Digital karyotyping: an update of its applications in cancer

DNA copy number alterations, including entire chromosomal changes and small interstitial DNA amplifications and deletions, characterize the development of cancer. These changes usually affect the expression of target genes and subsequently the function of the target proteins. Since the completion of the human genome project, the capacity to comprehensively analyze the human cancer genome has expanded significantly. Techniques such as digital karyotyping have been developed to allow for the detection of DNA copy number alterations in cancer at the whole-genome scale. When compared with conventional methods such as spectral karyotyping, representational difference analysis, comparative genomic hybridization (CGH), or the more recent array CGH; digital karyotyping provides an evaluation of copy number of genetic material at higher resolution. Digital karyotyping has therefore promised to enhance our understanding of the cancer genome. This article provides an overview of digital karyotyping including the principle of the technology and its applications in identifying potential oncogenes and tumor suppressor genes.     

Dielectric studies of wheat in powder form at microwave frequencies

Dielectric constant and loss factor of Raj-4120 variety of Indian wheat were determined in powder form (grain size 125 to 150 micron) at room temperature. Microwaves at three different frequencies were employed in C-band, X-band and Ku-band respectively for investigating frequency dependence of dielectric parameters of the sample. Bulk dielectric values of the sample were determined by employing the dielectric mixture relations, such as, half power mixture equation, Landau and Lifshitz, Looyenga equation etc.     

Overexpression of prothymosin alpha predicts poor disease outcome in head and neck cancer

Background

In our recent study, tissue proteomic analysis of oral pre-malignant lesions (OPLs) and normal oral mucosa led to the identification of a panel of biomarkers, including prothymosin alpha (PTMA), to distinguish OPLs from histologically normal oral tissues. This study aimed to determine the clinical significance of PTMA overexpression in oral squamous cell hyperplasia, dysplasia and head and neck squamous cell carcinoma (HNSCC).

Methodology

Immunohistochemistry of PTMA protein was performed in HNSCCs (n = 100), squamous cell hyperplasia (n = 116), dysplasia (n = 50) and histologically normal oral tissues (n = 100). Statistical analysis was carried out to determine the association of PTMA overexpression with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients.

Results

Our immunohistochemical analysis demonstrated significant overexpression of nuclear PTMA in squamous cell hyperplasia (63.8%), dysplasia (50%) and HNSCC (61%) in comparison with oral normal mucosa (p_trend<0.001). Chi-square analysis showed significant association of nuclear PTMA with advanced tumor stages (III+IV). Kaplan Meier survival analysis indicated reduced disease free survival (DFS) in HNSCC patients (p<0.001; median survival 11 months). Notably, Cox-multivariate analysis revealed nuclear PTMA as an independent predictor of poor prognosis of HNSCC patients (p<0.001, Hazard''s ratio, HR = 5.2, 95% CI = 2.3–11.8) in comparison with the histological grade, T-stage, nodal status and tumor stage.

Conclusions

Nuclear PTMA may serve as prognostic marker in HNSCC to determine the subset of patients that are likely to show recurrence of the disease.     

Adenovirus serotype 5-specific neutralizing antibodies target multiple hexon hypervariable regions

The immunogenicity of adenovirus serotype 5 (Ad5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against the hexon hypervariable regions (HVRs). We previously reported that replacing all seven HVRs with those from the rare serotype virus Ad48 resulted in a chimeric Ad5HVR48(1-7) vector that largely evaded preexisting Ad5 immunity in mice and rhesus monkeys. In this study, we evaluated the extent to which Ad5-specific NAbs are directed against various HVRs. We constructed partial HVR-chimeric Ad5 vectors with only a subset of HVRs exchanged, and we utilized these vectors in both NAb assays and murine immunogenicity studies with and without baseline Ad5 immunity. Our results demonstrate that Ad5-specific NAbs target multiple HVRs, suggesting that replacing all HVRs is required to optimize evasion of anti-Ad5 immunity. These data have important implications for the development of novel vectors for both vaccines and gene therapy.     

Cytologic features of granulosa cell tumors in fluids and fine needle aspiration specimens

OBJECTIVE: To describe the cytologic features of granulosa cell tumors in fluids and fine needle aspiration specimens, with histologic confirmation. STUDY DESIGN: Histologically confirmed granulosa cell tumors, 6 adult type and 1 juvenile type, were identified. All patients had local recurrences or metastases. Eleven specimens from 7 patients, including cytologic samples, cell blocks and histology, were reviewed. Inhibin immunostaining was performed on cell blocks to aid identification of this group of tumors in the cytologic and histologic samples. RESULTS: The patients were 22-72 years old. Sites included ovary and peritoneum; there were pelvic recurrences and metastatic lesions in the spleen, liver, perirectum and cervical lymph node. Cytologic features of adult granulosa cell tumors included 3-dimensional clusters, resettes loose monolayers and individual cells. Other features were Call-Exner bodies, vacuolated cytoplasm, exuberant capillaries associated with papillarylike fronds, a second population of elongated theca cells, and prominent or rare nuclear grooves. In juvenile granulosa cell tumor the features observed were monolayers, loosely cohesive sheets, single cells, occasional larger pleomorphic cells with nuclear clefting and nuclear protrusions, vacuolated cytoplasm, finely granular chromatin and frequent mitoses. The overall cytologic and histologic correlation was good. Inhibin was focally positive in one peritoneal fluid, correlating with the focal pattern of staining seen on histology. CONCLUSION: A definitive cytologic diagnosis of granulosa cell tumor can be made based on the above criteria. Aggressive tumors are discohesive and show pleomorphism and nuclear protrusions. Inhibin stain may be helpful in identifying granulosa cell tumors in cell block specimens.     

Reclassification of "atypical" diagnoses in endoscopic retrograde cholangiopancreaticography-guided biliary brushings

OBJECTIVE: To evaluate the usefulness of reclassifying "atypical" diagnoses in reporting biliary cytology using strict morphologic criteria. STUDY DESIGN: Cytologic specimens from 139 patients (direct, alcohol-fixed smears or cytocentrifuge preparations) were evaluated. Diagnoses were benign (70), atypical (36) and malignant (33). Using strict criteria--major (nuclear contour, chromatin pattern) and minor (polarity, cell types, nuclear size, nuclear grooves, nucleoli, mitosis, nuclear/cytoplasmic [N/C] ratio)--atypical cases were reevaluated and reclassified. Follow-up (F/U) was available on all cases. RESULTS: Atypical cases, (36) were reclassified as malignant (26), atypical favor benign (2)/reactive (3) and atypical, not otherwise specified (NOS) (5). Cases reclassified as malignant showed irregular nuclear contours, chromatin irregularities and rare mitosis. Nuclear enlargement, nucleoli and cellularity varied widely in all groups. N/C ratio was increased in most reclassified malignant cases. All 26 malignant reclassifications correlated with F/U of malignancy. Benign and reactive cases (5) were negative for malignancy on F/U (4), and in 1 case a metastatic carcinoma involving the biliary tree was found. In the 5 atypical (NOS) cases, F/U showed malignancy (3) and pancreatitis (2). Cytocentrifuge preparations made in our laboratory were of superior quality when compared to other methods of cell preparation. CONCLUSION: Irregularities in nuclear membrane and abnormal chromatin pattern were the most consistently useful features correlating with malignancy. The sensitivity and specificity of biliary brush cytology can be enhanced by using strict cytomorphologic criteria and proper collection and fixation, all of which decrease atypical diagnoses.     

Purification and concentration of alkaline phosphatase by selective permeabilization of Escherichia coli using reverse micellar solutions

Recovery of alkaline phosphatase (AP) from the periplasm of Escherichia coli using reverse micellar solutions (RMSs) of sodium dioctyl sulfosuccinate (AOT) in aliphatic hydrocarbons has been attempted. A variety of surface-active agents, solvents, and reverse micellar conditions were screened, and an excellent recovery of the enzyme in a concentrated form, with a high purification factor, was obtained in a single-step process. The permeabilization process strongly depended on the water content of the RMS as well as on the amount of water coating the microbial cell surface. The product was almost free from nucleic acids. In addition, because of the low affinity of AOT and the organic solvent for the aqueous phase, contamination by the permeabilizing agents would also be negligible.     

Molecular pathology of dityrosine cross-links in proteins: Structural and functional analysis of four proteins

The dityrosine bond (DT) is an oxidative covalent cross-link between two tyrosines. DT cross-linking is increasingly identified as a marker of oxidative stress, aging and disease, and has been detected in diverse pathologies. While DT cross- linked proteins have been documented, the consequences of the DT link on the structure and function of the so modified proteins are yet to be understood. With this in view, we have studied the properties of intermolecular DT-dimers of four proteins of diverse functions, namely the enzyme ribonuclease A, the signal protein calmodulin, and the eye lens proteins alpha- and gamma B-crystallins. We find that DT is formed through radical reactions and type I photosensitization (including OH, O₂ ^– and OONO^–), but not by ¹O₂ and NO₂ ^– (which modify his, trp and met more readily). Tyr residues on the surface of the protein make DT bonds (intra- and intermolecular) most readily and preferentially. The conformation of each of these DT-dimers, monitored by spectroscopy, is seen not to be significantly altered in comparison to that of the parent monomer, but the structural stability of the DT cross-linked molecule is lower than that of the parent native monomer. The DT-dimer is denatured at a lower temperature, and at lower concentrations of urea or guanidinium chloride. The effect of DT-cross-linking on the biological activities of these proteins was next studied. The enzymatic activity of the DT-dimer of ribonuclease A is not lost but lowered. DT-dimerization of lens alpha-crystallin did not significantly affect the chaperone-like ability; it inhibits the self-aggregation and precipitation of target proteins just as well as the parent, unmodified alpha-crystallin does. DT-dimerization of gamma B-crystallin is however seen to lead to more ready aggregation and precipitation, a point of interest in cataract. In the case of calmodulin, we could generate both intermolecular and intramolecular DT cross-linking, and study both the DT-dimer and DT-monomer. The DT-dimer binds smooth muscle light chain kinase and also Ca²⁺, but less efficiently and over a broad concentration range than the native monomer. The intramolecular DT-monomer is weaker in all these respects, presumably since it is structurally more constrained. These results suggest that DT cross-linking of globular proteins weakens their structural stability and compromises (though does not abolish) their biological activity, both of which are pathologically relevant. The intramolecular DT cross-link would appear to lead to more severe structural and functional consequences.     

Molecular pathology of dityrosine cross-links in proteins: structural and functional analysis of four proteins

The dityrosine bond (DT) is an oxidative covalent cross-link between two tyrosines. DT cross-linking is increasingly identified as a marker of oxidative stress, aging and disease, and has been detected in diverse pathologies. While DT cross- linked proteins have been documented, the consequences of the DT link on the structure and function of the so modified proteins are yet to be understood. With this in view, we have studied the properties of intermolecular DT-dimers of four proteins of diverse functions, namely the enzyme ribonuclease A, the signal protein calmodulin, and the eye lens proteins alpha- and gamma B-crystallins. We find that DT is formed through radical reactions and type I photosensitization (including .OH, O2- and OONO-), but not by 1O2 and NO, (which modify his, trp and met more readily). Tyr residues on the surface of the protein make DT bonds (intra- and intermolecular) most readily and preferentially. The conformation of each of these DT-dimers, monitored by spectroscopy, is seen not to be significantly altered in comparison to that of the parent monomer, but the structural stability of the DT cross-linked molecule is lower than that of the parent native monomer. The DT-dimer is denatured at a lower temperature, and at lower concentrations of urea or guanidinium chloride. The effect of DT-cross-linking on the biological activities of these proteins was next studied. The enzymatic activity of the DT-dimer of ribonuclease A is not lost but lowered. DT-dimerization of lens alpha-crystallin did not significantly affect the chaperone-like ability; it inhibits the self-aggregation and precipitation of target proteins just as well as the parent, unmodified alpha-crystallin does. DT-dimerization of gamma B-crystallin is however seen to lead to more ready aggregation and precipitation, a point of interest in cataract. In the case of calmodulin, we could generate both intermolecular and intramolecular DT cross-linking, and study both the DT-dimer and DT-monomer. The DT-dimer binds smooth muscle light chain kinase and also Ca2+, but less efficiently and over a broad concentration range than the native monomer. The intramolecular DT-monomer is weaker in all these respects, presumably since it is structurally more constrained. These results suggest that DT cross-linking of globular proteins weakens their structural stability and compromises (though does not abolish) their biological activity, both of which are pathologically relevant. The intramolecular DT cross-link would appear to lead to more severe structural and functional consequences.