A brief, patient- and proxy-reported outcome measure in advanced illness:Validity, reliability and responsiveness of the Integrated Palliative care Outcome Scale (IPOS)

Background: Few measures capture the complex symptoms and concerns of those receiving palliative care. Aim: To validate the Integrated Palliative care Outcome Scale, a measure underpinned by extensive psychometric development, by evaluating its validity, reliability and responsiveness to change. Design: Concurrent, cross-cultural validation study of the Integrated Palliative care Outcome Scale – both (1) patient self-report and (2) staff proxy-report versions. We tested construct validity (factor analysis, known-group comparisons, and correlational analysis), reliability (internal consistency, agreement, and test–retest reliability), and responsiveness (through longitudinal evaluation of change). Setting/participants: In all, 376 adults receiving palliative care, and 161 clinicians, from a range of settings in the United Kingdom and Germany Results: We confirm a three-factor structure (Physical Symptoms, Emotional Symptoms and Communication/Practical Issues). Integrated Palliative care Outcome Scale shows strong ability to distinguish between clinically relevant groups; total Integrated Palliative care Outcome Scale and Integrated Palliative care Outcome Scale subscale scores were higher – reflecting more problems – in those patients with ‘unstable’ or ‘deteriorating’ versus ‘stable’ Phase of Illness (F = 15.1, p &lt; 0.001). Good convergent and discriminant validity to hypothesised items and subscales of the Edmonton Symptom Assessment System and Functional Assessment of Cancer Therapy–General is demonstrated. The Integrated Palliative care Outcome Scale shows good internal consistency (α = 0.77) and acceptable to good test–retest reliability (60% of items kw &gt; 0.60). Longitudinal validity in form of responsiveness to change is good. Conclusion: The Integrated Palliative care Outcome Scale is a valid and reliable outcome measure, both in patient self-report and staff proxy-report versions. It can assess and monitor symptoms and concerns in advanced illness, determine the impact of healthcare interventions, and demonstrate quality of care. This represents a major step forward internationally for palliative care outcome measurement.</p

Initial activation of EpCAM cleavage via cell-to-cell contact

<p>Abstract</p> <p>Background</p> <p>Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is frequently over-expressed in simple epithelia, progenitors, embryonic and tissue stem cells, carcinoma and cancer-initiating cells. Besides functioning as a homophilic adhesion protein, EpCAM is an oncogenic receptor that requires regulated intramembrane proteolysis for activation of its signal transduction capacity. Upon cleavage, the extracellular domain EpEX is released as a soluble ligand while the intracellular domain EpICD translocates into the cytoplasm and eventually into the nucleus in combination with four-and-a-half LIM domains protein 2 (FHL2) and β-catenin, and drives cell proliferation.</p> <p>Methods</p> <p>EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were investigated under varying density conditions using confocal laser scanning microscopy, immunoblotting, cell counting, and conditional cell systems.</p> <p>Results</p> <p>EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were dependent on adequate cell-to-cell contact. If cell-to-cell contact was prohibited EpCAM did not provide growth advantages. If cells were allowed to undergo contact to each other, EpCAM transmitted proliferation signals based on signal transduction-related cleavage processes. Accordingly, the pre-cleaved version EpICD was not dependent on cell-to-cell contact in order to induce <it>c-myc </it>and cell proliferation, but necessitated nuclear translocation. For the case of contact-inhibited cells, although cleavage of EpCAM occurred, nuclear translocation of EpICD was reduced, as were EpCAM effects.</p> <p>Conclusion</p> <p>Activation of EpCAM's cleavage and oncogenic capacity is dependent on cellular interaction (juxtacrine) to provide for initial signals of regulated intramembrane proteolysis, which then support signalling via soluble EpEX (paracrine).</p

Effects of Plant Biomass, Plant Diversity, and Water Content on Bacterial Communities in Soil Lysimeters: Implications for the Determinants of Bacterial Diversity▿ †

Soils may comprise tens of thousands to millions of bacterial species. It is still unclear whether this high level of diversity is governed by functional redundancy or by a multitude of ecological niches. In order to address this question, we analyzed the reproducibility of bacterial community composition after different experimental manipulations. Soil lysimeters were planted with four different types of plant communities, and the water content was adjusted. Group-specific phylogenetic fingerprinting by PCR-denaturing gradient gel electrophoresis revealed clear differences in the composition of Alphaproteobacteria, Betaproteobacteria, Bacteroidetes, Chloroflexi, Planctomycetes, and Verrucomicrobia populations in soils without plants compared to that of populations in planted soils, whereas no influence of plant species composition on bacterial diversity could be discerned. These results indicate that the presence of higher plant species affects the species composition of bacterial groups in a reproducible manner and even outside of the rhizosphere. In contrast, the environmental factors tested did not affect the composition of Acidobacteria, Actinobacteria, Archaea, and Firmicutes populations. One-third (52 out of 160) of the sequence types were found to be specifically and reproducibly associated with the absence or presence of plants. Unexpectedly, this was also true for numerous minor constituents of the soil bacterial assemblage. Subsequently, one of the low-abundance phylotypes (beta10) was selected for studying the interdependence under particular experimental conditions and the underlying causes in more detail. This so-far-uncultured phylotype of the Betaproteobacteria species represented up to 0.18% of all bacterial cells in planted lysimeters compared to 0.017% in unplanted systems. A cultured representative of this phylotype exhibited high physiological flexibility and was capable of utilizing major constituents of root exudates. Our results suggest that the bacterial species composition in soil is determined to a significant extent by abiotic and biotic factors, rather than by mere chance, thereby reflecting a multitude of distinct ecological niches

MYC Translocations In Mature B-Cell Neoplasms: Single, Double, Triple and Quadruple Hit Lymphoma

Abstract Background According to the WHO classification from 2008 the subclassification of mature B-cell neoplasms is based mainly on cytomorphological features. So far entities are not defined by genetics, although some close associations between morphology and genetic abnormalities exist like MYC translocation and Burkitt lymphoma/leukemia, CCND1-translocation and mantle cell lymphoma, BCL6 translocation and diffuse large B-cell lymphoma and BCL2 translocation and follicular lymphoma. However, most of the mentioned genetic abnormalities are characteristic but not specific for the respective morphological subtype. Further, these genetic lesions can occur concomitantly. For lymphomas harboring MYC translocations as well as one, two or three additional translocations, respectively, involving BCL2, BCL6 and/or CCND1 the terms “double hit”, “triple hit” and “quadruple hit” lymphoma have been introduced. Aims To characterize a large cohort of patients with mature B-cell neoplasms and MYC translocation with respect to phenotypic presentation and additional cytogenetic abnormalities with a special focus on concomitantly occurring BCL2, BCL6 and CCND1 rearrangements. Patients and Methods 214 patients with mature B-cell neoplasms harboring a MYC translocation were included in this study. For all cases chromosome banding analysis and FISH data verifying the MYC rearrangements were available. Results 141 (65.9%) were male, 73 (34.1%) female, median age was 66.4 years (range: 5.5-88.2 years). Patients were diagnosed as ALL (n=38, 17.8%), Burkitt lymphoma (n=37, 17.3%), CLL (n=43, 20.1%), and other mature B-cell neoplasms including follicular lymphoma, mantle cell lymphoma and PLL (n=96, 44.9%). 134/214 (62.6%) patients showed a MYC translocation but no translocation involving BCL2, BCL6 or CCND1 (single hit), while 59 (27.6%), 19 (8.9%) and 2 (0.9%) patients had so-called double, triple and quadruple hits. Multiple hit lymphomas displayed more frequently a complex karyotype (defined as ≥5 abnormalities in addition to MYC, BCL2, BCL6 and/or CCND1 translocation) in comparison to single hit lymphomas (61.3% vs 28.4%, p&lt;0.001). In mean 3.8 chromosome abnormalities in addition to the MYC translocation (ACA) were observed in single hit cases as compared to 9.2 in multiple hit cases (p&lt;0.001). The most frequently translocated gene in addition to MYC in double hit lymphomas was BCL2 (n=41/59; 69.5%) and in triple hit lymphomas BCL2 together with BCL6 (n=18/19; 94.7%). While in ALL, Burkitt lymphoma and other mature B-cell neoplasms MYC was involved in a translocation with one of the immunoglobulin loci (IGH, IGK, IGL) in the majority of cases (86.8%, 94.6%, 83.3%, respectively), in CLL MYC was translocated to other loci/genes in 62.8% of cases. Additionally, patients presenting as CLL showed most frequently single hit lymphoma (93%) and only 4.1 ACA in mean compared to all others (6.2, p=0.035). Also the pattern of ACA differed between CLL and other entities. The most frequently observed ACA in CLL resemble the spectrum of abnormalities typically found in CLL such as del(13q) (n=16/43, 37.2%), del(11q) (n=11/43, 25.6%), del(17p) (n=12/43, 27.9%) and +12 (n=2/43, 4.7%). In contrast in ALL, Burkitt lymphoma and other mature B-cell neoplasms the most frequently observed ACA were gains of 1q (n=83/171, 48.5%), del(6q) (n=41/171, 24.0%), gains of chromosome 7 (n=49/171, 28.7%), del(17p) (n=31/171, 18.1%), gains of 18q (55/171, 32.2%) and +12 (n=29/171, 17.0%). MYC translocations occurred in Burkitt lymphoma and ALL always within the primary clone, while in mature B-cell neoplasms and in CLL it was also present within subclones exclusively. Conclusions 1. MYC translocations occured either as single hit or multiple hit lymphoma and presented as ALL or different mature B-cell neoplasms. 2. Multiple hit lymphomas more often harbored a complex karyotype in comparison to single hit lymphomas. 3. In double, triple and quadruple hit cases MYC translocations were most frequently accompanied by BCL2 rearrangements. 4. While in Burkitt lymphoma and ALL MYC translocations were always present in the primary clone, MYC translocations could also occur as secondary events in CLL and other mature B-cell neoplasms. 5. MYC translocations in CLL differed from those found in other lymphatic malignancies with respect to their translocation partners and their additional chromosome aberrations. Disclosures: Denzel: MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. </jats:sec

Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1

Abstract The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5’-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for all glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and through phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interfered with both UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the relative positioning of the two GFAT-1 domains to control its activity. Of note, Ser205 phosphorylation had two discernible effects: It lowered baseline GFAT-1 activity and abolished UDP-GlcNAc feedback inhibition. Thus, PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.</jats:p

Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1

AbstractThe hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5’-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for all glycosylation processes in mammals. It modulates the ER stress response, is implicated in cancer and diabetes, and HP activation extends lifespan inCaenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the first and rate-limiting HP enzyme. GFAT-1 activity is modulated through UDP-GlcNAc feedback inhibition and by kinase signaling, including Ser205 phosphorylation by protein kinase A (PKA). The consequence and molecular mechanism of GFAT-1 phosphorylation, however, remains poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels inC. elegans, leading to ER stress resistance. In human GFAT-1, the R203H substitution interfered with both UDP-GlcNAc inhibition and PKA-mediated Ser205 phosphorylation. Of note, Ser205 phosphorylation had two discernible effects: It lowered baseline GFAT-1 activity and abolished UDP-GlcNAc feedback inhibition. Thus, GFAT-1 phosphorylation by PKA uncoupled the feedback loop of the HP and, depending on UDP-GlcNAc availability, phosphorylation by PKA results in lower or enhanced GFAT-1 activityin vivo. Mechanistically, our data indicate that the relative positioning of the two GFAT-1 domains might be affected by phosphorylation and we propose a model how Ser205 phosphorylation modulates the activity and feedback inhibition of GFAT-1.</jats:p

Loss of GFAT-1 feedback regulation activates the hexosamine pathway that modulates protein homeostasis

Glutamine fructose-6-phosphate amidotransferase (GFAT) is the key enzyme in the hexosamine pathway (HP) that produces uridine 5′-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), linking energy metabolism with posttranslational protein glycosylation. In Caenorhabditis elegans, we previously identified gfat-1 gain-of-function mutations that elevate UDP-GlcNAc levels, improve protein homeostasis, and extend lifespan. GFAT is highly conserved, but the gain-of-function mechanism and its relevance in mammalian cells remained unclear. Here, we present the full-length crystal structure of human GFAT-1 in complex with various ligands and with important mutations. UDP-GlcNAc directly interacts with GFAT-1, inhibiting catalytic activity. The longevity-associated G451E variant shows drastically reduced sensitivity to UDP-GlcNAc inhibition in enzyme activity assays. Our structural and functional data point to a critical role of the interdomain linker in UDP-GlcNAc inhibition. In mammalian cells, the G451E variant potently activates the HP. Therefore, GFAT-1 gain-of-function through loss of feedback inhibition constitutes a potential target for the treatment of age-related proteinopathies

Loss of GFAT-1 feedback regulation activates the hexosamine pathway that modulates protein homeostasis

AbstractGlutamine fructose-6-phosphate amidotransferase (GFAT) is the key enzyme in the hexosamine pathway (HP) that produces uridine 5′-diphospho-N-acetyl-d-glucosamine (UDP-GlcNAc), linking energy metabolism with posttranslational protein glycosylation. In Caenorhabditis elegans, we previously identified gfat-1 gain-of-function mutations that elevate UDP-GlcNAc levels, improve protein homeostasis, and extend lifespan. GFAT is highly conserved, but the gain-of-function mechanism and its relevance in mammalian cells remained unclear. Here, we present the full-length crystal structure of human GFAT-1 in complex with various ligands and with important mutations. UDP-GlcNAc directly interacts with GFAT-1, inhibiting catalytic activity. The longevity-associated G451E variant shows drastically reduced sensitivity to UDP-GlcNAc inhibition in enzyme activity assays. Our structural and functional data point to a critical role of the interdomain linker in UDP-GlcNAc inhibition. In mammalian cells, the G451E variant potently activates the HP. Therefore, GFAT-1 gain-of-function through loss of feedback inhibition constitutes a potential target for the treatment of age-related proteinopathies.</jats:p

Molecular Characterization of B-Cell Neoplasms Harboring MYC Translocations

Abstract Background MYC translocations are observed in a variety of mature B-cell neoplasms and are also found in ALL. A clear assignment to an entity is not always possible in terms of immunophenotyping and morphology alone. So far, neither cytogenetics nor molecular genetics have been used to classify this group of diseases. Aims Analyze cytogenetic aberrations and molecular mutations in a large cohort of patients with B-cell neoplasms and MYC rearrangements to evaluate their respective value for classification. Patients and Methods 155 patients with B-cell neoplasms harboring a MYC translocation were included in this study. Chromosome banding analysis, FISH verifying the MYC rearrangement and mutation screening of the genes ID3, MYC, TP53 and SF3B1 (only in CLL) was performed. Results The cohort comprised 103 (66.5%) males and 52 (33.5%) females with a median age of 66.2 yrs (range: 5.5-87.1 yrs). Cytomorphologic and flow cytometric findings were heterogeneous with patients presenting as ALL (n=33, 21.3%), Burkitt lymphoma (n=29, 18.7%), CLL (n=29, 18.7%), and other mature B-cell neoplasms including follicular lymphoma, mantle cell lymphoma and PLL (n=64, 41.3%). MYC translocations occurred either as a single translocation event (“single hit”) or in addition to one, two or three other translocations involving BCL2, BCL6 and/or CCND1 (“double hit”, “triple hit” and “quadruple hit”, respectively (combined: “multiple hit”). Accordingly, the cohort was subdivided into 98/155 (63.2%) patients with single hit and 57/155 (36.8%) patients with multiple hit lymphoma (double hit: 39 (25.2%), triple hit: 16 (10.3%) and quadruple hit: 2 (1.3%)). Results of mutation analysis are depicted in the figure. TP53 mutations were identified in 57/155 (36.8%) patients and were significantly more frequent in Burkitt lymphoma compared to all other entities (18/29 (62.1%) vs. 39/126 (31.0%), p=0.003). MYC mutations were identified with a frequency of 48/155 (31.0%), without significant differences between entities. However, in patients with CLL we observed a slightly lower frequency than in all other entities (17.2% vs 34.1%, p=0.117). ID3 mutations were found in 20/155 (12.9%) cases and were significantly more frequent in Burkitt lymphoma compared to all other entities (10/29 (34.5%) vs. 10/126 (7.9%), p=0.001). Remarkably, in the subgroup of patients presenting as CLL no ID3 mutation was detected (0/29). Single hit lymphoma showed higher mutation frequencies of TP53 and ID3 compared to multiple hit lymphoma (TP53mut: 45.9% vs. 21.1%, p=0.002, ID3mut: 19.4% vs. 1.8%, p=0.001). In contrast, in single hit lymphoma the number of cytogenetic abnormalities observed in addition to MYC-translocation (ACA) was lower than in multiple hit lymphoma (mean: 3.5 vs. 8.5, p&lt;0.001). Accordingly, cases with BCL2 rearrangements were associated with significantly lower mutation frequencies in TP53 (18.6% vs. 43.8%, p=0.005). Of note, single hit Burkitt lymphoma were characterized by significantly higher mutation frequencies of TP53, MYC and ID3 compared to Burkitt lymphoma with multiple hit (75% vs. 33.3%, p=0.048, 60% vs 11.1%, p=0.020; 50% vs 0%, p=0.011). CLL patients were additionally analyzed for mutations in SF3B1. Interestingly, this subgroup showed a higher frequency of SF3B1 mutations compared to published data (Cazzola et al. Blood 2013,121:260-9) (11/29; 37.9% vs. 5-17%). Conclusions 1. Mutations in ID3 and TP53 were most frequently observed in cases presenting as Burkitt lymphoma and especially associated with single hit lymphoma. 2. CLL patients with MYC translocation showed a specific mutation pattern with no ID3 mutations, a low frequency of MYC mutations, but a very high frequency of SF3B1 mutations. Further, MYC translocated CLL is characterized by a low frequency of additional chromosome abnormalities and presents predominantly as single hit lymphoma. 3. Thus far, B-cell neoplasms are mainly classified based on morphological criteria and the immunophenotype. Our data suggest that the cytogenetic and molecular genetic profile might help to establish an improved classification system. The differentiation between single hit lymphoma and multiple hit lymphoma as well as the separation of the category of CLL with MYC-rearrangements is anticipated to be clinically relevant and should be further studied. Disclosures: Denzel: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kienast:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. </jats:sec