Photochemically modified diamond-like carbon surfaces for neural interfaces.

Diamond-like carbon (DLC) was modified using a UV functionalization method to introduce surface-bound amine and aldehyde groups. The functionalization process rendered the DLC more hydrophilic and significantly increased the viability of neurons seeded to the surface. The amine functionalized DLC promoted adhesion of neurons and fostered neurite outgrowth to a degree indistinguishable from positive control substrates (glass coated with poly-L-lysine). The aldehyde-functionalized surfaces performed comparably to the amine functionalized surfaces and both additionally supported the adhesion and growth of primary rat Schwann cells. DLC has many properties that are desirable in biomaterials. With the UV functionalization method demonstrated here it may be possible to harness these properties for the development of implantable devices to interface with the nervous system.

Amine functionalized nanodiamond promotes cellular adhesion, proliferation and neurite outgrowth.

In this study, we report the production of amine functionalized nanodiamond. The amine functionalized nanodiamond forms a conformal monolayer on a negatively charged surface produced via plasma polymerization of acrylic acid. Nanodiamond terminated surfaces were studied as substrates for neuronal cell culture. NG108-15 neuroblastoma-glioma hybrid cells were successfully cultured upon amine functionalized nanodiamond coated surfaces for between 1 and 7 d. Additionally, primary dorsal root ganglion (DRG) neurons and Schwann cells isolated from Wistar rats were also successfully cultured over a period of 21 d illustrating the potential of the coating for applications in the treatment of peripheral nerve injury.

Constrained global optimization for estimating molecular structure from atomic distances.

Finding optimal three-dimensional molecular configurations based on a limited amount of experimental and/or theoretical data requires efficient nonlinear optimization algorithms. Optimization methods must be able to find atomic configurations that are close to the absolute, or global, minimum error and also satisfy known physical constraints such as minimum separation distances between atoms (based on van der Waals interactions). The most difficult obstacles in these types of problems are that 1) using a limited amount of input data leads to many possible local optima and 2) introducing physical constraints, such as minimum separation distances, helps to limit the search space but often makes convergence to a global minimum more difficult. We introduce a constrained global optimization algorithm that is robust and efficient in yielding near-optimal three-dimensional configurations that are guaranteed to satisfy known separation constraints. The algorithm uses an atom-based approach that reduces the dimensionality and allows for tractable enforcement of constraints while maintaining good global convergence properties. We evaluate the new optimization algorithm using synthetic data from the yeast phenylalanine tRNA and several proteins, all with known crystal structure taken from the Protein Data Bank. We compare the results to commonly applied optimization methods, such as distance geometry, simulated annealing, continuation, and smoothing. We show that compared to other optimization approaches, our algorithm is able combine sparse input data with physical constraints in an efficient manner to yield structures with lower root mean squared deviation.

Disparate E-cadherin mutations in LCIS and associated invasive breast carcinomas.

AIMS: The relation between lobular carcinoma in situ (LCIS) and invasive breast cancer is unresolved. In an attempt to establish whether LCIS is a precursor of invasive cancer the mutational status and the expression of E-cadherin was analysed in LCIS and associated invasive breast carcinoma in 23 patients. METHODS: Foci of LCIS and associated invasive carcinoma were individually microdissected from tissue from 23 patients. Exons 4-16 of the E-cadherin gene were analysed using single strand conformation polymorphism (SSCP); protein expression and the localisation of E-cadherin and beta-catenin were assessed with the use of immunohistochemistry. RESULTS: Immunohistochemistry revealed a lack of expression of E-cadherin and beta-catenin in most LCIS samples and invasive foci. In all but four cases, the staining pattern was identical in the LCIS and associated invasive areas. When E-cadherin was absent, beta-catenin was also undetected, suggesting a lack of expression of alternative classic cadherin members in these lesions. Coincident E-cadherin mutations in LCIS and associated invasive carcinoma were not identified in this series of patients. However, mutational analysis of E-cadherin in multiple foci of carcinoma in situ surrounding an invasive lesion provided evidence to support ductal carcinoma in situ as a precursor of invasive ductal carcinoma. CONCLUSION: These data support the hypothesis that LCIS is not a precursor of invasive breast carcinoma but a marker of increased risk of developing invasive disease.

Expression of classic cadherins type I in urothelial neoplastic progression.

Loss or reduced expression of E-cadherin has been shown to be associated with poor survival in patients with bladder cancer. In numerous cases, loss of E-cadherin expression in bladder tumors has been accompanied by continued association of catenins with the membrane, suggestive of the expression of an alternative cadherin member. In this study we examined 75 bladder tumors using immunohistochemistry for the expression of E-, P-cadherin, and alpha-, beta-, and gamma-catenins. As reported previously, loss or reduced E-cadherin expression is a frequent event in late stage bladder cancer, accompanied by less frequent alterations associated with different catenin family members. Analysis of 51 tumors for expression of E-, P-, and N-cadherin showed P-cadherin localized to the basal cell layers of normal urothelium, with retention of expression in the majority of tumors. In low-grade tumors P-cadherin was found localized to an expanded basal cell compartment, contrasting with the more extensive staining observed in late stage tumors. Membranous P-cadherin staining was often found in the absence of E-cadherin staining. N-cadherin is not expressed in normal bladder mucosa, but detection of this cadherin member was recorded in 39% (20/51) of bladder tumors. Unlike P-cadherin, membranous N-cadherin was detected in focal regions within tumors, representing novel expression in urothelial neoplastic progression. Although focal N-cadherin staining was observed in 3 noninvasive lesions, the majority of tumors expressing N-cadherin were invasive (17/20). Coexpression of E-, P-, and N-cadherin was recorded in 5 grade 2 bladder tumors. Expression of P-cadherin is maintained throughout bladder tumorigenesis, accompanied by aberrant expression of N-cadherin. Clearly, neither P- nor N-cadherin act in an invasive-suppressor mode in bladder cancer, but whether they have a primary role to play in urothelial neoplastic progression has yet to be established.

Somatic mutation of PTEN in vulvar cancer.

PTEN, a candidate tumor suppressor gene located at chromosome 10q23.3, has been shown to be mutated in approximately 40% of endometrial cancers. Such mutations have also been identified in endometrial hyperplasia, indicating that inactivation of the PTEN tumor suppressor gene is an early event in the genesis of some endometrial cancers. In this study, we have extended the analysis of PTEN in gynecological cancer to include adenocarcinoma of the cervix and vulvar carcinomas. Microdissected tissue (including normal tissues), preneoplastic, and neoplastic lesions were analyzed from 9 patients with cervical cancer and 10 patients with vulvar cancer. Only 1 cervical adenocarcinoma displayed a PTEN mutation. In contrast, five of eight vulvar carcinomas studied harbored PTEN mutations. Alterations were identified in carcinoma in situ as well as squamous cell carcinoma of the vulva. In two patients, PTEN mutations were identified in mucosal regions with mild or focal dysplasia. These results suggest that PTEN is frequently altered in vulvar carcinomas and can be found associated with early dysplastic changes in vulvar mucosa.

Management of hilar cholangiocarcinoma: comparison of an American and a Japanese experience.

OBJECTIVE: To compare the experience and outcome in the management of hilar cholangiocarcinoma at one American and one Japanese medical center. SUMMARY BACKGROUND DATA: Controversies surround the issues of extent of resection for hilar cholangiocarcinoma and whether the histopathology of such cancers are similar between patients treated in America and in Japan. METHODS: Records were reviewed of 100 patients treated between 1980 and 1995 at the Lahey Clinic in the United States, and of 155 patients treated between 1977 and 1995 at Nagoya University Hospital in Japan. Selected pathologic slides of resected cancers were exchanged between the two institutions and reviewed for diagnostic concordance. RESULTS: In the Lahey cohort, there were 25 resections, 53 cases of surgical exploration with biliary bypass or intubation, and 22 cases of percutaneous transhepatic biliary drainage or endoscopic biliary drainage without surgery. In the Nagoya cohort, the respective figures were 122, 10, and 23. The overall 5-year survival rate of all patients treated (surgical and nonsurgical) during the study periods was 7% in the Lahey cohort and 16% in the Nagoya cohort. The overall 10-year survival rates were 0% and 12%, respectively. In patients who underwent resection with negative margins, the 5- and 10-year survival rates were 43% and 0% for the Lahey cohort and 25% and 18% for the Nagoya cohort. The surgical death rate for patients undergoing resection was 4% for Lahey patients and 8% for Nagoya patients. Of the patients who underwent resection, en bloc caudate lobectomy was performed in 8% of the Lahey patients and 89% of the Nagoya patients. Histopathologic examination of resected cancers showed that the Nagoya patients had a higher stage of disease than the Lahey patients. CONCLUSIONS: In both Lahey and Nagoya patients, survival was most favorable when resection of hilar cholangiocarcinoma was accomplished with margin-negative resections. Combined bile duct and liver resection with caudate lobectomy contributed to a higher margin-negative resection rate in the Nagoya cohort.

Automation and integration of components for generalized semantic markup of electronic medical texts.

Our group has built an information retrieval system based on a complex semantic markup of medical textbooks. We describe the construction of a set of web-based knowledge-acquisition tools that expedites the collection and maintenance of the concepts required for text markup and the search interface required for information retrieval from the marked text. In the text markup system, domain experts (DEs) identify sections of text that contain one or more elements from a finite set of concepts. End users can then query the text using a predefined set of questions, each of which identifies a subset of complementary concepts. The search process matches that subset of concepts to relevant points in the text. The current process requires that the DE invest significant time to generate the required concepts and questions. We propose a new system--called ACQUIRE (Acquisition of Concepts and Queries in an Integrated Retrieval Environment)--that assists a DE in two essential tasks in the text-markup process. First, it helps her to develop, edit, and maintain the concept model: the set of concepts with which she marks the text. Second, ACQUIRE helps her to develop a query model: the set of specific questions that end users can later use to search the marked text. The DE incorporates concepts from the concept model when she creates the questions in the query model. The major benefit of the ACQUIRE system is a reduction in the time and effort required for the text-markup process. We compared the process of concept- and query-model creation using ACQUIRE to the process used in previous work by rebuilding two existing models that we previously constructed manually. We observed a significant decrease in the time required to build and maintain the concept and query models.

Changes in protein prenylation and prenyltransferase activity in the rat seminiferous epithelium during early stages of spermatogenesis.

Changes in protein prenyltransferase activity, levels of prenylated protein, and the type of isoprenoid modification was described in cells of rat seminifereous epithelium and correlated with differentiative events of spermatogenesis. The activity of protein farnesyltransferase (PFT) was at least 10-fold higher than that for protein geranylgeranyltransferase-I (PGGT-I) in seminiferous epithelium and spermatogenic cells of prepubertal rats of different ages. Both activities increased during the meiotic stages of differentiation and peaked at 23 days of age. The activity of farnesyltransferase in seminiferous epithelium was the same as that in mixed spermatogenic cell populations from animals aged 9 and 23 days, indicating that the activity of this enzyme in somatic cells and germ cells was similar at these ages. Farnesyltransferase activities were similar and low in both pachytene spermatocytes and round spermatids from adult rats; however, the activity in pachytene spermatocytes from 23-day old animals was 2-fold higher than in adults. The highest activity was associated with intermediate-sized spermatocytes appearing late during meiosis. PGGT-I activity was at least 10-fold lower than farnesyltransferase activity and was not significantly different among all cell populations. Differentiation-dependent in vivo protein prenylation was demonstrated by labeling of seminiferous epithelium with [3H]mevalonic acid at different prepubertal ages. Total protein prenylation and the ratio of geranylgeranylated to farnesylated protein, in contrast to prenyltransferase activity, decreased with increasing age. Although 20-30-kDa proteins were the most highly labeled at all ages, [3H]-proteins from different-aged prepubertal rats showed age-dependent changes in the level of prenylation of at least 14 proteins as determined by two-dimensional (2D) electrophoresis. Prenylated proteins of round spermatids were distinguished from those of the spermatocytes by the lack of many 20-30-kDa proteins and by low geranylgeranyl/farnesyl (GG/F) ratios. These results show that independent changes in prenyltransferase activity and protein prenylation accompany the differentiation events during the premeiotic and meiotic stages of spermatogenesis. This suggests that prenylation in the seminiferous epithelium may be more dependent on available protein substrate than on protein prenyltransferase activity.

Cytologic diagnosis of basal cell (basaloid) carcinoma of the lung. A report of two cases.

Basal cell (basaloid) carcinoma (BC) of the lung is a recently described variant of bronchogenic carcinoma reported to behave as an aggressive, non-small cell lung carcinoma. It may exist in pure form or be mixed with other non-small cell carcinoma patterns. We report the cytologic features in two cases of BC and compare the findings with those in five cases of biopsy-confirmed anaplastic small cell carcinoma, which BC closely mimics.

The Ras-related GTP-binding protein, Rab1B, regulates early steps in exocytic transport and processing of beta-amyloid precursor protein.

The role of the Ras-related GTP-binding protein, Rab1B, in intracellular trafficking of beta-amyloid precursor protein (beta APP) was studied in cultured 293 cells. beta APP is processed via one of two alternative routes. In the major secretory pathway, beta APP is cleaved by alpha-secretase within the region comprising the beta-amyloid peptide (A beta), resulting in release of a soluble NH2-terminal exodomain (APP alpha) and a 3-kDa peptide (p3) derived from the carboxyl-terminal tail. In the alternative amyloidogenic pathway, beta APP is cleaved by beta-secretase, with the release of a truncated exodomain (APP beta) and an intact A beta peptide. When beta APP751 was coexpressed with Rab1B(wt) or dominant-negative Rab1B mutants (Rab1BN121I or Rab1BS22N) there was a marked decrease in conversion of the immature Endo-H sensitive form of beta APP751 (108 kDa) to the mature O-glycosylated form of beta APP751 (130 kDa) in cells expressing the mutant forms of Rab1B. The block in Golgi-dependent processing of beta APP was accompanied by inhibition of secretion of APPS (APP alpha). A similar decrease in secretion of APPS (APP alpha+APP beta) was observed in cells that were coexpressing Rab1BN121I with the "Swedish" variant of beta APP751 (i.e. beta APPSW751), which undergoes increased amyloidogenic processing. Coincident with the decline in APPS secretion, the cells coexpressing beta APPSW751 with Rab1BN121I showed a 90% decrease in A beta secretion. The data indicate that Rab1B plays a key role in endoplasmic reticulum-->Golgi transport of beta APP, and that beta APP must pass through a late Golgi compartment before entering either the alpha-secretase or the amyloidogenic beta-secretase pathway. The results also suggest that mutant versions of other Rab proteins that function in different parts of the exocytic and endocytic pathways may be useful in defining the specific routes of beta APP transport involved in the biogenesis of A beta.

Cytologic diagnosis of measles pneumonia in a bronchoalveolar lavage specimen. A case report.

An immunocompromised employee of our medical center contracted measles during an epidemic and subsequently died of giant cell pneumonia. In this report we discuss the cytologic diagnosis of measles pneumonia and the differential diagnosis of multinucleate giant cells in lung cytology specimens. In the appropriate clinical setting, inclusion-bearing syncytial epithelial giant cells in lung are virtually pathognomonic of measles infection. Cytologic specimens can provide an important early diagnosis.

Dispersion compensation for high bit rate fiber-optic communication using a dynamically tunable optical filter.

A detailed analysis of dispersion-compensating fiber-optic filter is provided. Design parameters of the filter that could provide dispersion compensation for wide spectral sources are discussed. The dispersion-compensating fiber filter designed here can be dynamically tuned to compensate for the drift in the laser center frequency caused by temperature change and other deteriorations. The filter can be fabricated by exposing the photorefractive fibers to ultraviolet radiation. A design methodology for fabricating the refractive-index variation along the longitudinal direction of the fiber with appropriate chirp and taper coefficients is also given here. Dispersion-compensation capability of the fiber filter is demonstrated by simulating pulse broadening after transmission in a long fiber and the regeneration of the pulse after dispersion compensation.

Papillary fibroelastoma of the mitral valve.

A case report of a 73-year-old woman with mitral regurgitation secondary to papillary fibroelastoma and prolapse of the mitral valve is described. The tumor was excised, and the valve repaired with a Duran annuloplasty ring. The clinicopathologic features and the surgical management of this rare tumor are reviewed.

Quantitative DNA analysis and proliferation in breast carcinomas. A comparison between image analysis and flow cytometry.

The DNA content and proliferation in 100 invasive breast carcinomas were evaluated by computerized image analysis (IA) and flow cytometry (FCM). For DNA content, image analysis of Feulgen-stained slides of fresh tumor imprints were compared with flow cytometry of propidium iodide-stained disaggregated fresh tumor tissue. The DNA indices obtained by the two methods showed close correlation by linear regression analysis (r = 0.89, p less than .001). There were 44 (44%) diploid and 56 (56%) aneuploid tumors. There was agreement between the two methods in detection of aneuploidy in 81% of tumors. Image analysis required smaller tissue samples, permitted direct visualization and selection of tumor cells, and was more sensitive in detecting tetraploid and highly aneuploid cell populations. In contrast, flow cytometry histograms provided better resolution, and were more effective in detecting multiploid tumors and near-diploid aneuploid tumors. Aneuploidy was significantly related to various adverse prognostic parameters, namely, negative estrogen receptor, high mitotic rate, high histologic and nuclear grades. Proliferation was evaluated by measuring the FCM S phase fraction (SPF), and by image analysis quantitation of immunohistochemical staining using Ki-67 monoclonal antibody. SPF and Ki-67 count showed modest correlation (r = 0.42). Both SPF and Ki-67 count were significantly related to the mitotic rate, histologic and nuclear grades. Our results indicate that the two methods provide comparable results, but offer individual advantages and are complementary techniques in analyzing DNA ploidy and proliferation in breast carcinomas.

DNA ploidy, proliferation, and neu-oncogene protein overexpression in breast carcinoma.

DNA content, proliferative activity (Ki-67 immuno-staining and S-phase fraction by flow cytometry), and neu-oncogene overexpression were studied in 135 patients with invasive breast carcinoma. Image analysis and flow cytometry of fresh tumors showed good correlation between the two methods and yielded 39% diploid tumors and 61% aneuploid tumors. Aneuploidy, including tetraploidy, was significantly related to the loss of estrogen (p = 0.0002) and progesterone (p = 0.03) receptors, high histologic (p = 0.014) and nuclear (p less than 0.0001) grades, and mitotic rate (p = 0.0001). Immunohistochemical evaluation of proliferation by staining with Ki-67 monoclonal antibody and of neu-oncogene protein overexpression was performed in fresh frozen tissue from 83 tumors. The Ki-67 score, quantitated by the CAS-200 image analyzer, correlated only moderately with S-phase fraction obtained by flow cytometry by linear regression analysis (r = 0.39, p less than 0.001). However, both of these proliferation markers correlated strongly with the mitotic rate (p less than 0.0001). Aneuploid and tetraploid tumors demonstrated higher Ki-67 scores and S-phase fractions than diploid tumors. Neu-oncogene protein overexpression was seen in 24 tumors (29%) overall and was much higher in aneuploid tumors (38%) and tetraploid tumors (50%) than in diploid tumors (7%). However, the concentration of neu-oncogene protein positive tumors in the tetraploid region reported by others was not observed. Neu-oncogene protein overexpression was also associated with higher Ki-67 scores (p = 0.016) and S-phase fractions (p = 0.037).(ABSTRACT TRUNCATED AT 250 WORDS)

DNA measurement by image analysis of paraffin-embedded breast carcinoma tissue. A comparative investigation.

Measurement of cellular DNA content may provide useful prognostic information in several human neoplasms. The DNA content by image analysis of fresh tissue has been compared with flow cytometry with good correlation, but the use of paraffin-embedded tissue for image analysis has not been studied widely. This study reports the DNA content of 54 breast carcinomas and compares the results of image analysis of paraffin-embedded tissue and results of image analysis and flow cytometry of corresponding fresh tumors. Image analysis of paraffin blocks and fresh tumors showed comparable results in 51 tumors (94%), whereas 47 tumors (87%) were similar by all three methods. The DNA indices from image analysis of fresh and paraffin-embedded tumors showed significant correlation by linear regression analysis (r = 0.96; P less than 0.001). Discordances between image analysis of fresh and paraffin-embedded tumors are the result of technical problems, such as staining and tissue preservation. Discordances between image analysis and flow cytometry reflect the advantages and pitfalls of the two techniques. These data suggest that image analysis of paraffin-embedded tissue is a viable technique that will permit the performance of retrospective studies with long-term follow-up data for the evaluation of the prognostic significance of DNA content.

Diagnosis of Pneumocystis carinii pneumonia by cytologic evaluation of Papanicolaou-stained bronchial specimens.

To evaluate the sensitivity and specificity of diagnosing Pneumocystis carinii pneumonia (PCP) by Papanicolaou-stained bronchial brushing and wash/lavage specimens obtained by fiberoptic bronchoscopy, the cytologic preparations and clinical records from 58 immunocompromised patients were reviewed. Bronchial brushings and wash/lavage specimens were examined using methenamine silver (Grocott) and Papanicolaou stains. Pneumocystis carinii pneumonia was recognized with Papanicolaou stain by identifying distinctive alveolar casts, which frequently contained collections of encysted sporozoites. Thirty cases of PCP were identified, and Grocott-stained bronchial wash/lavage specimens were positive in 29 instances (97%). Grocott staining of the transbronchial biopsy was positive for PCP in 18 of 22 specimens (82%). Bronchial brushings were insensitive, yielding a positive specimen in only 30% of cases of PCP. Alveolar casts of PCP were identified by Papanicolaou-stained slides of wash/lavage specimens in 83% of cases of Pneumocystis pneumonia. These proteinaceous alveolar casts were not seen in other pulmonary disorders. Encysted sporozoites were found in 56% of cases in which Papanicolaou-stained alveolar casts were identified. We conclude that the diagnosis of PCP can be made rapidly and reliably on the Papanicolaou-stained bronchial wash/lavage or bronchial brush specimens by detecting the characteristic alveolar casts, which contain P. carinii-encysted sporozoites. The presence of encysted sporozoites within alveolar casts is pathognomonic for PCP, and methenamine silver stains can be eliminated in those cases in which encysted sporozoites are identified.