic primaries, strongly positive SSTR-2a was associated with longer survival (univariate analysis, p = 0.02). A significantly lower Ki67 proliferation index was found in patients with a heterogeneously positive p53, a positive SSTR-2a and CgA expression. CONCLUSION: Our results suggest that abnormal p53-expression is an independent negative prognostic marker in GEP-NEN with a Ki67-index > 20%. Patients with heterogeneously positive p53 had the best prognosis. SSTR-2a was a positive prognostic marker in pancreatic NEN. Negative CgA was associated with a significantly worse OS compared to heterogeneously positive CgA-expression in a multivariate sub-analysis. Lower Ki67 index correlated significantly with heterogeneously positive p53, positive SSTR-2a and CgA expression.

Hepatitis B virus upregulates host microRNAs that target apoptosis-regulatory genes in an in vitro cell model.

Chronic hepatitis B (CHB) infection increases the risk of developing severe liver disease including cirrhosis and hepatocellular carcinoma (HCC). As microRNAs may modulate host - virus interactions, we here investigated if hepatitis B virus (HBV) infection modulate microRNA expression using an in vitro HepG2 cell model system with inducible HBV replication. We found that HBV replication was associated with upregulation of miR-192-5p, miR-194-5p and miR-215-5p, of which miR-192-5p and miR-215-5p have identical seed sequences. Bioinformatics analyses revealed a significant enrichment of potential target genes involved in apoptosis signaling of all three microRNAs. In line with this, transfection with a mimic of miR-192-5p suppressed the protein level of pro-apoptotic BIM and reduced endoplasmic reticulum (ER) stress-induced apoptosis in HepG2 cells. In contrast, transfection with a mimic of miR-194-5p downregulated the anti-apoptotic proteins SODD and cFLIP, and sensitized HepG2 cells to both ER stress- and cytokine-induced apoptosis. In conclusion, our study suggests that HBV upregulates the expression of miR-192-5p and miR-194-5p in the host cell. These microRNAs target important apoptosis-regulatory proteins, and may thus contribute to the development of HBV-related liver disease.

Hepatitis B virus suppresses the secretion of insulin-like growth factor binding protein 1 to facilitate anti-apoptotic IGF-1 effects in HepG2 cells.

Hepatitis B virus (HBV) infection is a major global health burden as chronic hepatitis B (CHB) is associated with the development of liver diseases including hepatocellular carcinoma (HCC). To gain insight into the mechanisms causing HBV-related HCC, we investigated the effects of HBV replication on global host cell gene expression using human HepG2 liver cells. By microarray analysis, we identified 54 differentially expressed genes in HBV-replicating HepG2 cells. One of the differentially-expressed genes was insulin-like growth factor binding protein 1 (IGFBP1) which was downregulated in HBV-replicating cells. Consistent with the gene expression data, IGFBP1 was suppressed at both the cellular and secreted protein levels in the presence of HBV replication. Transient transfection experiments with an inducible plasmid encoding the HBV X protein (HBx) revealed that HBx alone was sufficient to modulate IGFBP1 expression. Small interference RNA (siRNA)-mediated loss of function studies revealed that knockdown of IGFBP1 reduced apoptosis induced by either thapsigargin (TG) or staurosporine (STS). Treatment of cells with recombinant insulin-like growth factor 1 (IGF-1) decreased both TG- or STS-induced apoptosis. Interestingly, addition of recombinant IGFBP1 reversed the anti-apoptotic effect of IGF-1 on TG-induced, but not STS-induced, apoptosis. In conclusion, our results suggest an anti-apoptotic autocrine function of HBV-mediated downregulation of IGFBP1 in HepG2 cells. Such an effect may contribute to the development of HBV-mediated HCC by increasing pro-survival and anti-apoptotic IGF-1 effects.

A Metabolomics Study of Retrospective Forensic Data from Whole Blood Samples of Humans Exposed to 3,4-Methylenedioxymethamphetamine: A New Approach for Identifying Drug Metabolites and Changes in Metabolism Related to Drug Consumption.

The illicit drug 3,4-methylenedioxymethamphetamine (MDMA) has profound physiological cerebral, cardiac, and hepatic effects that are reflected in the blood. Screening of blood for MDMA and other narcotics are routinely performed in forensics analysis using ultra-performance liquid chromatography with high-resolution time-of-flight mass spectrometry (UPLC-HR-TOFMS). The aim of this study was to investigate whether such UPLC-HR-TOFMS data collected over a two-year period could be used for untargeted metabolomics to determine MDMA metabolites as well as endogenous changes related to drug response and toxicology. Whole blood samples from living Danish drivers' positive for MDMA in different concentrations were compared to negative control samples using various statistical methods. The untargeted identification of known MDMA metabolites was used to validate the methods. The results further revealed changes of several acylcarnitines, adenosine monophosphate, adenosine, inosine, thiomorpholine 3-carboxylate, tryptophan, S-adenosyl-l-homocysteine (SAH), and lysophospatidylcholine (lysoPC) species in response to MDMA. These endogenous metabolites could be implicated in an increased energy demand and mechanisms related to the serotonergic syndrome as well as drug induced neurotoxicity. The findings showed that it was possible to extract meaningful results from retrospective UPLC-HR-TOFMS screening data for metabolic profiling in relation to drug metabolism, endogenous physiological effects, and toxicology.

Untargeted Metabolomics Analysis of ABCC6-Deficient Mice Discloses an Altered Metabolic Liver Profile.

Loss-of-function mutations in the transmembrane ABCC6 transport protein cause pseudoxanthoma elasticum (PXE), an ectopic, metabolic mineralization disorder that affects the skin, eye, and vessels. ABCC6 is assumed to mediate efflux of one or several small molecule compounds from the liver cytosol to the circulation. Untargeted metabolomics using liquid chromatography-mass spectrometry was employed to inspect liver cytosolic extracts from mice with targeted disruption of the Abcc6 gene. Absence of the ABCC6 protein induced an altered profile of metabolites in the liver causing accumulation of compounds as more features were upregulated than downregulated in ABCC6-deficient mice. However, no differences of the identified metabolites in liver could be detected in plasma, whereas urine reflected some of the changes. Of note, N-acetylated amino acids and pantothenic acid (vitamin B5), which is involved in acetylation reactions, were accumulated in the liver. None of the identified metabolites seems to explain mineralization in extrahepatic tissues, but the present study now shows that abrogated ABCC6 function does cause alterations in the metabolic profile of the liver in accordance with PXE being a metabolic disease originating from liver disturbance. Further studies of these changes and the further identification of yet unknown metabolites may help to clarify the liver-related pathomechanism of PXE.

Non-interpersonal traumatic events in patients with eating disorders: a systematic review.

Objectives: The purpose of the systematic review was to synthesize literature on eating disorders (ED) and non-interpersonal traumatic events (NTE) and consolidate the reported prevalence of NTE in patients with an ED. Methods: The literature search was performed in Embase, PsycInfo, and PubMed. The keywords in the search were "eating disorder," "trauma" and "non-interpersonal," using index-terms and free-search keywords related to NTE and ED. The PRISMA guidelines were followed. Relevant studies were screened using Rayyan. Results: Of the 16 studies included in the quantitative synthesis, five overall types of NTE were identified: accidents, illness, injury, natural disaster and war. Findings provided tentative evidence for illness and injury being more prevalent in patients suffering from an ED compared to controls. The remaining subtypes of NTE did not show a higher prevalence in patients with an ED when compared to controls. Findings also suggest that those with binge/purge subtype of anorexia nervosa (AN) had a higher prevalence of non-interpersonal traumatic events compared to the restrictive subtype of AN. Discussion: This systematic review provided a clear synthesis of previous findings related to NTE among patients with an ED. Noteworthy, is that many studies do not take into account if the trauma happened prior or after to ED onset, which may affect the association. Furthermore, the body of research on NTE in patients with ED is exceedingly limited, and more research is needed.

Molecular Mechanisms of Temperature Tolerance Plasticity in an Arthropod.

How species thrive in a wide range of environments is a major focus of evolutionary biology. For many species, limited genetic diversity or gene flow among habitats means that phenotypic plasticity must play an important role in their capacity to tolerate environmental heterogeneity and to colonize new habitats. However, we have a limited understanding of the molecular components that govern plasticity in ecologically relevant phenotypes. We examined this hypothesis in a spider species (Stegodyphus dumicola) with extremely low species-wide genetic diversity that nevertheless occupies a broad range of thermal environments. We determined phenotypic responses to temperature stress in individuals from four climatic zones using common garden acclimation experiments to disentangle phenotypic plasticity from genetic adaptations. Simultaneously, we created data sets on multiple molecular modalities: the genome, the transcriptome, the methylome, the metabolome, and the bacterial microbiome to determine associations with phenotypic responses. Analyses of phenotypic and molecular associations reveal that acclimation responses in the transcriptome and metabolome correlate with patterns of phenotypic plasticity in temperature tolerance. Surprisingly, genes whose expression seemed to be involved in plasticity in temperature tolerance were generally highly methylated contradicting the idea that DNA methylation stabilizes gene expression. This suggests that the function of DNA methylation in invertebrates varies not only among species but also among genes. The bacterial microbiome was stable across the acclimation period; combined with our previous demonstrations that the microbiome is temporally stable in wild populations, this is convincing evidence that the microbiome does not facilitate plasticity in temperature tolerance. Our results suggest that population-specific variation in temperature tolerance among acclimation temperatures appears to result from the evolution of plasticity in mainly gene expression.

Large-Scale metabolomics: Predicting biological age using 10,133 routine untargeted LC-MS measurements.

Untargeted metabolomics is the study of all detectable small molecules, and in geroscience, metabolomics has shown great potential to describe the biological age-a complex trait impacted by many factors. Unfortunately, the sample sizes are often insufficient to achieve sufficient power and minimize potential biases caused by, for example, demographic factors. In this study, we present the analysis of biological age in ~10,000 toxicologic routine blood measurements. The untargeted screening samples obtained from ultra-high pressure liquid chromatography-quadruple time of flight mass spectrometry (UHPLC- QTOF) cover + 300 batches and + 30 months, lack pooled quality controls, lack controlled sample collection, and has previously only been used in small-scale studies. To overcome experimental effects, we developed and tested a custom neural network model and compared it with existing prediction methods. Overall, the neural network was able to predict the chronological age with an rmse of 5.88 years (r2  = 0.63) improving upon the 6.15 years achieved by existing normalization methods. We used the feature importance algorithm, Shapley Additive exPlanations (SHAP), to identify compounds related to the biological age. Most importantly, the model returned known aging markers such as kynurenine, indole-3-aldehyde, and acylcarnitines along with a potential novel aging marker, cyclo (leu-pro). Our results validate the association of tryptophan and acylcarnitine metabolism to aging in a highly uncontrolled large-s cale sample. Also, we have shown that by using robust computational methods it is possible to deploy large LC-MS datasets for metabolomics studies to reduce the risk of bias and empower aging studies.

[VEXAS gene variants explain previously unrecognized clinical syndrome].

This review aims to make clinicians aware of the newly described syndrome, VEXAS. VEXAS should become an obvious differential diagnosis in cases of unexplained inflammation, anemia, and rheumatological and/or hematological manifestations. Patients with VEXAS are typically male aged > 60, with inflammation, and macrocytic anaemia. On suspicion of cancer or infections patients have frequently been exposed to extensive diagnostic procedures and hospital admissions. In this review, we summarise the current knowledge of VEXAS regarding pathogenesis, symptoms, diagnosis, and treatment.

Patients with VEXAS diagnosed in a Danish tertiary rheumatology setting have highly elevated inflammatory markers, macrocytic anaemia and negative autoimmune biomarkers.

BACKGROUND: Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) is an autoinflammatory condition with overlapping features of rheumatology and haematology caused by somatic mutations in the UBA1 gene. Patients present with highly variable symptoms and their path towards diagnosis are often complicated and characterised by extensive examinations. It is, therefore, pivotal that clinicians become familiar with the clinical presentation of VEXAS to advance identification of patients with the disease. OBJECTIVES: We aimed to (1) characterise patients diagnosed with VEXAS in a tertiary rheumatology referral centre, (2) identify common rheumatological biomarkers that may distinguish VEXAS from other rheumatic diseases and (3) suggest which clinical findings should motivate genetic testing for VEXAS. METHODS: Patients were identified and diagnosed at the department of Rheumatology, Aarhus University Hospital (AUH), Denmark. Blood samples were examined for VEXAS-associated UBA1 variants by Sanger sequencing at the department of Clinical Immunology, AUH. Clinical and biochemical data were retrieved from the hospital electronic patient chart. RESULTS: Eleven male patients with clinical suspicion of VEXAS underwent sequencing. Five of these carried known VEXAS-associated variants. Median age at diagnosis was 84 (75-87) years. All patients had significantly elevated inflammatory markers with a median C-reactive protein (CRP) of 297 (196-386) mg/L and macrocytic anaemia. None of the patients presented common biomarkers for autoimmunity. CONCLUSION: Danish patients with VEXAS syndrome are men with persistent inflammation, constitutional symptoms and heterogeneous clinical presentations. Shared features for all patients in this study were highly elevated inflammatory markers, macrocytic anaemia and negative autoimmune biomarkers.

A Retrospective Metabolomics Analysis of Gamma-Hydroxybutyrate in Humans: New Potential Markers and Changes in Metabolism Related to GHB Consumption.

GHB is an endogenous short-chain organic acid presumably also widely applied as a rape and knock out drug in cases of drug-facilitated crimes or sexual assaults (DFSA). Due to the endogenous nature of GHB and its fast metabolism in vivo, the detection window of exogenous GHB is however narrow, making it challenging to prove use of GHB in DFSA cases. Alternative markers of GHB intake have recently appeared though none has hitherto been validated for forensic use. UHPLC-HRMS based screening of blood samples for drugs of abuse is routinely performed in several forensic laboratories which leaves an enormous amount of unexploited data. Recently we devised a novel metabolomics approach to use archived data from such routine screenings for elucidating both direct metabolites from exogenous compounds, but potentially also regulation of endogenous metabolism and metabolites. In this paper we used UHPLC-HRMS data acquired over a 6-year period from whole blood analysis of 51 drivers driving under the influence of GHB as well as a matched control group. The data were analyzed using a metabolomics approach applying a range of advanced analytical methods such as OPLS-DA, LASSO, random forest, and Pearson correlation to examine the data in depth and demonstrate the feasibility and potential power of the approach. This was done by initially detecting a range of potential biomarkers of GHB consumption, some that previously have been found in controlled GHB studies, as well as several new potential markers not hitherto known. Furthermore, we investigate the impact of GHB intake on human metabolism. In aggregate, we demonstrate the feasibility to extract meaningful information from archived data here exemplified using GHB cases. Hereby we hope to pave the way for more general use of the principle to elucidate human metabolites of e.g. new legal or illegal drugs as well as for applications in more global and large scale metabolomics studies in the future.

Super-Resolution Nanolithography of Two-Dimensional Materials by Anisotropic Etching.

Nanostructuring allows altering of the electronic and photonic properties of two-dimensional (2D) materials. The efficiency, flexibility, and convenience of top-down lithography processes are, however, compromised by nanometer-scale edge roughness and resolution variability issues, which especially affect the performance of 2D materials. Here, we study how dry anisotropic etching of multilayer 2D materials with sulfur hexafluoride (SF6) may overcome some of these issues, showing results for hexagonal boron nitride (hBN), tungsten disulfide (WS2), tungsten diselenide (WSe2), molybdenum disulfide (MoS2), and molybdenum ditelluride (MoTe2). Scanning electron microscopy and transmission electron microscopy reveal that etching leads to anisotropic hexagonal features in the studied transition metal dichalcogenides, with the relative degree of anisotropy ranked as: WS2 > WSe2 > MoTe2 ∼ MoS2. Etched holes are terminated by zigzag edges while etched dots (protrusions) are terminated by armchair edges. This can be explained by Wulff constructions, taking the relative stabilities of the edges and the AA' stacking order into account. Patterns in WS2 are transferred to an underlying graphite layer, demonstrating a possible use for creating sub-10 nm features. In contrast, multilayer hBN exhibits no lateral anisotropy but shows consistent vertical etch angles, independent of crystal orientation. Using an hBN crystal as the base, ultrasharp corners can be created in lithographic patterns, which are then transferred to a graphite crystal underneath. We find that the anisotropic SF6 reactive ion etching process makes it possible to downsize nanostructures and obtain smooth edges, sharp corners, and feature sizes significantly below the resolution limit of electron beam lithography. The nanostructured 2D materials can be used themselves or as etch masks to pattern other nanomaterials.

Neuroendocrine neoplasms of the appendix: Characterization of 335 patients referred to the Copenhagen NET Center of Excellence.

INTRODUCTION: Neuroendocrine neoplasms (NEN) of the appendix are often incidentally discovered after appendectomy. Appropriate management is debated. The purpose was to characterize a cohort of 335 appendix NEN and evaluate the risk of recurrence. METHODS: Retrospective collection of data from 335 patients referred to the Neuroendocrine Tumor Center at Rigshospitalet 2000-2019. Appendix goblet cell carcinoids and mixed neuroendocrine non-neuroendocrine neoplasms were excluded. Patients were followed until December 31st, 2019. No patients were lost to follow-up. RESULTS: Sixty-three percent of the patients were female. The median (range) age at diagnosis was 34 (9-92) years. Median follow-up was 66 (1-250) months. Median tumor size was 7 (1-45) mm with 10 (3%) tumors >20 mm. In 18 specimens (5%) resection margins were positive. Mesoappendiceal invasion was found in 113 (35%). Sixty-three (19%) patients underwent right-sided completion hemicolectomy (RHC) after appendectomy according to ENETS guidelines. Among these, 11 (17%) had lymph node metastases in the resected tissue. Further, one patient who underwent initial RHC due to colonic adenocarcinoma had lymph node metastases. All lymph node metastases were detected in patients with serotonin positive tumors. No patients with glucagon positive tumors (n = 85) had lymph node metastases. Mesoappendiceal invasion >3 mm and positive resection margins were associated with presence of lymph node metastases. No recurrences were recorded. CONCLUSION: Following ENETS guidelines may lead to overtreatment of patients with respect to completion RHC. The risk of over- and undertreatment needs to be further evaluated.

Surgery of the primary tumour in 201 patients with high-grade gastroenteropancreatic neuroendocrine and mixed neuroendocrine-non-neuroendocrine neoplasms.

The benefit of surgery in high-grade gastroenteropancreatic neuroendocrine neoplasms (GEP NEN) and mixed neuroendocrine-non-neuroendocrine neoplasms (MiNEN) is uncertain. The present study aimed to investigate outcomes after tumour surgery in patients with high-grade (Ki-67 > 20%) GEP NEN or MiNEN stage I-III or stage IV. We analysed data from patients treated in the period 2007-2015 at eight Nordic university hospitals. Overall survival (OS) and progression-free survival (PFS)/disease-free survival (DFS) were analysed by Kaplan-Meier estimates. Prognostic factors were evaluated using Cox regression. We included 201 surgically resected patients, 143 stage I-III and 58 stage IV, with 68% having neuroendocrine carcinoma, 23% MiNEN, 5% neuroendocrine tumour G3 and 4% uncertain NEN G3. Primary tumours were located in colon/rectum (52%), oesophagus/cardia (19%), pancreas (10%), stomach (7%), jejunum/ileum (5%), duodenum (4%), gallbladder (2%) and anal canal (1%). For patients with stage I-III, median DFS was 12 months (95% confidence interval [CI] = 5.5-18.5) and median OS was 32 months (95% CI = 24.0-40.0). For patients with stage I-III and an R0 resection, median DFS was 21 months (95% CI = 4.9-37.1) and median OS was 39 months (95% CI = 25.0-53.0). For patients with stage IV, median PFS/DFS was 4 months (95% CI = 1.9-6.1) and median OS was 11 months (95% CI = 4.8-17.2). For patients with stage IV and an R0 resection, median DFS was 6 months (95% CI = 0-16.4) and median OS was 32 months (95% CI = 25.5-38.5). Performance status > 1 and colorectal primary were associated with poor prognosis. There was no difference in survival between patients with high-grade GEP NEN and MiNEN. Surgery of the primary tumour in patients with loco-regional high-grade GEP NEN or MiNEN led to good long-term results and should be considered if an R0 resection is considered achievable. Highly selected patients with stage IV disease may also benefit from surgery.