Mass Spectrometric Identification of a Novel Factor XIIIa Cross-Linking Site in Fibrinogen.

Transglutaminases are a class of enzymes that catalyze the formation of a protein:protein cross-link between a lysine and a glutamine residue. These cross-links play important roles in diverse biological processes. Analysis of cross-linking sites in target proteins is required to elucidate their molecular action on target protein function and the molecular specificity of different transglutaminase isozymes. Mass-spectrometry using settings designed for linear peptide analysis and software designed for the analysis of disulfide bridges and chemical cross-links have previously been employed to identify transglutaminase cross-linking sites in proteins. As no control peptide with which to assess and improve the mass spectrometric analysis of TG cross-linked proteins was available, we developed a method for the enzymatic synthesis of a well-defined transglutaminase cross-linked peptide pair that mimics a predicted tryptic digestion product of collagen I. We then used this model peptide to determine optimal score thresholds for correct peptide identification from y- and b-ion series of fragments produced by collision-induced dissociation. We employed these settings in an analysis of fibrinogen cross-linked by the transglutaminase Factor XIIIa. This approach resulted in identification of a novel cross-linked peptide in the gamma subunit. We discuss the difference in behavior of ions derived from different cross-linked peptide sequences and the consequent demand for a more tailored mass spectrometry approach for cross-linked peptide identification compared to that routinely used for linear peptide analysis.

TGFß-1 Induced Cross-Linking of the Extracellular Matrix of Primary Human Dermal Fibroblasts.

Excessive cross-linking is a major factor in the resistance to the remodelling of the extracellular matrix (ECM) during fibrotic progression. The role of TGFß signalling in impairing ECM remodelling has been demonstrated in various fibrotic models. We hypothesised that increased ECM cross-linking by TGFß contributes to skin fibrosis in Systemic Sclerosis (SSc). Proteomics was used to identify cross-linking enzymes in the ECM of primary human dermal fibroblasts, and to compare their levels following treatment with TGFß-1. A significant upregulation and enrichment of lysyl-oxidase-like 1, 2 and 4 and transglutaminase 2 were found. Western blotting confirmed the upregulation of lysyl hydroxylase 2 in the ECM. Increased transglutaminase activity in TGFß-1 treated ECM was revealed from a cell-based assay. We employed a mass spectrometry-based method to identify alterations in the ECM cross-linking pattern caused by TGFß-1. Cross-linking sites were identified in collagens I and V, fibrinogen and fibronectin. One cross-linking site in fibrinogen alpha was found only in TGFß-treated samples. In conclusion, we have mapped novel cross-links between ECM proteins and demonstrated that activation of TGFß signalling in cultured dermal fibroblasts upregulates multiple cross-linking enzymes in the ECM.

Early-onset prostate cancer is associated with increased risks of disease progression and cancer-specific mortality.

OBJECTIVE: Prostate cancer (PCa) incidence has stabilized but not in patients at a young age. We assessed patient characteristics and disease progression in early-onset PCa. METHODS: A retrospective cohort of 28,039 newly diagnosed PCa patients aged ≥35 years was constructed using the Taiwan Cancer Registry in 2008-2016. Patients were categorized by age at diagnosis (≤54, 55-59, 60-69, 70-74, and ≥75 years). The clinical stage at diagnosis, Gleason score, prostate-specific antigen level at diagnosis, Charlson's comorbidity index, and primary and secondary treatments for PCa were included in the analysis. All-cause mortality and prostate cancer-specific mortality (PCSM) were reported. Hazard ratios (HRs) and 95% confidence intervals (CIs) estimating the risks of death and of receiving secondary cancer treatment were generated by Cox hazard models. RESULTS: In patients aged ≤54, 55-59, and 60-69 years, about 60% of them in each group were classified into the high-risk, very high-risk, or metastatic group. However, young patients ≤54 years had a higher risk of PCSM than patients aged 60-69 years (HR = 1.22; 95% CI = 1.10-1.49). This trend of an increased risk in PCSM remained for high-risk, very high-risk, or metastatic patients (HR = 1.24; 95% CI = 1.01-1.51), but not in low- or intermediate-risk patients. Besides, young patients diagnosed with high-risk diseases had the highest risk of receiving secondary cancer treatment within 180 days after completing primary treatment among all age groups (HR = 1.32; 95% CI = 1.07-1.63). CONCLUSIONS: PCa arising in young patients ≤54 years of age, especially those with a high risk or metastatic form, might be more aggressive than that in other age groups.

Modelling Pancreatic Neuroendocrine Cancer: From Bench Side to Clinic.

Pancreatic neuroendocrine tumours (pNETs) are a heterogeneous group of epithelial tumours with neuroendocrine differentiation. Although rare (incidence of <1 in 100,000), they are the second most common group of pancreatic neoplasms after pancreatic ductal adenocarcinoma (PDAC). pNET incidence is however on the rise and patient outcomes, although variable, have been linked with 5-year survival rates as low as 40%. Improvement of diagnostic and treatment modalities strongly relies on disease models that reconstruct the disease ex vivo. A key constraint in pNET research, however, is the absence of human pNET models that accurately capture the original tumour phenotype. In attempts to more closely mimic the disease in its native environment, three-dimensional culture models as well as in vivo models, such as genetically engineered mouse models (GEMMs), have been developed. Despite adding significant contributions to our understanding of more complex biological processes associated with the development and progression of pNETs, factors such as ethical considerations and low rates of clinical translatability limit their use. Furthermore, a role for the site-specific extracellular matrix (ECM) in disease development and progression has become clear. Advances in tissue engineering have enabled the use of tissue constructs that are designed to establish disease ex vivo within a close to native ECM that can recapitulate tumour-associated tissue remodelling. Yet, such advanced models for studying pNETs remain underdeveloped. This review summarises the most clinically relevant disease models of pNETs currently used, as well as future directions for improved modelling of the disease.

The effectiveness of long-acting injectable antipsychotics versus oral antipsychotics in the maintenance treatment of outpatients with chronic schizophrenia.

OBJECTIVE: To compare the psychiatric service utilization between patients who only received long-acting injectable antipsychotics (LAIAs) and those who only received oral antipsychotics (OAPs) in the maintenance treatment of chronic schizophrenia. METHODS: We constructed a cohort of chronic schizophrenia patients who underwent maintenance treatment from the Taiwan National Health Insurance Research Database in 2011 and followed these patients for 12 months. We included patients who had been diagnosed with schizophrenia for at least 3 years, were not hospitalized in 2011, and had received 1 year of maintenance treatment. Inverse probability of treatment weighting logistic, linear, and negative binomial regression models were used to estimate associated psychiatric services utilization and adjust for covariate imbalances between the LAIAs and OAPs groups. RESULTS: Among 40,194 patients, 948 (2.36%) received only LAIAs and 39,246 (97.64%) received only OAPs. Compared with those who received only OAPs, the sole LAIAs users were associated with a lower percentage of psychiatric hospitalization (8.4% and 5.8%, respectively; odds ratio: 0.63, p < .01), shorter lengths of hospitalization days (82.8 and 65.9, respectively; coefficient [b]: -16.87, p = .03), and fewer emergency room visits (2.3 and 1.8, respectively; b: -0.24, p < .01) per patient. CONCLUSIONS: Chronic schizophrenia patients who received only LAIs had a lower risk of disease relapse and a reduction in psychiatric service utilization than those receiving only OAPs.

The current standard of Foot and Ankle Fellowship in the UK.

BACKGROUND: The aim of this study was to evaluate the current standards of Fellowship training in Foot and Ankle Surgery Fellowship in the UK. METHODS: Thirteen UK post-FRCS (Tr&Orth) or equivalent Fellows completed a questionnaire detailing their outpatient, surgical, teaching and research experience, along with documenting their supervision and terms of employment. RESULTS: A Fellow attended a mean of 2.5 (0.5-4) clinics and 3.84 (2-7) theatre sessions per week. 62% of Fellows had independent clinics. The three largest sub-specialty areas experienced were forefoot surgery, mid or/hindfoot arthritis and deformity correction. 82% of Fellows had a regular MDT meeting. All were involved in both teaching and research, but only 64% had timetabled research sessions. All Fellows were satisfied with their experience and would recommended the Fellowship. CONCLUSIONS: The current standard of a post FRCS (Tr&Orth) Fellowship in Foot & Ankle surgery in the UK has been defined. Further improvement will require all Fellows to be involved in a regular MDT meetings, work in an independent clinic, have guaranteed timetabled research time and a ring fenced study leave budget.

Ageing in India: Financial hardship from health expenditures.

India's rapidly ageing population raises concerns about the burden of health care payments among older individuals who may have both limited income and greater health care needs. Using a nationally representative household survey, we investigate the association between age and financial hardship due to health expenditures. We find that both the probability of experiencing health problems and mean total out-of-pocket health expenditures increase with age. Second, the probability of households experiencing catastrophic health expenditures increases with each additional member aged 60 and above-33% of households with one 60+ member and 38% of households with 2 or more 60+ members experienced catastrophic health expenditures, compared to only 20% in households with all members under the age of 60 years. Lastly, we show that individuals aged 60 and above had a much higher probability of becoming impoverished as a result of health expenditures-the probability of impoverishment for 60+ individuals was 3 percentage points higher than for individuals under the age of 60. Overall, around 4.8% of the older population, representing 4.1 million people, fell into poverty. The results suggest that there is an urgent need for public investments in financial protection programs for older people in India.

Use of Patterned Collagen Coated Slides to Study Normal and Scleroderma Lung Fibroblast Migration.

Systemic sclerosis (SSc) is a spreading fibrotic disease affecting the skin and internal organs. We aimed to model pathogenic fibroblast migration in SSc in order to identify enhancing factors, measure the effect of migrating cells on underlying extracellular matrix (ECM) and test possible therapeutic inhibitors. Novel patterned collagen substrates were used to investigate alignment and migration of skin and lung fibroblasts from SSc patients and healthy controls. Normal lung but not skin fibroblasts consistently elongated and aligned with underlying collagen and migrated dependent on PDGF or serum. SSc lung fibroblasts remained growth factor dependent, did not migrate more rapidly and were less restricted to alignment of the collagen. Multiple collagen proline and lysine-modifying enzymes were identified in SSc but not control fibroblast extracellular matrix preparations, indicating differential levels of ECM modification by the diseased cells. Profiling of migrating cells revealed a possible SCF/c-Kit paracrine mechanism contributing to migration via a subpopulation of cells. Heparin, which binds ligands including PDGF and SCF, and imatininib which blocks downstream tyrosine kinase receptors, both inhibited lung fibroblast migration individually but showed synergy in SSc cells. Pathologic lung fibroblasts from SSc patients modify ECM during migration but remain growth factor dependent and sensitive to inhibitors.

Clinical utility of serum biomarkers in Duchenne muscular dystrophy.

Assessments of disease progression and response to therapies in Duchenne muscular dystrophy (DMD) patients remain challenging. Current DMD patient assessments include complex physical tests and invasive procedures such as muscle biopsies, which are not suitable for young children. Defining alternative, less invasive and objective outcome measures to assess disease progression and response to therapy will aid drug development and clinical trials in DMD. In this review we highlight advances in development of non-invasive blood circulating biomarkers as a means to assess disease progression and response to therapies in DMD.

Dysbindin-associated proteome in the p2 synaptosome fraction of mouse brain.

The gene DTNBP1 encodes the protein dysbindin and is among the most promising and highly investigated schizophrenia-risk genes. Accumulating evidence suggests that dysbindin plays an important role in the regulation of neuroplasticity. Dysbindin was reported to be a stable component of BLOC-1 complex in the cytosol. However, little is known about the endogenous dysbindin-containing complex in the brain synaptosome. In this study, we investigated the associated proteome of dysbindin in the P2 synaptosome fraction of mouse brain. Our data suggest that dysbindin has three isoforms associating with different complexes in the P2 fraction of mouse brain. To facilitate immunopurification, BAC transgenic mice expressing a tagged dysbindin were generated, and 47 putative dysbindin-associated proteins, including all components of BLOC-1, were identified by mass spectrometry in the dysbindin-containing complex purified from P2. The interactions of several selected candidates, including WDR11, FAM91A1, snapin, muted, pallidin, and two proteasome subunits, PSMD9 and PSMA4, were verified by coimmunoprecipitation. The specific proteasomal activity is significantly reduced in the P2 fraction of the brains of the dysbindin-null mutant (sandy) mice. Our data suggest that dysbindin is functionally interrelated to the ubiquitin-proteasome system and offer a molecular repertoire for future study of dysbindin functional networks in brain.

Stem-cell-based, tissue engineered tracheal replacement in a child: a 2-year follow-up study.

BACKGROUND: Stem-cell-based, tissue engineered transplants might offer new therapeutic options for patients, including children, with failing organs. The reported replacement of an adult airway using stem cells on a biological scaffold with good results at 6 months supports this view. We describe the case of a child who received a stem-cell-based tracheal replacement and report findings after 2 years of follow-up. METHODS: A 12-year-old boy was born with long-segment congenital tracheal stenosis and pulmonary sling. His airway had been maintained by metal stents, but, after failure, a cadaveric donor tracheal scaffold was decellularised. After a short course of granulocyte colony stimulating factor, bone marrow mesenchymal stem cells were retrieved preoperatively and seeded onto the scaffold, with patches of autologous epithelium. Topical human recombinant erythropoietin was applied to encourage angiogenesis, and transforming growth factor ß to support chondrogenesis. Intravenous human recombinant erythropoietin was continued postoperatively. Outcomes were survival, morbidity, endoscopic appearance, cytology and proteomics of brushings, and peripheral blood counts. FINDINGS: The graft revascularised within 1 week after surgery. A strong neutrophil response was noted locally for the first 8 weeks after surgery, which generated luminal DNA neutrophil extracellular traps. Cytological evidence of restoration of the epithelium was not evident until 1 year. The graft did not have biomechanical strength focally until 18 months, but the patient has not needed any medical intervention since then. 18 months after surgery, he had a normal chest CT scan and ventilation-perfusion scan and had grown 11 cm in height since the operation. At 2 years follow-up, he had a functional airway and had returned to school. INTERPRETATION: Follow-up of the first paediatric, stem-cell-based, tissue-engineered transplant shows potential for this technology but also highlights the need for further research. FUNDING: Great Ormond Street Hospital NHS Trust, The Royal Free Hampstead NHS Trust, University College Hospital NHS Foundation Trust, and Region of Tuscany.

CDP-diacylglycerol phospholipid synthesis in detergent-soluble, non-raft, membrane microdomains of the endoplasmic reticulum.

Phosphatidylinositol (PI) is essential for numerous cell functions and is generated by consecutive reactions catalyzed by CDP-diacylglycerol synthase (CDS) and PI synthase. In this study, we investigated the membrane organization of CDP-diacylglycerol synthesis. Separation of mildly disrupted A431 cell membranes on sucrose density gradients revealed cofractionation of CDS and PI synthase activities with cholesterol-poor, endoplasmic reticulum (ER) membranes and partial overlap with plasma membrane caveolae. Cofractionation of CDS activity with caveolae was also observed when low-buoyant density caveolin-enriched membranes were prepared using a carbonate-based method. However, immunoisolation studies determined that CDS activity localized to ER membrane fragments containing calnexin and type III inositol (1,4,5)-trisphosphate receptors but not to caveolae. Membrane fragmentation in neutral pH buffer established that CDP-diacylglycerol and PI syntheses were restricted to a subfraction of the calnexin-positive ER. In contrast to lipid rafts enriched for caveolin, cholesterol, and GM1 glycosphingolipids, the CDS-containing ER membranes were detergent soluble. In cell imaging studies, CDS and calnexin colocalized in microdomain-sized patches of the ER and also unexpectedly at the plasma membrane. These results demonstrate that key components of the PI pathway localize to nonraft, phospholipid-synthesizing microdomains of the ER that are also enriched for calnexin.

Detergent-free isolation and characterization of cholesterol-rich membrane domains from trans-Golgi network vesicles.

Cholesterol is an abundant lipid of the trans-Golgi network (TGN) and of certain endosomal membranes where cholesterol-rich microdomains are important in the organization and compartmentalization of vesicular trafficking. Here we describe the development of a rapid method to isolate a cholesterol-rich endomembrane fraction. We show that widely used subcellular fractionation techniques incompletely separate cholesterol-rich membranes, such as the TGN, from organelles, such as late endosomes and lysosomes. To address this issue, we devised a new subcellular fractionation scheme involving two rounds of velocity centrifugation, membrane sonication, and discontinuous sucrose density gradient centrifugation. This strategy resulted in the isolation of a cholesterol and GM1 glycosphingolipid-enriched membrane fraction that was completely cleared of plasma membrane, endoplasmic reticulum, and mitochondria. This buoyant fraction was enriched for the TGN and recycling endosome proteins Rab11 and syntaxin-6, and it was well resolved from cis-Golgi and early and late endosomal membranes. We demonstrate that this technique can give useful insights into the compartmentation of phosphoinositide synthesis, and it facilitates the isolation of cholesterol-rich membranes from a population of TGN-trafficking vesicles.

Relationship between phosphatidylinositol 4-phosphate synthesis, membrane organization, and lateral diffusion of PI4KIIalpha at the trans-Golgi network.

Type II phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha) is the dominant phosphatidylinositol kinase activity measured in mammalian cells and has important functions in intracellular vesicular trafficking. Recently PI4KIIalpha has been shown to have important roles in neuronal survival and tumorigenesis. This study focuses on the relationship between membrane cholesterol levels, phosphatidylinositol 4-phosphate (PI4P) synthesis, and PI4KIIalpha mobility. Enzyme kinetic measurements, sterol substitution studies, and membrane fragmentation analyses all revealed that cholesterol regulates PI4KIIalpha activity indirectly through effects on membrane structure. In particular, we found that cholesterol levels determined the distribution of PI4KIIalpha to biophysically distinct membrane domains. Imaging studies on cells expressing enhanced green fluorescent protein (eGFP)-tagged PI4KIIalpha demonstrated that cholesterol depletion resulted in morphological changes to the juxtanuclear membrane pool of the enzyme. Lateral membrane diffusion of eGFP-PI4KIIalpha was assessed by fluorescence recovery after photobleaching (FRAP) experiments, which revealed the existence of both mobile and immobile pools of the enzyme. Sterol depletion decreased the size of the mobile pool of PI4KIIalpha. Further measurements revealed that the reduction in the mobile fraction of PI4KIIalpha correlated with a loss of trans-Golgi network (TGN) membrane connectivity. We conclude that cholesterol modulates PI4P synthesis through effects on membrane organization and enzyme diffusion.

Differential effects of the phosphatidylinositol 4-kinases, PI4KIIα and PI4KIIIß, on Akt activation and apoptosis.

In this study, we investigated the role of PI4P synthesis by the phosphatidylinositol 4-kinases, PI4KIIα and PI4KIIIß, in epidermal growth factor (EGF)-stimulated phosphoinositide signaling and cell survival. In COS-7 cells, knockdown of either isozyme by RNA interference reduced basal levels of PI4P and PI(4,5)P(2), without affecting receptor activation. Only knockdown of PI4KIIα inhibited EGF-stimulated Akt phosphorylation, indicating that decreased PI(4,5)P(2) synthesis observed by loss of either isoform could not account for this PI4KIIα-specific effect. Phospholipase Cγ activation was also differentially affected by knockdown of either PI4K isozyme. Overexpression of kinase-inactive PI4KIIα, which induces defective endosomal trafficking without reducing PI(4,5)P(2) levels, also reduced Akt activation. Furthermore, PI4KIIα knockdown profoundly inhibited cell proliferation and induced apoptosis as evidenced by the cleavage of caspase-3 and its substrate poly(ADP-ribose) polymerase. However, in MDA-MB-231 breast cancer cells, apoptosis was observed subsequent to knockdown of either PI4KIIα or PI4KIIIß and this correlated with enhanced proapoptotic Akt phosphorylation. The differential effects of phosphatidylinositol 4-kinase knockdown in the two cell lines lead to the conclusion that phosphoinositide turnover is inhibited through PI4P substrate depletion, whereas impaired antiapoptotic Akt signaling is an indirect consequence of dysfunctional endosomal trafficking.

Identification of Mac-2-binding protein as a putative marker of neuroendocrine tumors from the analysis of cell line secretomes.

Neuroendocrine tumors (NETs) can arise from a variety of organs. They can vary widely in clinical behavior; consequently, optimizing their treatment plan can be problematic. NETs display diverse tumor biology; however, most secrete peptides such as chromogranin A into the circulation, consistent with their neuroendocrine origin. In this study, we sought to identify other potential markers for NETs by analyzing the secreted proteomes of three neuroendocrine cell lines. BON-1, NCI-H727, and SHP-77 cells were grown in serum-free media, and the secreted proteins were separated by SDS-PAGE and identified by LC-MS/MS. We identified 205 proteins of which 61 were secreted by two or more of the cell lines and 19 were secreted by all three lines. Mac-2-binding protein (Mac-2BP) was found to be secreted by all three cell lines, and this was confirmed by Western blotting. Immunohistochemical analysis found 29 of 33 NET cases from different primary sites to be positive for Mac-2BP. Serum Mac-2BP was significantly elevated in NET patients compared with healthy controls (p < 0.001). This study demonstrated that analysis of the secreted proteomes of neuroendocrine cell lines can identify potential biomarkers for NET. Initial assessment showed that serum Mac-2BP is significantly elevated in patients with NET and is expressed by the majority of NET tissues.

The role of general practitioners in managing and treating hepatitis C.

General practitioners hold the key to expanding access to treatment.

Loss of phosphatidylinositol 4-kinase 2alpha activity causes late onset degeneration of spinal cord axons.

Phosphoinositide (PI) lipids are intracellular membrane signaling intermediates and effectors produced by localized PI kinase and phosphatase activities. Although many signaling roles of PI kinases have been identified in cultured cell lines, transgenic animal studies have produced unexpected insight into the in vivo functions of specific PI 3- and 5-kinases, but no mammalian PI 4-kinase (PI4K) knockout has previously been reported. Prior studies using cultured cells implicated the PI4K2alpha isozyme in diverse functions, including receptor signaling, ion channel regulation, endosomal trafficking, and regulated secretion. We now show that despite these important functions, mice lacking PI4K2alpha kinase activity initially appear normal. However, adult Pi4k2a(GT/GT) animals develop a progressive neurological disease characterized by tremor, limb weakness, urinary incontinence, and premature mortality. Histological analysis of aged Pi4k2a(GT/GT) animals revealed lipofuscin-like deposition and gliosis in the cerebellum, and loss of Purkinje cells. Peripheral nerves are essentially normal, but massive axonal degeneration was found in the spinal cord in both ascending and descending tracts. These results reveal a previously undescribed role for aberrant PI signaling in neurological disease that resembles autosomal recessive hereditary spastic paraplegia.

Papilloedema secondary to venous sinus thrombosis following glomus jugulare tumour surgery.

OBJECTIVE: We present a case of a patient who had undergone embolisation and resection of a left glomus jugulare tumour, who presented three weeks post-operatively with magnetic resonance venography confirmed symptomatic cerebral venous sinus thrombosis. METHOD: We present a case report and a review of the world literature concerning glomus jugulare tumours and cerebral venous sinus thrombosis. CASE REPORT: A 42-year-old man presented with blurred vision and reduced Snellen visual acuity just three weeks after glomus jugulare tumour surgery. Fundoscopy revealed bilateral haemorrhagic optic disc oedema. Urgent magnetic resonance venography confirmed a left lateral venous sinus thrombosis. It was felt that this was responsible for inadequate cerebrospinal fluid drainage, resulting in raised intracranial pressure and papilloedema. CONCLUSION: To the authors' knowledge, this is the first account of a magnetic resonance venography confirmed venous sinus thrombosis and secondary papilloedema following glomus jugulare tumour surgery. Patients undergoing surgery involving resection or manipulation of the internal jugular vein may be at higher risk of developing thrombosis superior to the level of resection, and magnetic resonance venography ought to be considered an important diagnostic adjunct.

Quantification of multiple phosphatidylinositol 4-kinase isozyme activities in cell extracts.

A wide spectrum of intracellular signaling events mediated by up to seven different phosphorylated forms of phosphatidylinositol (PtdIns) occurs in all eukaryotic cells. The activities of multiple, nondegenerate PI kinases and phosphatases control these signaling events. The PI 4-kinase isozymes account for the major PI kinase activity in many different cell types, and the activity of each isozyme is differentially regulated. The ability to measure and distinguish the activity of individual enzymes is therefore important and forms the subject of the methods in this chapter. We describe the use and application of a versatile radiometric assay to measuring PI 4-kinase activity in a variety of biochemical contexts, from purified enzymes to membrane preparations and permeabilized cells. Until a suitable nonradioactive reagent becomes available, this assay is destined to remain the most widely used method.