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The article discusses bleeding mechanisms focusing on coagulation disorders. The case of an adult male experiencing pain and swelling is reviewed revealing a diagnosis of thromboplastin inhibitory factor. The case offers a perspective on the three main mechanisms involved in the control of bleeding but the discussion will be limited to coagulation or fibrin formation. Types of circulating anticoagulants which are encountered in clinical practice are identified. The principle of the cascade theory of the mechanism of coagulation is discussed. There are four screening tests which effectively can screen this entire mechanism including the one-stage prothrombin time.

Aims: The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter region is essential in evaluating the prognosis and predicting the drug response in patients with glioblastoma. In this study, we evaluated the utility of using nanopore long-read sequencing as a method for assessing methylation levels throughout the MGMT CpG-island, compared its performance to established techniques and demonstrated its clinical applicability., Methods: We analysed 165 samples from CNS tumours, focusing on the MGMT CpG-island using nanopore sequencing. Oxford Nanopore Technologies (ONT) MinION and PromethION flow cells were employed for single sample or barcoded assays, guided by a CRISPR/Cas9 protocol, adaptive sampling or as part of a whole genome sequencing assay. Methylation data obtained through nanopore sequencing were compared to results obtained via pyrosequencing and methylation bead arrays. Hierarchical clustering was applied to nanopore sequencing data for patient stratification., Results: Nanopore sequencing displayed a strong correlation (R 2  = 0.91) with pyrosequencing results for the four CpGs of MGMT analysed by both methods. The MGMT-STP27 algorithm's classification was effectively reproduced using nanopore data. Unsupervised hierarchical clustering revealed distinct patterns in methylated and unmethylated samples, providing comparable survival prediction capabilities. Nanopore sequencing yielded high-confidence results in a rapid timeframe, typically within hours of sequencing, and extended the analysis to all 98 CpGs of the MGMT CpG-island., Conclusions: This study presents nanopore sequencing as a valid and efficient method for determining MGMT promotor methylation status. It offers a comprehensive view of the MGMT promoter methylation landscape, which enables the identification of potentially clinically relevant subgroups of patients. Further exploration of the clinical implications of patient stratification using nanopore sequencing of MGMT is warranted., (© 2024 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

Breast cancer cells that have undergone partial epithelial-mesenchymal transition (EMT) are believed to be more invasive than cells that have completed EMT. To study metabolic reprogramming in different mesenchymal states, we analyzed protein expression following EMT in the breast epithelial cell model D492 with single-shot LFQ supported by a SILAC proteomics approach. The D492 EMT cell model contains three cell lines: the epithelial D492 cells, the mesenchymal D492M cells, and a partial mesenchymal, tumorigenic variant of D492 that overexpresses the oncogene HER2. The analysis classified the D492 and D492M cells as basal-like and D492HER2 as claudin-low. Comparative analysis of D492 and D492M to tumorigenic D492HER2 differentiated metabolic markers of migration from those of invasion. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) was one of the top dysregulated enzymes in D492HER2. Gene expression analysis of the cancer genome atlas showed that GFPT2 expression was a characteristic of claudin-low breast cancer. siRNA-mediated knockdown of GFPT2 influenced the EMT marker vimentin and both cell growth and invasion in vitro and was accompanied by lowered metabolic flux through the hexosamine biosynthesis pathway (HBP). Knockdown of GFPT2 decreased cystathionine and sulfide:quinone oxidoreductase (SQOR) in the transsulfuration pathway that regulates H 2 S production and mitochondrial homeostasis. Moreover, GFPT2 was within the regulation network of insulin and EGF, and its expression was regulated by reduced glutathione (GSH) and suppressed by the oxidative stress regulator GSK3-β. Our results demonstrate that GFPT2 controls growth and invasion in the D492 EMT model, is a marker for oxidative stress, and associated with poor prognosis in claudin-low breast cancer., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Brain tumor surgery must balance the benefit of maximal resection against the risk of inflicting severe damage. The impact of increased resection is diagnosis-specific. However, the precise diagnosis is typically uncertain at surgery due to limitations of imaging and intraoperative histomorphological methods. Novel and accurate strategies for brain tumor classification are necessary to support personalized intraoperative neurosurgical treatment decisions. Here, we describe a fast and cost-efficient workflow for intraoperative classification of brain tumors based on DNA methylation profiles generated by low coverage nanopore sequencing and machine learning algorithms., Methods: We evaluated 6 independent cohorts containing 105 patients, including 50 pediatric and 55 adult patients. Ultra-low coverage whole-genome sequencing was performed on nanopore flow cells. Data were analyzed using copy number variation and ad hoc random forest classifier for the genome-wide methylation-based classification of the tumor., Results: Concordant classification was obtained between nanopore DNA methylation analysis and a full neuropathological evaluation in 93 of 105 (89%) cases. The analysis demonstrated correct diagnosis in 6/6 cases where frozen section evaluation was inconclusive. Results could be returned to the operating room at a median of 97 min (range 91-161 min). Precise classification of the tumor entity and subtype would have supported modification of the surgical strategy in 12 out of 20 patients evaluated intraoperatively., Conclusion: Intraoperative nanopore sequencing combined with machine learning diagnostics was robust, sensitive, and rapid. This strategy allowed DNA methylation-based classification of the tumor to be returned to the surgeon within a timeframe that supports intraoperative decision making., (© The Author(s) 2021. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Epithelial-to-mesenchymal transition (EMT) is fundamental to both normal tissue development and cancer progression. We hypothesized that EMT plasticity defines a range of metabolic phenotypes and that individual breast epithelial metabolic phenotypes are likely to fall within this phenotypic landscape. To determine EMT metabolic phenotypes, the metabolism of EMT was described within genome-scale metabolic models (GSMMs) using either transcriptomic or proteomic data from the breast epithelial EMT cell culture model D492. The ability of the different data types to describe breast epithelial metabolism was assessed using constraint-based modeling which was subsequently verified using 13 C isotope tracer analysis. The application of proteomic data to GSMMs provided relatively higher accuracy in flux predictions compared to the transcriptomic data. Furthermore, the proteomic GSMMs predicted altered cholesterol metabolism and increased dependency on argininosuccinate lyase (ASL) following EMT which were confirmed in vitro using drug assays and siRNA knockdown experiments. The successful verification of the proteomic GSMMs afforded iBreast2886, a breast GSMM that encompasses the metabolic plasticity of EMT as defined by the D492 EMT cell culture model. Analysis of breast tumor proteomic data using iBreast2886 identified vulnerabilities within arginine metabolism that allowed prognostic discrimination of breast cancer patients on a subtype-specific level. Taken together, we demonstrate that the metabolic reconstruction iBreast2886 formalizes the metabolism of breast epithelial cell development and can be utilized as a tool for the functional interpretation of high throughput clinical data., (© 2021. The Author(s).)

Epithelial-to-mesenchymal transition (EMT) is a fundamental developmental process with strong implications in cancer progression. Understanding the metabolic alterations associated with EMT may open new avenues of treatment and prevention. Here we used 13 C carbon analogs of glucose and glutamine to examine differences in their utilization within central carbon and lipid metabolism following EMT in breast epithelial cell lines. We found that there are inherent differences in metabolic profiles before and after EMT. We observed EMT-dependent re-routing of the TCA-cycle, characterized by increased mitochondrial IDH2-mediated reductive carboxylation of glutamine to lipid biosynthesis with a concomitant lowering of glycolytic rates and glutamine-dependent glutathione (GSH) generation. Using weighted correlation network analysis, we identified cancer drugs whose efficacy against the NCI-60 Human Tumor Cell Line panel is significantly associated with GSH abundance and confirmed these in vitro . We report that EMT-linked alterations in GSH synthesis modulate the sensitivity of breast epithelial cells to mTOR inhibitors. IMPLICATIONS: EMT in breast cells causes an increased demand for glutamine for fatty acid biosynthesis, altering its contribution to glutathione biosynthesis, which sensitizes the cells to mTOR inhibitors., (©2021 American Association for Cancer Research.)

The lipid-enveloped influenza C virus contains a single surface glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, that mediates receptor binding, receptor destruction, and membrane fusion at the low pH of the endosome. Here we apply electron cryotomography and subtomogram averaging to describe the structural basis for hexagonal lattice formation by HEF on the viral surface. The conformation of the glycoprotein in situ is distinct from the structure of the isolated trimeric ectodomain, showing that a splaying of the membrane distal domains is required to mediate contacts that form the lattice. The splaying of these domains is also coupled to changes in the structure of the stem region which is involved in membrane fusion, thereby linking HEF's membrane fusion conformation with its assembly on the virus surface. The glycoprotein lattice can form independent of other virion components but we show a major role for the matrix layer in particle formation.

Background: Understanding how dysregulation in lipid metabolism relates to the severity of Alzheimer's disease (AD) pathology might be critical in developing effective treatments., Objective: To identify lipid species in cerebrospinal fluid (CSF) associated with signature AD pathology and to explore their relationships with measures reflecting AD-related processes (neurodegeneration, inflammation, deficits in verbal episodic memory) among subjects at the pre- and early symptomatic stages of dementia., Methods: A total of 60 subjects that had been referred to an Icelandic memory clinic cohort were classified as having CSF AD (n = 34) or non-AD (n = 26) pathology profiles. Untargeted CSF lipidomic analysis was performed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) for the detection of mass-to-charge ratio (m/z) features. CSF proteins reflecting neurodegeneration (neurofilament light [NFL]) and inflammation (chitinase-3-like protein 1 [YKL-40], S100 calcium-binding protein B [S100B], glial fibrillary acidic protein [GFAP]) were also measured. Rey Auditory Verbal Learning (RAVLT) and Story tests were used for the assessment of verbal episodic memory., Results: Eight out of 1008 features were identified as best distinguishing between the CSF profile groups. Of those, only the annotation of the m/z feature assigned to lipid species C18 ceramide was confirmed with a high confidence. Multiple regression analyses, adjusted for age, gender, and education, demonstrated significant associations of CSF core AD markers (Aβ42: st.β= -0.36, p = 0.007; T-tau: st.β= 0.41, p = 0.005) and inflammatory marker S100B (st.β= 0.51, p = 0.001) with C18 ceramide levels., Conclusion: Higher levels of C18 ceramide associated with increased AD pathology and inflammation, suggesting its potential value as a therapeutic target.

Background: Azithromycin (Azm) is a macrolide recognized for its disease-modifying effects and reduction in exacerbation of chronic airway diseases. It is not clear whether the beneficial effects of Azm are due to its anti-microbial activity or other pharmacological actions. We have shown that Azm affects the integrity of the bronchial epithelial barrier measured by increased transepithelial electrical resistance. To better understand these effects of Azm on bronchial epithelia we have investigated global changes in gene expression., Methods: VA10 bronchial epithelial cells were treated with Azm and cultivated in air-liquid interface conditions for up to 22 days. RNA was isolated at days 4, 10 and 22 and analyzed using high-throughput RNA sequencing. qPCR and immunostaining were used to confirm key findings from bioinformatic analyses. Detailed assessment of cellular changes was done using microscopy, followed by characterization of the lipidomic profiles of the multivesicular bodies present., Results: Bioinformatic analysis revealed that after 10 days of treatment genes encoding effectors of sterol and cholesterol metabolism were prominent. Interestingly, expression of genes associated with epidermal barrier differentiation, KRT1, CRNN, SPINK5 and DSG1, increased significantly at day 22. Together with immunostaining, these results suggest an epidermal differentiation pattern. We also found that Azm induced the formation of multivesicular and lamellar bodies in two different airway epithelial cell lines. Lipidomic analysis revealed that Azm was entrapped in multivesicular bodies linked to different types of lipids, most notably palmitate and stearate. Furthermore, targeted analysis of lipid species showed accumulation of phosphatidylcholines, as well as ceramide derivatives., Conclusions: Taken together, we demonstrate how Azm might confer its barrier enhancing effects, via activation of epidermal characteristics and changes to intracellular lipid dynamics. These effects of Azm could explain the unexpected clinical benefit observed during Azm-treatment of patients with various lung diseases affecting barrier function.

Epithelial to mesenchymal transition (EMT) is a developmental event characterized by phenotypic switching from a polarized epithelial phenotype to an unpolarized mesenchymal phenotype. Changes to plasma membrane function accompany EMT yet the differences in lipid composition of cells that have undergone EMT are relatively unexplored. To address this the lipidome of two cell models of EMT in breast epithelial tissue, D492 and HMLE, were analyzed by untargeted LC-MS. Detected masses were identified and their abundance was compared through multivariate statistical analysis. Considerable concordance was observed in eight lipid components between epithelial and mesenchymal cells in both cell models. Specifically, an increase in phosphatidylcholine and triacylglycerol were found to accompany EMT while phosphatidylcholine- and phosphatidylethanolamine plasmalogens, as well as diacylglycerols decreased. The most abundant fatty acid lengths were C16 and C18 but mesenchymal cells had on average shorter and more unsaturated fatty acids. The results are consistent with enhanced cell mobility post EMT and reflect a consequence of oxidative stress pre- and post EMT in breast epithelial tissue., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Entry of enveloped viruses relies on insertion of hydrophobic residues of the viral fusion protein into the host cell membrane. However, the intermediate conformations during fusion remain unknown. Here, we address the fusion mechanism of Rift Valley fever virus. We determine the crystal structure of the Gn glycoprotein and fit it with the Gc fusion protein into cryo-electron microscopy reconstructions of the virion. Our analysis reveals how the Gn shields the hydrophobic fusion loops of the Gc, preventing premature fusion. Electron cryotomography of virions interacting with membranes under acidic conditions reveals how the fusogenic Gc is activated upon removal of the Gn shield. Repositioning of the Gn allows extension of Gc and insertion of fusion loops in the outer leaflet of the target membrane. These data show early structural transitions that enveloped viruses undergo during host cell entry and indicate that analogous shielding mechanisms are utilized across diverse virus families.

Epithelial to mesenchymal transition (EMT) has implications in tumor progression and metastasis. Metabolic alterations have been described in cancer development but studies focused on the metabolic re-wiring that takes place during EMT are still limited. We performed metabolomics profiling of a breast epithelial cell line and its EMT derived mesenchymal phenotype to create genome-scale metabolic models descriptive of both cell lines. Glycolysis and OXPHOS were higher in the epithelial phenotype while amino acid anaplerosis and fatty acid oxidation fueled the mesenchymal phenotype. Through comparative bioinformatics analysis, PPAR-γ1, PPAR- γ2 and AP-1 were found to be the most influential transcription factors associated with metabolic re-wiring. In silico gene essentiality analysis predicts that the LAT1 neutral amino acid transporter is essential for mesenchymal cell survival. Our results define metabolic traits that distinguish an EMT derived mesenchymal cell line from its epithelial progenitor and may have implications in cancer progression and metastasis. Furthermore, the tools presented here can aid in identifying critical metabolic nodes that may serve as therapeutic targets aiming to prevent EMT and inhibit metastatic dissemination., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

Protein tyrosine phosphatase 1B (PTP1B) is a well-known inhibitor of insulin signaling pathways and inhibitors against PTP1B are being developed as promising drug candidates for treatment of obesity. PTP1B has also been linked to breast cancer both as a tumor suppressor and as an oncogene. Furthermore, PTP1B has been shown to be a regulator of cell adhesion and migration in normal and cancer cells. In this study, we analyzed the PTP1B expression in normal breast tissue, primary breast cells and the breast epithelial cell line D492. In normal breast tissue and primary breast cells, PTP1B is widely expressed in both epithelial and stromal cells, with highest expression in myoepithelial cells and fibroblasts. PTP1B is widely expressed in branching structures generated by D492 when cultured in 3D reconstituted basement membrane (3D rBM). Inhibition of PTP1B in D492 and another mammary epithelial cell line HMLE resulted in reduced cell proliferation and induction of anoikis. These changes were seen when cells were cultured both in monolayer and in 3D rBM. PTP1B inhibition affected cell attachment, expression of cell adhesion proteins and actin polymerization. Moreover, epithelial to mesenchymal transition (EMT) sensitized cells to PTP1B inhibition. A mesenchymal sublines of D492 and HMLE (D492M and HMLEmes) were more sensitive to PTP1B inhibition than D492 and HMLE. Reversion of D492M to an epithelial state using miR-200c-141 restored resistance to detachment induced by PTP1B inhibition. In conclusion, we have shown that PTP1B is widely expressed in the human breast gland with highest expression in myoepithelial cells and fibroblasts. Inhibition of PTP1B in D492 and HMLE affects cell-cell adhesion and induces anoikis-like effects. Finally, cells with an EMT phenotype are more sensitive to PTP1B inhibitors making PTP1B a potential candidate for further studies as a target for drug development in cancer involving the EMT phenotype.

An emergent viral pathogen termed severe fever with thrombocytopenia syndrome virus (SFTSV) is responsible for thousands of clinical cases and associated fatalities in China, Japan, and South Korea. Akin to other phleboviruses, SFTSV relies on a viral glycoprotein, Gc, to catalyze the merger of endosomal host and viral membranes during cell entry. Here, we describe the postfusion structure of SFTSV Gc, revealing that the molecular transformations the phleboviral Gc undergoes upon host cell entry are conserved with otherwise unrelated alpha- and flaviviruses. By comparison of SFTSV Gc with that of the prefusion structure of the related Rift Valley fever virus, we show that these changes involve refolding of the protein into a trimeric state. Reverse genetics and rescue of site-directed histidine mutants enabled localization of histidines likely to be important for triggering this pH-dependent process. These data provide structural and functional evidence that the mechanism of phlebovirus-host cell fusion is conserved among genetically and patho-physiologically distinct viral pathogens.

Epithelial to mesenchymal transition (EMT) is an important event during development and cancer metastasis. There is limited understanding of the metabolic alterations that give rise to and take place during EMT. Dysregulation of signalling pathways that impact metabolism, including epidermal growth factor receptor (EGFR), are however a hallmark of EMT and metastasis. In this study, we report the investigation into EGFR signalling and metabolic crosstalk of EMT through constraint-based modelling and analysis of the breast epithelial EMT cell model D492 and its mesenchymal counterpart D492M. We built an EGFR signalling network for EMT based on stoichiometric coefficients and constrained the network with gene expression data to build epithelial (EGFR_E) and mesenchymal (EGFR_M) networks. Metabolic alterations arising from differential expression of EGFR genes was derived from a literature review of AKT regulated metabolic genes. Signaling flux differences between EGFR_E and EGFR_M models subsequently allowed metabolism in D492 and D492M cells to be assessed. Higher flux within AKT pathway in the D492 cells compared to D492M suggested higher glycolytic activity in D492 that we confirmed experimentally through measurements of glucose uptake and lactate secretion rates. The signaling genes from the AKT, RAS/MAPK and CaM pathways were predicted to revert D492M to D492 phenotype. Follow-up analysis of EGFR signaling metabolic crosstalk in three additional breast epithelial cell lines highlighted variability in in vitro cell models of EMT. This study shows that the metabolic phenotype may be predicted by in silico analyses of gene expression data of EGFR signaling genes, but this phenomenon is cell-specific and does not follow a simple trend.

Many phleboviruses (family Bunyaviridae) are emerging as medically important viruses. These viruses enter target cells by endocytosis and low pH-dependent membrane fusion in late endosomes. However, the necessary and sufficient factors for fusion have not been fully characterized. We have studied the minimal fusion requirements of a prototypic phlebovirus, Uukuniemi virus, in an in vitro virus-liposome assay. We show that efficient lipid mixing between viral and liposome membranes requires close to physiological temperatures and phospholipids with negatively charged headgroups, such as the late endosomal phospholipid bis(monoacylglycero)phosphate. We further demonstrate that bis(monoacylglycero)phosphate increases Uukuniemi virus fusion beyond the lipid mixing stage. By using electron cryotomography of viral particles in the presence or absence of liposomes, we observed that the conformation of phlebovirus glycoprotein capsomers changes from the native conformation toward a more elongated conformation at a fusion permissive pH. Our results suggest a rationale for phlebovirus entry in late endosomes., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Culture of cells using various microfluidic devices is becoming more common within experimental cell biology. At the same time, a technological radiation of microfluidic cell culture device designs is currently in progress. Ultimately, the utility of microfluidic cell culture will be determined by its capacity to permit new insights into cellular function. Especially insights that would otherwise be difficult or impossible to obtain with macroscopic cell culture in traditional polystyrene dishes, flasks or well-plates. Many decades of heuristic optimization have gone into perfecting conventional cell culture devices and protocols. In comparison, even for the most commonly used microfluidic cell culture devices, such as those fabricated from polydimethylsiloxane (PDMS), collective understanding of the differences in cellular behavior between microfluidic and macroscopic culture is still developing. Moving in vitro culture from macroscopic culture to PDMS based devices can come with unforeseen challenges. Changes in device material, surface coating, cell number per unit surface area or per unit media volume may all affect the outcome of otherwise standard protocols. In this review, we outline some of the advantages and challenges that may accompany a transition from macroscopic to microfluidic cell culture. We focus on decisive factors that distinguish macroscopic from microfluidic cell culture to encourage a reconsideration of how macroscopic cell culture principles might apply to microfluidic cell culture., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Glycan analysis of virion-derived glycoproteins is challenging due to the difficulties in glycoprotein isolation and low sample abundance. Here, we describe how ion mobility mass spectrometry can be used to obtain spectra from virion samples. We also describe how negative ion fragmentation of glycans can be used to probe structural features of virion glycans.

Uukuniemi virus (UUKV) is a model system for investigating the genus Phlebovirus of the Bunyaviridae. We report the UUKV glycome, revealing differential processing of the Gn and Gc virion glycoproteins. Both glycoproteins display poly-N-acetyllactosamines, consistent with virion assembly in the medial Golgi apparatus, whereas oligomannose-type glycans required for DC-SIGN-dependent cellular attachment are predominant on Gc. Local virion structure and the route of viral egress from the cell leave a functional imprint on the phleboviral glycome., (Copyright © 2014 Crispin et al.)

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.