Disaccharide compositional analysis of chondroitin sulphate using WAX HILIC-MS with pre-column procainamide labelling; application to the placenta in pre-eclampsia.

Chondroitin sulphate (CS) and dermatan sulphate are negatively charged linear heteropolysaccharides. These glycosaminoglycans (GAG) are involved in cellular signalling via binding to growth factors. CS is expressed in a range of tissue and biological fluids and is highly expressed in the placenta. There is evidence that decorin; a CS proteoglycan is significantly decreased in pre-eclampsia and fetal growth restriction. It is considered that GAG chain composition may influence cellular processes that are altered in pre-eclampsia. The goal of the present study was to develop an LC-MS method with precolumn procainamide labelling for the disaccharide compositional analysis of CS. The method was used to investigate whether the disaccharide composition of placenta-extracted CS is altered in pre-eclampsia. The study revealed differential disaccharide compositions of placental chondroitin sulphate between pre-eclampsia and other pregnancy conditions. This suggests that the method may have diagnostic potential for pregnancy disorders. Furthermore, the findings suggest that CS sulphation might play a significant role in maternal labour.

Invariant Natural Killer T cells coordinate removal of senescent cells.

BACKGROUND: The failure of immune surveillance to remove senescent cells drive age-related diseases. Here, we target an endogenous immune surveillance mechanism that can promote elimination of senescent cells and reverse disease progression. METHODS: We identify a class of lipid-activated T cells, invariant natural killer T cells (iNKTs) are involved in the removal of pathologic senescent cells. We use two disease models in which senescent cells accumulate to test whether activation of iNKT cells was sufficient to eliminate senescent cells in vivo. FINDINGS: Senescent preadipocytes accumulate in white adipose tissue of chronic high-fat diet (HFD) fed mice, and activation of iNKT cells with the prototypical glycolipid antigen alpha-galactosylceramide (αGalCer) led to a reduction of these cells with improved glucose control. Similarly, senescent cells accumulate within the lungs of mice injured by inhalational bleomycin, and αGalCer-induced activation of iNKT cells greatly limited this accumulation, decreased the lung fibrosis and improved survival. Furthermore, co-culture experiments showed that the preferential cytotoxic activity of iNKT cells to senescent cells is conserved in human cells. CONCLUSIONS: These results uncover a senolytic capacity of tissue-resident iNKT cells and pave the way for anti-senescence therapies that target these cells and their mechanism of activation.

Antibacterial apple cider vinegar eradicates methicillin resistant Staphylococcus aureus and resistant Escherichia coli.

Methicillin-resistant Staphylococcus aureus (MRSA) and resistant Escherichia coli (rE.coli) infections can spread rapidly. Further they are associated with high morbidity and mortality from treatment failure. Therapy involves multiple rounds of ineffective antibiotics alongside unwanted side effects, alternative treatments are crucial. Apple cider vinegar (ACV) is a natural, vegan product that has been shown to have powerful antimicrobial activity hence we investigated whether ACV could ameliorate these resistant bacteria. The minimum dilution of ACV required for growth inhibition was comparable for both bacteria (1/25 dilution of ACV liquid and ACV tablets at 200 µg/ml were effective against rE. coli and MRSA). Monocyte co-culture with microbes alongside ACV resulted in an increase in monocyte phagocytosis by 21.2% and 33.5% compared to non-ACV treated but MRSA or rE. coli stimulated monocytes, respectively. Label free quantitative proteomic studies of microbial protein extracts demonstrated that ACV penetrated microbial cell membranes and organelles, altering the expression of key proteins. This resulted in significant reductions in total protein expression, moreover we could only detect ribosomal proteins; 50 s 30 s, enolase, phosphenol pyruvate and the ATP synthase subunit in rE. coli. Elongation factor iNOS and phosphoglycerate kinase OS were the only proteins present in MRSA samples following ACV treatment.

Detection of Extended-Spectrum ß-Lactamase and Carbapenemase Activity in Gram-Negative Bacilli Using Liquid Chromatography - Tandem Mass Spectrometry.

PURPOSE: Several mass spectrometry-based methods for antimicrobial sensitivity testing have been described in recent years. They offer an alternative to commercially available testing systems which were considered to have disadvantages in terms of cost- and time-efficiency. The aim of this study was to develop an LC-MS/MS-based antibiotic hydrolysis assay for evaluating antimicrobial resistance (AMR) of Gram-negative bacteria. MATERIALS AND METHODS: Four species of Gram-negative bacilli (Klebsiella pneumoniae, Escherichia coli, Providencia stuartii and Acinetobacter baumannii) were tested against six antibiotics from three different classes: ampicillin, meropenem, imipenem, ceftazidime, ceftriaxone and cefepime. Bacterial suspensions from each species were incubated with a mixture of the six antibiotics. Any remaining antibiotic following incubation was measured using LC-MS/MS. The results were interpreted using measurements obtained for an E. coli strain sensitive to all antibiotics and expressed as percentage of hydrolyzed antibiotic. These were subsequently compared to commercially-available system for the bacteria identification and susceptibility testing. RESULTS: Overall, LC-MS/MS assay and commercial antimicrobial susceptibility platform results showed good agreement in terms of an organism being resistant/sensitive to an antibiotic. The time required to complete the LC-MS/MS-based hydrolysis test was under 5 h, significantly shorter that commercially available susceptibility testing platforms. CONCLUSION: By using a sensitive strain for results interpretation and simultaneous use of multiple antibiotics, the proposed protocol offers improved robustness and multiplexing over previously described methods for antibiotic sensitivity testing. Nevertheless, further research is needed before routine assimilation of the method, especially for strains with intermediate resistance.

SRL pathogenicity island contributes to the metabolism of D-aspartate via an aspartate racemase in Shigella flexneri YSH6000.

In recent years, multidrug resistance of Shigella strains associated with genetic elements like pathogenicity islands, have become a public health problem. The Shigella resistance locus pathogenicity island (SRL PAI) of S. flexneri 2a harbors a 16Kbp region that contributes to the multidrug resistance phenotype. However, there is not much information about other functions such as metabolic, physiologic or ecological ones. For that, wild type S. flexneri YSH6000 strain, and its spontaneous SRL PAI mutant, 1363, were used to study the contribution of the island in different growth conditions. Interestingly, when both strains were compared by the Phenotype Microarrays, the ability to metabolize D-aspartic acid as a carbon source was detected in the wild type strain but not in the mutant. When D-aspartate was added to minimal medium with other carbon sources such as mannose or mannitol, the SRL PAI-positive strain was able to metabolize it, while the SRL PAI-negative strain did not. In order to identify the genetic elements responsible for this phenotype, a bioinformatic analysis was performed and two genes belonging to SRL PAI were found: orf8, coding for a putative aspartate racemase, and orf9, coding for a transporter. Thus, it was possible to measure, by an indirect analysis of racemization activity in minimal medium supplemented only with D-aspartate, that YSH6000 strain was able to transform the D-form into L-, while the mutant was impaired to do it. When the orf8-orf9 region from SRL island was transformed into S. flexneri and S. sonnei SRL PAI-negative strains, the phenotype was restored. Although, when single genes were cloned into plasmids, no complementation was observed. Our results strongly suggest that the aspartate racemase and the transporter encoded in the SRL pathogenicity island are important for bacterial survival in environments rich in D-aspartate.

Effect of berberine on in vitro metabolism of sulfonylureas: A herb-drug interactions study.

Patients with type 2 diabetes may co-ingest herbal and prescription medicines to control their blood sugar levels. Competitive binding of drug and herb may mutually affect their metabolism. This can alter the level of drug and its kinetics in the body, potentially causing toxicities or loss of efficacy. Understanding how the metabolism of sulfonylureas like glyburide and gliclazide can be affected by the presence of berberine and vice versa can provide valuable information on the possible risk of toxicities caused by co-ingestion of drugs. METHODS: Berberine and sulfonylureas (glyburide and gliclazide) were co-incubated with rat liver microsomes in the presence of a NADPH-regenerating system. The metabolites of berberine and sulfonylureas were analysed using liquid chromatography with high-resolution mass spectrometry in the positive ion mode. The role of individual isozymes in the metabolism of berberine, glyburide and gliclazide was investigated by using specific inhibitors. RESULTS: In vitro metabolism of berberine led to the formation of demethyleneberberine (B1a) and its isomer B1b through demethylenation. Berberrubine (B2a) and its isomer B2b were formed through demethylation. The isozymes CYP3A and CYP2D were found to be involved in the metabolism of berberine. In vitro metabolism of glyburide and gliclazide led to the formation of hydroxylated metabolites. The isozymes CYP3A and CYP2C were found to be involved in the metabolism of glyburide. Gliclazide was metabolised by CYP2C. In vitro co-incubation of glyburide or gliclazide with berberine showed that each drug's metabolism was compromised as they share a common isozyme. A strong negative linear correlation of glyburide or gliclazide metabolite levels and the concentration of berberine confirmed the effect of berberine on the metabolism of sulfonylureas. CONCLUSIONS: The metabolism of sulfonylureas and berberine was affected when these compounds were co-incubated with each other. This may be attributable to competitive binding of the herb and drug to the catalytic sites of the same isozymes.

Comparative Proteomic Profiling of Methicillin-Susceptible and Resistant Staphylococcus aureus.

Staphylococcus aureus is a highly successful human pathogen responsible for a wide range of infections. This study provides insights into the virulence, pathogenicity, and antimicrobial resistance determinants of methicillin-susceptible and methicillin-resistant S. aureus (MSSA; MRSA) recovered from non-healthcare environments. Three environmental MSSA and three environmental MRSA are selected for proteomic profiling using isobaric tag for relative and absolute quantitation tandem mass spectrometry (iTRAQ MS/MS). Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway annotation are applied to interpret the functions of the proteins detected. 792 proteins are identified in MSSA and MRSA. Comparative analysis of MRSA and MSSA reveals that 8 of out 792 proteins are upregulated and 156 are downregulated. Proteins that have differences in abundance are predominantly involved in catalytic and binding activity. Among 164 differently abundant proteins, 29 are involved in pathogenesis, antimicrobial resistance, stress response, mismatch repair, and cell wall synthesis. Twenty-two proteins associated with pathogenicity including SPA, SBI, CLFA, and DLT are upregulated in MRSA. Moreover, the upregulated pathogenic protein ENTC2 in MSSA is determined to be a super antigen, potentially capable of triggering toxic shock syndrome in the host. Enhanced pathogenicity, antimicrobial resistance, and stress response are observed in MRSA compared to MSSA.

A method to assess bioavailability of antibiotics in anthropogenic polluted ecosystems by using a bacterial fitness test.

Antibiotics released in the environment exert a selective pressure on the resident microbiota. It is well accepted that the mere measurement of antibiotics does not reflect the actual bioavailability. In fact, antibiotics can be adsorbed or complexed to particles and/or chemicals in water and soil. Bioavailable concentrations of antibiotics in soil and water are subjected to great uncertainty, therefore biological assays are increasingly recognized as that allow an indirect determination of the residual antibiotic activity. Here we propose how a fitness test for bacteria can be used to qualitatively assess the bioavailability of a specific antibiotic in the environment. The findings show that by using a pair of resistant and sensitive bacterial strains, the resulting fitness can indirectly reflect antibiotic bioavailability. Hence, this test can be used as a complementary assay to other biological and chemical tests to assess bioavailability of antibiotics.

BET Proteins Are Required for Transcriptional Activation of the Senescent Islet Cell Secretome in Type 1 Diabetes.

Type 1 diabetes (T1D) results from the progressive loss of pancreatic beta cells as a result of autoimmune destruction. We recently reported that during the natural history of T1D in humans and the female nonobese diabetic (NOD) mouse model, beta cells acquire a senescence-associated secretory phenotype (SASP) that is a major driver of disease onset and progression, but the mechanisms that activate SASP in beta cells were not explored. Here, we show that the SASP in islet cells is transcriptionally controlled by Bromodomain ExtraTerminal (BET) proteins, including Bromodomain containing protein 4 (BRD4). A chromatin analysis of key beta cell SASP genes in NOD islets revealed binding of BRD4 at active regulatory regions. BET protein inhibition in NOD islets diminished not only the transcriptional activation and secretion of SASP factors, but also the non-cell autonomous activity. BET protein inhibition also decreased the extent of SASP induction in human islets exposed to DNA damage. The BET protein inhibitor iBET-762 prevented diabetes in NOD mice and also attenuated SASP in islet cells in vivo. Taken together, our findings support a crucial role for BET proteins in the activation of the SASP transcriptional program in islet cells. These studies suggest avenues for preventing T1D by transcriptional inhibition of SASP.

Stratification of inflammatory bowel disease outpatients by disease activity and risk of complications to guide out-of-hospital monitoring: a patient-centred quality improvement project.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic relapsing-remitting condition affecting 600 000 people in the UK. Traditionally, patients attend outpatient clinics for monitoring regardless of their symptoms or risk of developing complications. This can lead to a mismatch between need and access: patients in remission given elective appointments displace those in need of urgent specialist attention. Novel initiatives implemented in the UK to improve outpatient monitoring have often required a well-maintained patient registry, empowered patients and significant information technology support. DESIGN AND STRATEGY: In this large-scale quality improvement project at St Mark's Hospital, a tertiary centre for IBD, we stratified over 1000 patients attending three non-complex IBD clinics over 12 months according to disease activity and risk profile. The aim was to offer a choice and subsequently transfer 50% of eligible patients to specialist nurse-led telephone clinics and demonstrate non-inferior satisfaction levels to existing outpatient follow-up. We also sought to ensure there was timely access to a newly established rapid access clinic for patients requiring urgent specialist attention.A core project team consisting of healthcare professionals, patients and quality improvement scientists met regularly. The team tested and scaled up interventions using 'Plan-Do-Study-Act' cycles within the 'Model for Improvement' framework and analysed data continuously using statistical process charts. RESULTS: Over 12 months, the average number of eligible patients transferred to telephone clinics rose from 17.6% (42/239) using a questionnaire method to 59.3% (73/123) using active discussion in clinic. Patient satisfaction scores remained high and non-inferior to baseline scores in face-to-face clinics. The median waiting time to be seen in the rapid access clinic was 6.5 days. CONCLUSION: This is the first published study to report on the successful stratification of patients with IBD based on disease activity and risk of complications to create a more responsive, sustainable and patient-centred model for IBD monitoring.

mTORC1 to AMPK switching underlies ß-cell metabolic plasticity during maturation and diabetes.

Pancreatic beta cells (ß-cells) differentiate during fetal life, but only postnatally acquire the capacity for glucose-stimulated insulin secretion (GSIS). How this happens is not clear. In exploring what molecular mechanisms drive the maturation of ß-cell function, we found that the control of cellular signaling in ß-cells fundamentally switched from the nutrient sensor target of rapamycin (mTORC1) to the energy sensor 5'-adenosine monophosphate-activated protein kinase (AMPK), and that this was critical for functional maturation. Moreover, AMPK was activated by the dietary transition taking place during weaning, and this in turn inhibited mTORC1 activity to drive the adult ß-cell phenotype. While forcing constitutive mTORC1 signaling in adult ß-cells relegated them to a functionally immature phenotype with characteristic transcriptional and metabolic profiles, engineering the switch from mTORC1 to AMPK signaling was sufficient to promote ß-cell mitochondrial biogenesis, a shift to oxidative metabolism, and functional maturation. We also found that type 2 diabetes, a condition marked by both mitochondrial degeneration and dysregulated GSIS, was associated with a remarkable reversion of the normal AMPK-dependent adult ß-cell signature to a more neonatal one characterized by mTORC1 activation. Manipulating the way in which cellular nutrient signaling pathways regulate ß-cell metabolism may thus offer new targets to improve ß-cell function in diabetes.

Targeted Elimination of Senescent Beta Cells Prevents Type 1 Diabetes.

Type 1 diabetes (T1D) is an organ-specific autoimmune disease characterized by hyperglycemia due to progressive loss of pancreatic beta cells. Immune-mediated beta cell destruction drives the disease, but whether beta cells actively participate in the pathogenesis remains unclear. Here, we show that during the natural history of T1D in humans and the non-obese diabetic (NOD) mouse model, a subset of beta cells acquires a senescence-associated secretory phenotype (SASP). Senescent beta cells upregulated pro-survival mediator Bcl-2, and treatment of NOD mice with Bcl-2 inhibitors selectively eliminated these cells without altering the abundance of the immune cell types involved in the disease. Significantly, elimination of senescent beta cells halted immune-mediated beta cell destruction and was sufficient to prevent diabetes. Our findings demonstrate that beta cell senescence is a significant component of the pathogenesis of T1D and indicate that clearance of senescent beta cells could be a new therapeutic approach for T1D.

Antimicrobial activity of apple cider vinegar against Escherichia coli, Staphylococcus aureus and Candida albicans; downregulating cytokine and microbial protein expression.

The global escalation in antibiotic resistance cases means alternative antimicrobials are essential. The aim of this study was to investigate the antimicrobial capacity of apple cider vinegar (ACV) against E. coli, S. aureus and C. albicans. The minimum dilution of ACV required for growth inhibition varied for each microbial species. For C. albicans, a 1/2 ACV had the strongest effect, S. aureus, a 1/25 dilution ACV was required, whereas for E-coli cultures, a 1/50 ACV dilution was required (p < 0.05). Monocyte co-culture with microbes alongside ACV resulted in dose dependent downregulation of inflammatory cytokines (TNFα, IL-6). Results are expressed as percentage decreases in cytokine secretion comparing ACV treated with non-ACV treated monocytes cultured with E-coli (TNFα, 99.2%; IL-6, 98%), S. aureus (TNFα, 90%; IL-6, 83%) and C. albicans (TNFα, 83.3%; IL-6, 90.1%) respectively. Proteomic analyses of microbes demonstrated that ACV impaired cell integrity, organelles and protein expression. ACV treatment resulted in an absence in expression of DNA starvation protein, citrate synthase, isocitrate and malate dehydrogenases in E-coli; chaperone protein DNak and ftsz in S. aureus and pyruvate kinase, 6-phosphogluconate dehydrogenase, fructose bisphosphate were among the enzymes absent in C.albican cultures. The results demonstrate ACV has multiple antimicrobial potential with clinical therapeutic implications.

Heme promotes transcriptional and demethylase activities of Gis1, a member of the histone demethylase JMJD2/KDM4 family.

The yeast Gis1 protein is a transcriptional regulator belonging to the JMJD2/KDM4 subfamily of demethylases that contain a JmjC domain, which are highly conserved from yeast to humans. They have important functions in histone methylation, cellular signaling and tumorigenesis. Besides serving as a cofactor in many proteins, heme is known to directly regulate the activities of proteins ranging from transcriptional regulators to potassium channels. Here, we report a novel mechanism governing heme regulation of Gis1 transcriptional and histone demethylase activities. We found that two Gis1 modules, the JmjN + JmjC domain and the zinc finger (ZnF), can bind to heme specifically in vitro. In vivo functional analysis showed that the ZnF, not the JmjN + JmjC domain, promotes heme activation of transcriptional activity. Likewise, measurements of the demethylase activity of purified Gis1 proteins showed that full-length Gis1 and the JmjN + JmjC domain both possess demethylase activity. However, heme potentiates the demethylase activity of full-length Gis1, but not that of the JmjN + JmjC domain, which can confer heme activation of transcriptional activity in an unrelated protein. These results demonstrate that Gis1 represents a novel class of multi-functional heme sensing and signaling proteins, and that heme binding to the ZnF stimulates Gis1 demethylase and transcriptional activities.

Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry.

Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6-4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA-GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biology. Graphical abstract Extraction and HILIC UPLC-MS analysis of procainamide-labelled heparan sulphate disaccharides.

Classification of cancer cell lines using matrix-assisted laser desorption/ionization time­of­flight mass spectrometry and statistical analysis.

Over the past decade, matrix-assisted laser desorption/ionization time­of­flight mass spectrometry (MALDI­TOF MS) has been established as a valuable platform for microbial identification, and it is also frequently applied in biology and clinical studies to identify new markers expressed in pathological conditions. The aim of the present study was to assess the potential of using this approach for the classification of cancer cell lines as a quantifiable method for the proteomic profiling of cellular organelles. Intact protein extracts isolated from different tumor cell lines (human and murine) were analyzed using MALDI­TOF MS and the obtained mass lists were processed using principle component analysis (PCA) within Bruker Biotyper® software. Furthermore, reference spectra were created for each cell line and were used for classification. Based on the intact protein profiles, we were able to differentiate and classify six cancer cell lines: two murine melanoma (B16­F0 and B164A5), one human melanoma (A375), two human breast carcinoma (MCF7 and MDA­MB­231) and one human liver carcinoma (HepG2). The cell lines were classified according to cancer type and the species they originated from, as well as by their metastatic potential, offering the possibility to differentiate non­invasive from invasive cells. The obtained results pave the way for developing a broad­based strategy for the identification and classification of cancer cells.

Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring.

Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 µg GAG. Limits of detection and quantitation were 0.02-0.15 and 0.07-0.31 µg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences.

Factors determining the balance between the wish to die and the wish to live in older adults.

BACKGROUND: The "Internal Struggle Hypothesis" (Kovacs and Beck, ) suggests that suicidal persons may have both a wish to live (WTL) and a wish to die (WTD). The current study investigates whether the three-group typology - "WTL", "ambivalent (AMB)", and "WTD" - is determined by common correlates of suicidality and whether these groups can be ordinally ranked. METHODS: The sample comprised 113 older inpatients. Discriminant analysis was used to create two functions (combining social, psychiatric, psychological, and somatic variables) to predict the assignment of older inpatients into the groups WTL, AMB, and WTD. RESULTS: The functions "Subjective Well-being" and "Social Support" allowed us to assign patients into these three distinct groups with good accuracy (66.1%). "Subjective Well-being" contrasted the groups WTD and WTL and "Social Support" discriminated between the groups WTD and AMB. "Social Support" was highest in the AMB group. CONCLUSIONS: Our results suggest a simultaneous presence of a WTL and a WTD in older inpatients, and also that the balance between them is determined by "Subjective Well-being" and "Social Support". Unexpectedly, the AMB group showed the highest scores on "Social Support". We hypothesize that higher social support might function as an important determinant of a remaining WTL when a WTD is present because of a lower sense of well-being. The study suggests that the groups WTL-AMB-WTD can not situated on a one-dimensional continuum. Copyright © 2016 John Wiley & Sons, Ltd.