Cellular cohesiveness in benign and malignant thyroid follicular tumours varies significantly,but the difference is not useful in diagnosis of individual cases

T. Rozkos, A. Ryska, J. Cap, F. Sobande and J. Laco
Cellular cohesiveness in benign and malignant thyroid follicular tumours varies significantly, but the difference is not useful in diagnosis of individual cases Introduction: The aim of our study was to search for new, readily available and statistically reliable cytological markers for differentiating benign and malignant follicular thyroid neoplasms pre‐operatively. Methods: Cohesiveness of tumour cells in cytology slides from a series of 58 follicular tumours diagnosed between 1998 and 2004 inclusive was studied, including 48 follicular adenomas, and eight minimally invasive and two widely invasive follicular carcinomas. Photomicrographs of the cytology slides were taken and the digital images were analysed using computer image analysis software. We evaluated the relative proportions of cells arranged in groups of various sizes. The cohesiveness of the cells in cytological smears was then correlated with the immunohistochemical expression of E‐cadherin in corresponding histological slides. Results: Cases from 15 men (26%) and 43 women (74%) with a mean age of 50 years (range, 19–79) were analysed. In follicular adenomas and carcinomas, respectively, isolated cells were seen in 16.8% and 24.7% (P = 0.028), groups of two to five cells in 9.7% and 11.5% (P = 0.145) and groups of more than five cells in 73.5% and 63.8% (P = 0.041). The mean cell count in groups with more than five cells was 46.5 and 27.0 in adenomas and carcinomas, respectively (P < 0.001). Cell cohesiveness, either as percentage of cells in groups of more than five (R² = 0.026) or as mean cell count per group of more than five (R² = 0.005), was not found to be dependent on the expression of E‐cadherin. Using a threshold of 13% isolated tumour cells in cytological smears, follicular adenomas and carcinomas could be distinguished with 90% sensitivity and 41% specificity. Conclusions: Although we demonstrated a statistically significant difference in cell cohesion between follicular adenomas and carcinomas, these could not be distinguished in the clinical setting by evaluation of the percentage of isolated cells in cytological smears because the specificity was too low. The absence of correlation of cellular cohesiveness with E‐cadherin expression indicates that other factors are probably responsible for the loss of cohesiveness observed in follicular thyroid malignancy.     

The Arabidopsis RING-type E3 ligase XBAT32 mediates the proteasomal degradation of the ethylene biosynthetic enzyme, 1-aminocyclopropane-1-carboxylate synthase 7

E3 ubiquitin ligases select specific proteins for ubiquitin conjugation, and the modified proteins are commonly degraded through the 26S proteasome. XBAT32 is a RING-type E3 ligase involved in maintaining appropriate levels of ethylene. Previous work has suggested that XBAT32 modulates ethylene production by ubiquitinating two ethylene biosynthesis enzymes, ACS4 (type-II isoform) and ACS7 (type-III isoform). In Arabidopsis, conserved sequences within the C-terminal tail of type-I and -II 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) isoforms influence ubiquitin-dependent proteolysis. ACS7, the sole Arabidopsis type-III ACS, contains a truncated C-terminal tail that lacks all known regulatory sequences, which suggests that this isoform may not be subject to ubiquitin-mediated proteasomal degradation. Here we demonstrate in planta that ACS7 is turned over in a 26S proteasome-dependent manner and that degradation of ACS7 requires the E3 ligase XBAT32. Furthermore, the ethylene-related phenotypes that result from overexpression of ACS7 in wild-type plants are greatly exaggerated in xbat32-1, suggesting that XBAT32 is required to attenuate the effect of overexpression of ACS7. This observation is consistent with a role for XBAT32 in the ubiquitin-mediated degradation of ACS7. The dark-grown phenotype of xbat32-1 seedlings overexpressing ACS7 can be effectively rescued by aminoethoxyvinylglycine, an inhibitor of ACS activity. The degradation rate of ACS4 is also significantly slower in the absence of XBAT32, further implicating XBAT32 in the ubiquitin-mediated degradation of ACS4. Altogether, these results demonstrate that XBAT32 targets ethylene biosynthetic enzymes for proteasomal degradation to maintain appropriate levels of hormone production.     

Solution structure of the E3 ligase HOIL-1 Ubl domain

The E3 ligases HOIL‐1 and parkin are each comprised of an N‐terminal ubiquitin‐like (Ubl) domain followed by a zinc‐binding region and C‐terminal RING–In‐between‐RING–RING domains. These two proteins, involved in the ubiquitin‐mediated degradation pathway, are the only two known E3 ligases to share this type of multidomain architecture. Further, the Ubl domain of both HOIL‐1 and parkin has been shown to interact with the S5a subunit of the 26S proteasome. The solution structure of the HOIL‐1 Ubl domain was solved using NMR spectroscopy to compare it with that of parkin to determine the structural elements responsible for S5a intermolecular interactions. The final ensemble of 20 structures had a β‐grasp Ubl‐fold with an overall backbone RMSD of 0.59 ± 0.10 Å in the structured regions between I55 and L131. HOIL‐1 had a unique extension of both β1 and β2 sheets compared to parkin and other Ubl domains, a result of a four‐residue insertion in this region. A similar 15‐residue hydrophobic core in the HOIL‐1 Ubl domain resulted in a comparable stability to the parkin Ubl, but significantly lower than that observed for ubiquitin. A comparison with parkin and other Ubl domains indicates that HOIL‐1 likely uses a conserved hydrophobic patch (W58, V102, Y127, Y129) found on the β1 face, the β3–β4 loop and β5, as well as a C‐terminal basic residue (R134) to recruit the S5a subunit as part of the ubiquitin‐mediated proteolysis pathway.     

Fibrinogen alpha chain O-glycopeptides as possible markers of urinary tract infection

Urinary tract infection (UTI) is the most common bacterial infection leading to substantial morbidity and considerable health care expenditures across all ages. Here we present an exploratory UPLC-MS study of human urine in the context of febrile, complicated urinary tract infection aimed to reveal and identify possible markers of a host response on infection. A UPLC-MS based workflow, taking advantage of Ultra High Resolution (UHR) Qq-ToF-MS, and multivariate data handling were applied to a carefully selected group of 39 subjects with culture-confirmed febrile Escherichia coli UTI. Using a combination of unsupervised and supervised multivariate modeling we have pinpointed a number of peptides specific for UTI. An unequivocal structural identification of these peptides, as O-glycosylated fragments of the human fibrinogen alpha 1 chain, required MS2 and MS3 experiments on two different MS platforms: ESI-UHR-Qq-ToF and ESI-ion trap, a blast search and, finally, confirmation was achieved by matching experimental tandem mass spectra with those of custom synthesized candidate-peptides.In conclusion, exploiting non-targeted UPLC-MS based approach for the investigation of UTI related changes in urine, we have identified and structurally characterized unique O-glycopeptides, which are, to our knowledge, the first demonstration of O-glycosylation of human fibrinogen alpha 1-chain.     

MFG-E8 activates proliferation of vascular smooth muscle cells via integrin signaling

An accumulation of milk fat globule EGF-8 protein (MFG-E8) occurs within the context of arterial wall inflammatory remodeling during aging, hypertension, diabetes mellitus, or atherosclerosis. MFG-E8 induces VSMC invasion, but whether it affects VSMC proliferation, a salient feature of arterial inflammation, is unknown. Here, we show that in the rat arterial wall in vivo, PCNA and Ki67, markers of cell cycle activation, increase with age between 8 and 30 months. In fresh and early passage VSMC isolated from old aortae, an increase in CDK4 and PCNA, an increase in the acceleration of cell cycle S and G2 phases, decrease in the G1/G0 phase, and an increase in PDGF and its receptors confer elevated proliferative capacity, compared to young VSMC. Increased coexpression and physical interaction of MFG-E8 and integrin αvβ5 occur with aging in both the rat aortic wall in vivo and in VSMC in vitro. In young VSMC in vitro, MFG-E8 added exogenously, or overexpressed endogenously, triggers phosphorylation of ERK1/2, augmented levels of PCNA and CDK4, increased BrdU incorporation, and promotes proliferation, via αvβ5 integrins. MFG-E8 silencing, or its receptor inhibition, or the blockade of ERK1/2 phosphorylation in these cells reduces PCNA and CDK4 levels and decelerates the cell cycle S phase, conferring a reduction in proliferative capacity. Collectively, these results indicate that MFG-E8 in a dose-dependent manner coordinates the expression of cell cycle molecules and facilitates VSMC proliferation via integrin/ERK1/2 signaling. Thus, an increase in MFG-E8 signaling is a mechanism of the age-associated increase in aortic VSMC proliferation.     

Staining protocols for human pancreatic islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region. The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&E and IHC stains. The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.     

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion ¹. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities ². Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab ^3,4 and we have used it in kidney tubular cell lines ^5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay⁸. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.     

Improved E. coli erythromycin A production through the application of metabolic and bioprocess engineering

In this report, small-scale culture and bioreactor experiments were used to compare and improve the heterologous production of the antibiotic erythromycin A across a series of engineered prototype Escherichia coli strains. The original strain, termed BAP1(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7), was designed to allow full erythromycin A biosynthesis from the exogenous addition of propionate. This strain was then compared against two alternatives hypothesized to increase final product titer. Strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) is a derivative of BAP1 designed to increase biosynthetic pathway carbon flow as a result of a ygfH deletion; whereas, strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4-2, pGro7) provided an extra copy of a key deoxysugar glycosyltransferase gene. Production was compared across the three strains with TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) showing significant improvement in erythronolide B (EB), 3-mycarosylerythronolide B (MEB), and erythromycin A titers. This strain was further tested in the context of batch bioreactor production experiments with time-course titers leveling at 4 mg/L, representing an approximately sevenfold increase in final erythromycin A titer.     

Dancing with the sterols: Critical roles for ABCG1, ABCA1, miRNAs, and nuclear and cell surface receptors in controlling cellular sterol homeostasis

ATP binding cassette (ABC) transporters represent a large and diverse family of proteins that transport specific substrates across a membrane. The importance of these transporters is illustrated by the finding that inactivating mutations within 17 different family members are known to lead to specific human diseases. Clinical data from humans and/or studies with mice lacking functional transporters indicate that ABCA1, ABCG1, ABCG4, ABCG5 and ABCG8 are involved in cholesterol and/or phospholipid transport. This review discusses the multiple mechanisms that control cellular sterol homeostasis, including the roles of microRNAs, nuclear and cell surface receptors and ABC transporters, with particular emphasis on recent findings that have provided insights into the role(s) of ABCG1. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).