Prevalence of BRCA1 Mutations in Familial and Sporadic Greek Ovarian Cancer Cases

Germline mutations in the BRCA1 and BRCA2 genes contribute to approximately 18% of hereditary ovarian cancers conferring an estimated lifetime risk from 15% to 50%. A variable incidence of mutations has been reported for these genes in ovarian cancer cases from different populations. In Greece, six mutations in BRCA1 account for 63% of all mutations detected in both BRCA1 and BRCA2 genes. This study aimed to determine the prevalence of BRCA1 mutations in a Greek cohort of 106 familial ovarian cancer patients that had strong family history or metachronous breast cancer and 592 sporadic ovarian cancer cases. All 698 patients were screened for the six recurrent Greek mutations (including founder mutations c.5266dupC, p.G1738R and the three large deletions of exon 20, exons 23–24 and exon 24). In familial cases, the BRCA1 gene was consequently screened for exons 5, 11, 12, 20, 21, 22, 23, 24. A deleterious BRCA1 mutation was found in 43/106 (40.6%) of familial cancer cases and in 27/592 (4.6%) of sporadic cases. The variant of unknown clinical significance p.V1833M was identified in 9/698 patients (1.3%). The majority of BRCA1 carriers (71.2%) presented a high-grade serous phenotype. Identifying a mutation in the BRCA1 gene among breast and/or ovarian cancer families is important, as it enables carriers to take preventive measures. All ovarian cancer patients with a serous phenotype should be considered for genetic testing. Further studies are warranted to determine the prevalence of mutations in the rest of the BRCA1 gene, in the BRCA2 gene, and other novel predisposing genes for breast and ovarian cancer.     

Accurate phylogenetic classification of variable-length DNA fragments

Metagenome studies have retrieved vast amounts of sequence data from a variety of environments leading to new discoveries and insights into the uncultured microbial world. Except for very simple communities, the encountered diversity has made fragment assembly and the subsequent analysis a challenging problem. A taxonomic characterization of metagenomic fragments is required for a deeper understanding of shotgun-sequenced microbial communities, but success has mostly been limited to sequences containing phylogenetic marker genes. Here we present PhyloPythia, a composition-based classifier that combines higher-level generic clades from a set of 340 completed genomes with sample-derived population models. Extensive analyses on synthetic and real metagenome data sets showed that PhyloPythia allows the accurate classification of most sequence fragments across all considered taxonomic ranks, even for unknown organisms. The method requires no more than 100 kb of training sequence for the creation of accurate models of sample-specific populations and can assign fragments >or=1 kb with high specificity.     

Cell cycle-regulated phosphorylation of hamartin, the product of the tuberous sclerosis complex 1 gene, by cyclin-dependent kinase 1/cyclin B

Tuberous sclerosis complex is a tumor suppressor gene syndrome whose manifestations can include seizures, mental retardation, and benign tumors of the brain, skin, heart, and kidneys. Hamartin and tuberin, the products of the TSC1 and TSC2 genes, respectively, form a complex and inhibit signaling by the mammalian target of rapamycin. Here, we demonstrate that endogenous hamartin is threonine-phosphorylated during nocodazole-induced G2/M arrest and during the G2/M phase of a normal cell cycle. In vitro assays showed that cyclin-dependent kinase 1 phosphorylates hamartin at three sites, one of which (Thr417) is in the hamartin-tuberin interaction domain. Tuberin interacts with phosphohamartin, and tuberin expression attenuates the phosphorylation of exogenous hamartin. Hamartin with alanine mutations in the three cyclin-dependent kinase 1 phosphorylation sites increased the inhibition of p70S6 kinase by the hamartin-tuberin complex. These findings support a model in which phosphorylation of hamartin regulates the function of the hamartin-tuberin complex during the G2/M phase of the cell cycle.     

Isolation of kappa-carrageenan oligosaccharides using ion-pair liquid chromatography--characterisation by electrospray ionisation mass spectrometry in positive-ion mode

Oligo-kappa-carrageenans participate as elicitors in the cell-cell recognition process in marine plants. Analytical methods can be usefully applied to gain insight into the biochemistry of these biological processes. Therefore, enzymatically digested oligomers of kappa-carrageenans have been separated and isolated on a Spherisorb ODS1 (250 x 4 mm i.d., particle size 5 microm) column using ion-pair liquid chromatography coupled with an evaporative light scattering detector. Heptylamine (5 mM, pH4) has been selected as the ion-pairing agent and MeOH as the organic modifier in a gradient mode. Overloading the column with 1mg of the mixture, the chromatographic mechanism presented adequate stability. The mobile phase of each isolated oligomer was evaporated and the residue was infused into an electrospray ionisation mass spectrometry (ESIMS) in positive-ion mode with 4:1 MeCN-water as mobile phase. Each ESIMS spectrum presented ions consisting of the oligomer attached with a number of heptylammonium ions depending on the molecule size. In addition, the different m/z values permitted direct detection of the oligomers in ESIMS positive-ion mode. The analytical method developed separated the oligomers up to dotriacontasaccharide.     

Loss of effector function of human cytolytic T lymphocytes is accompanied by major alterations in N- and O-glycosylation

Most human tumors are not eliminated by the immune system, and therapeutic vaccination shows poor results, a fact that can be explained at least partially by an immunosuppressive tumor microenvironment that is abundant in galectin-3. On cytolytic T lymphocyte (CTL) clones, maintained in culture by regular stimulation, recently activated CTLs present low effector functions. However, these functions are restored after a short treatment with LacNAc. The latter, which is in agreement with the glycoprotein-galectin lattice concept involving reduced motility, poses the question why galectin-3 ligands improve effector functions. We employed ultrasensitive MALDI-TOF-MS on resting and recently activated CTL clones combined with various glycosidase digestions and GC-MS linkage analyses. Our results showed that compared with the resting CTLs, the N-glycans of the recently activated CTLs consisted of (i) larger LacNAc oligomers of which a significant portion was longer than four-units and (ii) more multi-antennary structures. Interestingly, our results showed that the poly-LacNAc appeared to be equally distributed on all available N-glycan branches and not selectively enriched on a specific branch. The above structural alterations in the recently activated CTLs are expected to increase the galectin-3-LacNAc lattices and multivalent interactions and, therefore, reduce the motility of surface glycoproteins, such as the T-cell receptor. These findings suggest that the loss of effector functions on CTLs may be linked to reduced motility of surface glycoproteins. In addition, our results showed that recently activated CTLs had a reduced abundance of NeuAcα2,6-linked N-glycans and an increased abundance of disialylated core 1 and monosialylated core 2 O-glycan structures.     

Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2) Is Upregulated in Breast Epithelial–Mesenchymal Transition and Responds to Oxidative Stress

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 H₂S 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.     

Regulation of B-Raf kinase activity by tuberin and Rheb is mammalian target of rapamycin (mTOR)-independent

Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome with manifestations that can include seizures, mental retardation, autism, and tumors in the brain, retina, kidney, heart, and skin. The products of the TSC1 and TSC2 genes, hamartin and tuberin, respectively, heterodimerize and inhibit the mammalian target of rapamycin (mTOR). We found that tuberin expression increases p42/44 MAPK phosphorylation and B-Raf kinase activity. Short interfering RNA down-regulation of tuberin decreased the p42/44 MAPK phosphorylation and B-Raf activity. Expression of Rheb, the target of the GTPase-activating domain of tuberin, inhibited wild-type B-Raf kinase but not activated forms of B-Raf. The interaction of endogenous Rheb with B-Raf was enhanced by serum and by Ras overexpression. A farnesylation-defective mutant of Rheb co-immunoprecipitated with and inhibited B-Raf but did not activate ribosomal protein S6 kinase, indicating that farnesylation is not required for B-Raf inhibition by Rheb and that B-Raf inhibition and S6 kinase activation are separable activities of Rheb. Consistent with this, inhibition of B-Raf and p42/44 MAPK by Rheb was resistant to rapamycin in contrast to Rheb activation of S6 kinase, which is rapamycin-sensitive. Taken together these data demonstrate that inhibition of B-Raf kinase via Rheb is an mTOR-independent function of tuberin.     

Apical secretion and association of the Drosophila yellow gene product with developing larval cuticle structures during embryogenesis.

Summary The yellow (y) gene of Drosophila melanogaster is required for the pigmentation of larval and adult cuticle structures. The deduced y protein sequence includes two putative N-linked glycosylation sites and a putative signal peptide, suggesting that it might be a secreted molecule. Consistent with the characteristics of a secreted protein, our in vitro translation studies using RNA synthesised from the y cDNA demonstrate that the nascent y polypeptide is a preprotein that cotranslationally translocates into the endoplasmic reticulum (ER) membrane and becomes glycosylated. The N-terminal peptide is cleaved from the preprotein between the two alanine residues at positions 21 and 22, to release the final product into the lumen of the ER. Antibodies raised against the y polypeptide detect the protein starting at 13 h post-fertilization in epidermal cells and in the cuticle structures secreted by them that later become pigmented; in addition, yellow protein is detected in the cuticle structures associated with Keilin's organs. The embryonic -galactosidase staining pattern of a transgene, bearing a construct in which expression of the lacZ gene is driven by the y promoter, is also described and is similar to that of the y protein. Our results indicate that the y gene product is an apically secreted protein which becomes an immobilised structural component of the pigmented cuticle.     

Histology Verification Demonstrates That Biospectroscopy Analysis of Cervical Cytology Identifies Underlying Disease More Accurately than Conventional Screening: Removing the Confounder of Discordance

Background

Subjective visual assessment of cervical cytology is flawed, and this can manifest itself by inter- and intra-observer variability resulting ultimately in the degree of discordance in the grading categorisation of samples in screening vs. representative histology. Biospectroscopy methods have been suggested as sensor-based tools that can deliver objective assessments of cytology. However, studies to date have been apparently flawed by a corresponding lack of diagnostic efficiency when samples have previously been classed using cytology screening. This raises the question as to whether categorisation of cervical cytology based on imperfect conventional screening reduces the diagnostic accuracy of biospectroscopy approaches; are these latter methods more accurate and diagnose underlying disease? The purpose of this study was to compare the objective accuracy of infrared (IR) spectroscopy of cervical cytology samples using conventional cytology vs. histology-based categorisation.

Methods

Within a typical clinical setting, a total of n = 322 liquid-based cytology samples were collected immediately before biopsy. Of these, it was possible to acquire subsequent histology for n = 154. Cytology samples were categorised according to conventional screening methods and subsequently interrogated employing attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy. IR spectra were pre-processed and analysed using linear discriminant analysis. Dunn’s test was applied to identify the differences in spectra. Within the diagnostic categories, histology allowed us to determine the comparative efficiency of conventional screening vs. biospectroscopy to correctly identify either true atypia or underlying disease.

Results

Conventional cytology-based screening results in poor sensitivity and specificity. IR spectra derived from cervical cytology do not appear to discriminate in a diagnostic fashion when categories were based on conventional screening. Scores plots of IR spectra exhibit marked crossover of spectral points between different cytological categories. Although, significant differences between spectral bands in different categories are noted, crossover samples point to the potential for poor specificity and hampers the development of biospectroscopy as a diagnostic tool. However, when histology-based categories are used to conduct analyses, the scores plot of IR spectra exhibit markedly better segregation.

Conclusions

Histology demonstrates that ATR-FTIR spectroscopy of liquid-based cytology identifies the presence of underlying atypia or disease missed in conventional cytology screening. This study points to an urgent need for a future biospectroscopy study where categories are based on such histology. It will allow for the validation of this approach as a screening tool.