Basement membrane ligands initiate distinct signalling networks to direct cell shape

Cells have evolved mechanisms to sense the composition of their adhesive microenvironment. Although much is known about general mechanisms employed by adhesion receptors to relay signals between the extracellular environment and the cytoskeleton, the nuances of ligand-specific signalling remain undefined. Here, we investigated how glomerular podocytes, and four other basement membrane-associated cell types, respond morphologically to different basement membrane ligands. We defined the composition of the respective adhesion complexes using mass spectrometry-based proteomics. On type IV collagen, all epithelial cell types adopted a round morphology, with a single lamellipodium and large adhesion complexes rich in actin-binding proteins. On laminin (511 or 521), all cell types attached to a similar degree but were polygonal in shape with small adhesion complexes enriched in endocytic and microtubule-binding proteins. Consistent with their distinctive morphologies, cells on type IV collagen exhibited high Rac1 activity, while those on laminin had elevated PKCα. Perturbation of PKCα was able to interchange morphology consistent with a key role for this pathway in matrix ligand-specific signalling. Therefore, this study defines the switchable basement membrane adhesome and highlights two key signalling pathways within the systems that determine distinct cell morphologies. Proteomic data are availableviaProteomeXchange with identifier PXD017913

Three-dimensional electron microscopy reveals the evolution of glomerular barrier injury

Open access articleGlomeruli are highly sophisticated filters and glomerular disease is the leading cause of kidney failure. Morphological change in glomerular podocytes and the underlying basement membrane are frequently observed in disease, irrespective of the underlying molecular etiology. Standard electron microscopy techniques have enabled the identification and classification of glomerular diseases based on two-dimensional information, however complex three-dimensional ultrastructural relationships between cells and their extracellular matrix cannot be easily resolved with this approach. We employed serial block face-scanning electron microscopy to investigate Alport syndrome, the commonest monogenic glomerular disease, and compared findings to other genetic mouse models of glomerular disease (Myo1e−/−, Ptpro−/−). These analyses revealed the evolution of basement membrane and cellular defects through the progression of glomerular injury. Specifically we identified sub-podocyte expansions of the basement membrane with both cellular and matrix gene defects and found a corresponding reduction in podocyte foot process number. Furthermore, we discovered novel podocyte protrusions invading into the glomerular basement membrane in disease and these occurred frequently in expanded regions of basement membrane. These findings provide new insights into mechanisms of glomerular barrier dysfunction and suggest that common cell-matrix-adhesion pathways are involved in the progression of disease regardless of the primary insult

Global analysis reveals the complexity of the human glomerular extracellular matrix.

The glomerulus contains unique cellular and extracellular matrix (ECM) components, which are required for intact barrier function. Studies of the cellular components have helped to build understanding of glomerular disease; however, the full composition and regulation of glomerular ECM remains poorly understood. We used mass spectrometry-based proteomics of enriched ECM extracts for a global analysis of human glomerular ECM in vivo and identified a tissue-specific proteome of 144 structural and regulatory ECM proteins. This catalog includes all previously identified glomerular components plus many new and abundant components. Relative protein quantification showed a dominance of collagen IV, collagen I, and laminin isoforms in the glomerular ECM together with abundant collagen VI and TINAGL1. Protein network analysis enabled the creation of a glomerular ECM interactome, which revealed a core of highly connected structural components. More than one half of the glomerular ECM proteome was validated using colocalization studies and data from the Human Protein Atlas. This study yields the greatest number of ECM proteins relative to previous investigations of whole glomerular extracts, highlighting the importance of sample enrichment. It also shows that the composition of glomerular ECM is far more complex than previously appreciated and suggests that many more ECM components may contribute to glomerular development and disease processes. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000456

Is Dissonance Reduction a Special Case of Fluid Compensation? Evidence That Dissonant Cognitions Cause Compensatory Affirmation and Abstraction

Cognitive dissonance theory shares much in common with other perspectives that address anomalies, uncertainty, and general expectancy violations. This has led some theorists to argue that these theories represent overlapping psychological processes. If responding to dissonance and uncertainty occurs through a common psychological process, one should expect that the behavioral outcomes of feeling uncertain would also apply to feelings of dissonance, and vice versa. One specific prediction from the meaning maintenance model would be that cognitive dissonance, like other expectancy violations, should lead to the affirmation of unrelated beliefs, or the abstraction of unrelated schemas when the dissonant event cannot be easily accommodated. This article presents 4 studies (N ϭ 1124) demonstrating that the classic induced-compliance dissonance paradigm can lead not only to a change of attitudes (dissonance reduction), but also to (a) an increased reported belief in God (Study 2), (b) a desire to punish norm-violators (Study 1 and 3), (c) a motivation to detect patterns amid noise (Study 3), and (d) polarizing support of public policies among those already biased toward a particular side (Study 4). These results are congruent with theories that propose content-general fluid compensation following the experience of anomaly, a finding not predicted by dissonance theory. The results suggest that dissonance reduction behaviors may share psychological processes described by other theories addressing violations of expectations

Mixed-metal cluster chemistry. 39. Syntheses and X-ray structures of Mo 3 Ir 3 (μ 4 -η 2 -CO)(μ 3 -CO)(CO) 10 (η 5 -C 5 H 5 ) 3 and Mo 3 RhIr 3 (μ-CO) 4 (CO) 7 (η 5 -C 5 H 5 ) 3 (η 5 -C 5 Me 5 )

We thank the Australian Research Council (ARC) for support ofthis work. J.F. was the recipient of a China Scholarship Council ANUPostgraduate Scholarship and M.D.R. was the recipient of anAustralian Postgraduate Awar

Building a DMU e-Biology resource for health sciences’ students.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI linkThe BSc Biomedical Science (BMS) programme at De Montfort University (DMU, Leicester, UK) is accredited by the Institute of Biomedical Science (IBMS). Students enrolled within this programme acquire highly sought after skills related with human health sciences to work in: pathology departments in hospitals; research institutions; biotechnology and pharmaceutical industries; and the education sector to name a few. The degree recruits a large number of students with currently around 600 students enrolled on this programme at DMU. Despite pre-entry requirements of knowledge of subjects related to human biology, biology or chemistry, we have noted that first year students require basic support in STEM subjects (biology, chemistry and mathematics) in modules such as “Basic Microbiology”, “Basic Anatomy and Physiology” and “Chemistry for the Biosciences”. This support is especially necessary for students that come from non-traditional routes such as Business and Technology Education Council (BTEC) routes. Moreover, usually topics related with microbiology and human diseases are challenging for students, often causing stress impacting their overall performance and experience. A group of BMS academics at DMU in conjunction with universities in the European Union (EU; e.g. University of San Pablo CEU, Spain) have started to design, create and develop a series of e-learning resources or units in human biology and BMS for undergraduate students that study health sciences degrees in the EU. These units are being uploaded onto the DMU web server (http://parasitology.dmu.ac.uk/) and will be only accessible for students from participating universities during the first phase of this project (2017/18 course) in which comprehensive feedback will be collected. This web server space has three sections or modules (theoretical section, virtual laboratory and microscope) in which the new e-learning resources will be preliminary accommodated. These units will be interactive and easy to follow, and will cover basic human biology (e.g. cells, cell structure), human anatomy and physiology, histology and basic microbiology, which will be embedded in a theoretical module named DMU e-Biology within the above URL link. They will include formative assessments and case studies throughout each unit. In addition, a series of practical units are being developed which describe routine practical elements in any biomedical laboratory such as laboratory materials, pipetting, molecular techniques (e.g. PCR), cell culture (e.g. use of biological safety cabinet) and histological techniques (e.g. use of microtome, staining techniques). The development of this teaching and learning resource will cover a gap in the traditional teaching and learning methods that are currently used and provided in the participating universities. The DMU e-Biology will aid to our undergraduate students to gain knowledge in human biology and microbiology by promoting self-learning. We consider that the DMU e-Biology will help overcome spatiotemporal, equipment and resource barriers. Additionally, it may help student retention as currently about a 10% of our first year students fail to continue BMS at DMU. Finally, the creation of the DMU e-Biology will also provide support to the DMU Student Retention and Attainment Strategy 2016-2020 through the DMU Student Learning Hub, which is currently under development

SET-PP2A Complex as a New Therapeutic Target in KMT2A (MLL) Rearranged AML

KMT2A-rearranged (KMT2A-R) is an aggressive and chemo-refractory acute leukemia which mostly affects children. Transcriptomics-based characterization and chemical interrogation identified kinases as key drivers of survival and drug resistance in KMT2A-R leukemia. In contrast, the contribution and regulation of phosphatases is unknown. In this study we uncover the essential role and underlying mechanisms of SET, the endogenous inhibitor of Ser/Thr phosphatase PP2A, in KMT2A-R-leukemia. Investigation of SET expression in acute myeloid leukemia (AML) samples demonstrated that SET is overexpressed, and elevated expression of SET is correlated with poor prognosis and with the expression of MEIS and HOXA genes in AML patients. Silencing SET specifically abolished the clonogenic ability of KMT2A-R leukemic cells and the transcription of KMT2A targets genes HOXA9 and HOXA10. Subsequent mechanistic investigations showed that SET interacts with both KMT2A wild type and fusion proteins, and it is recruited to the HOXA10 promoter. Pharmacological inhibition of SET by FTY720 disrupted SET-PP2A interaction leading to cell cycle arrest and increased sensitivity to chemotherapy in KMT2A-R-leukemic models. Phospho-proteomic analyses revealed that FTY720 reduced the activity of kinases regulated by PP2A, including ERK1, GSK3β, AURB and PLK1 and led to suppression of MYC, supporting the hypothesis of a feedback loop among PP2A, AURB, PLK1, MYC, and SET. Our findings illustrate that SET is a novel player in KMT2A-R leukemia and they provide evidence that SET antagonism could serve as a novel strategy to treat this aggressive leukemia

SET-PP2A complex as a new therapeutic target in KMT2A (MLL) rearranged AML

KMT2A-rearranged (KMT2A-R) is an aggressive and chemo-refractory acute leukemia which mostly affects children. Transcriptomics-based characterization and chemical interrogation identified kinases as key drivers of survival and drug resistance in KMT2A-R leukemia. In contrast, the contribution and regulation of phosphatases is unknown. In this study we uncover the essential role and underlying mechanisms of SET, the endogenous inhibitor of Ser/Thr phosphatase PP2A, in KMT2A-R-leukemia. Investigation of SET expression in acute myeloid leukemia (AML) samples demonstrated that SET is overexpressed, and elevated expression of SET is correlated with poor prognosis and with the expression of MEIS and HOXA genes in AML patients. Silencing SET specifically abolished the clonogenic ability of KMT2A-R leukemic cells and the transcription of KMT2A targets genes HOXA9 and HOXA10. Subsequent mechanistic investigations showed that SET interacts with both KMT2A wild type and fusion proteins, and it is recruited to the HOXA10 promoter. Pharmacological inhibition of SET by FTY720 disrupted SET-PP2A interaction leading to cell cycle arrest and increased sensitivity to chemotherapy in KMT2A-R-leukemic models. Phospho-proteomic analyses revealed that FTY720 reduced the activity of kinases regulated by PP2A, including ERK1, GSK3β, AURB and PLK1 and led to suppression of MYC, supporting the hypothesis of a feedback loop among PP2A, AURB, PLK1, MYC, and SET. Our findings illustrate that SET is a novel player in KMT2A-R leukemia and they provide evidence that SET antagonism could serve as a novel strategy to treat this aggressive leukemia

A novel model of nephrotic syndrome results from a point mutation in Lama5 and is modified by genetic background

Nephrotic syndrome is characterised by severe proteinuria, hypoalbuminaemia, oedema and hyperlipidaemia. Genetic studies of nephrotic syndrome have led to the identification of proteins playing a crucial role in slit diaphragm signalling, regulation of actin cytoskeleton dynamics and cell-matrix interactions. The laminin α5 chain is essential for embryonic development and, in association with laminin β2 and laminin γ1, it is a major component of the glomerular basement membrane. Mutations in LAMA5 were recently identified in children with nephrotic syndrome. We have identified a novel missense mutation (E884G) in the uncharacterised L4a domain of LAMA5 where homozygous mice develop nephrotic syndrome with severe proteinuria with histological and ultrastructural changes in the glomerulus. The levels of LAMA5 are reduced in vivo and the assembly of the laminin 521 heterotrimer significantly reduced in vitro. Proteomic analysis of the glomerular extracellular fraction revealed changes in the matrix composition. Importantly, the genetic background had a significant effect on aspects of disease progression from proteinuria to changes in podocyte morphology. This novel model will provide insights into patho-mechanisms of nephrotic syndrome and pathways that influence the response to a dysfunctional glomerular basement membrane

The importance of clinician, patient and researcher collaborations in Alport syndrome

This is a post-peer-review, pre-copyedit version of an article published in Pediatric Nephrology. The final authenticated version is available online at: https://doi.org/10.1007/s00467-019-04241-7Alport syndrome (AS) is caused by mutations in the genes COL4A3, COL4A4 or COL4A5 and is characterised by progressive glomerular disease, sensorineural hearing loss and ocular defects. Occurring in less than 1:5000, AS is rare genetic disorder but still accounts for >1% of the prevalent population receiving renal replacement therapy. There is also increasing awareness about the risk of chronic kidney disease in individuals with heterozygous mutations in AS genes. The mainstay of current therapy is the use of angiotensin converting enzyme inhibitors and angiotensin receptor blockers, yet potential new therapies are now entering clinical trials. The 2017 International Workshop on Alport Syndrome in Glasgow was a preconference workshop ahead of the 50th anniversary meeting of the European Society for Pediatric Nephrology. It focussed on updates in clinical practice, genetics, basic science and also incorporated patient perspectives. More than 80 international experts including clinicians, geneticists, researchers from academia and industry, and patient representatives took part in panel discussions and breakout groups. This report summarises the workshop proceedings and the relevant contemporary literature. It highlights the unique clinician, patient and researcher collaborations achieved by regular engagement between the groups