An automated multiplex oligonucleotide synthesizer: development of high-throughput, low-cost DNA synthesis.

An automated oligonucleotide synthesizer has been developed that can simultaneously and rapidly synthesize up to 96 different oligonucleotides in a 96-well microtiter format using phosphoramidite synthesis chemistry. A modified 96-well plate is positioned under reagent valve banks, and appropriate reagents are delivered into individual wells containing the growing oligonucleotide chain, which is bound to a solid support. Each well has a filter bottom that enables the removal of spent reagents while retaining the solid support matrix. A seal design is employed to control synthesis environment and the entire instrument is automated via computer control. Synthesis cycle times for 96 couplings are < 11 min, allowing a plate of 96 20-mers to be synthesized in < 5 hr. Oligonucleotide synthesis quality is comparable to commercial machines, with average coupling efficiencies routinely > 98% across the entire 96-well plate. No significant well-to-well variations in synthesis quality have been observed in > 6000 oligonucleotides synthesized to date. The reduced reagent usage and increased capacity allow the overall synthesis cost to drop by at least a factor of 10. With the development of this instrument, it is now practical and cost-effective to synthesize thousands to tens of thousands of oligonucleotides.     

Retroperitoneal soft-tissue sarcoma: analysis of 500 patients treated and followed at a single institution.

OBJECTIVE: To analyze treatment and survival of a large cohort of patients with retroperitoneal soft-tissue sarcomas (STS) treated and prospectively followed at a single institution. SUMMARY BACKGROUND DATA: Retroperitoneal STS are relatively uncommon and constitute a difficult management problem. Although surgical resection is often difficult or impossible, current chemotherapy is not effective and radiation is limited by toxicity to adjacent structures. Thus, complete surgical resection remains the most effective modality for selected primary and recurrent disease. METHODS: Five hundred patients with retroperitoneal STS were admitted and treated between July 1, 1982, and September 30, 1997, and prospectively followed. Patient, tumor, and treatment variables were analyzed for disease-specific and disease-free survival. Survival was determined with the Kaplan-Meier method. Statistical significance was evaluated using the logrank test for univariate influence and Cox model stepwise regression for multivariate influence. RESULTS: Two hundred seventy-eight patients (56%) had primary disease and 222 (44%) recurrent disease. Median follow-up was 28 months (range 1 to 172 months), 40 months for survivors. Median survival was 72 months for patients with primary disease, 28 months for those with local recurrence, and 10 months for those with metastasis. For patients with primary or locally recurrent tumors, unresectable disease, incomplete resection, and high-grade tumors significantly reduced survival time. CONCLUSIONS: In this study of patients with retroperitoneal STS, stage at presentation, high histologic grade, unresectable primary tumor, and positive gross margin are strongly associated with the tumor mortality rate. Patients approached with curative intent should undergo aggressive attempts at complete surgical resection. Incomplete resection should be undertaken only for symptom relief.     

Use of an antigen-capture assay for characterisation of monoclonal antibodies to mycobacterial lipoarabinomannan

Monoclonal antibodies directed to six separate antigen molecules of Mycobacterium leprae have been tested in an antigen-capture assay based on combined use of polyclonal ("capture") and monoclonal ("detector") antibody reagents. This approach provides a potentially versatile, sensitive and specific assay for detection and relative quantitation of M. leprae antigens. Characterisation of monoclonal antibodies to mycobacterial lipoarabinomannan (LAM-B) by the antigen-capture assay indicates that some of the antigenic determinants present on LAM-B from M. leprae may be either absent altogether or present at much lower concentrations on the corresponding LAM-B structure form M. tuberculosis.     

Immunoglobulin G, A, and M Responses in Serum and Circulating Immune Complexes Elicited by the 16-Kilodalton Antigen of Mycobacterium tuberculosis

The 16-kDa cytosolic antigen of M. tuberculosis was purified to homogeneity by molecular sieving chromatography, and the diagnostic potential of the antigen was evaluated in various categories of patients by enzyme-linked immunosorbent assay (ELISA). The immunoglobulin G (IgG), IgA, and IgM antibody levels to 16-kDa antigen were estimated in the two polar groups, namely, smear- and culture-positive pulmonary tuberculosis (S⁺C⁺) patients and healthy subjects (HS). Sensitivities of 62, 52 and 11% with specificities of 100, 97, and 95% were obtained for the three isotypes, respectively. The total number of positives by a combination of the three isotypes was analyzed in the polar groups, and the sensitivity improved to 83% with a specificity of 93%. Even when a combination of IgG and IgA alone was considered, the sensitivity was 82% with a specificity of 97%. Polyethylene glycol precipitation of the circulating immune complex (CIC) in sera was carried out. The CIC-bound antibodies to 16-kDa antigen were assessed by ELISA in the S⁺C⁺, S⁻C⁺, and S⁻C⁻ categories of patients. Measuring the IgG-IgA-IgM combination positivities of the CIC-bound antibodies gave sensitivities of 97.5, 100, and 45.3%, respectively. The specificity of the assay with these combinations was maintained at 95.4%.     

Serological specificity of phenolic glycolipid I from Mycobacterium leprae and use in serodiagnosis of leprosy

The serological activities of the specific phenolic glycolipid I from Mycobacterium leprae, its dissected parts, and related glycolipids from other mycobacteria were examined by enzyme-linked immunosorbent assay against hyperimmune anti-M. leprae rabbit antiserum and sera from patients with leprosy and other mycobacterial diseases. High anti-phenolic glycolipid I immunoglobulin M antibodies were found in 23 of 24 (96%) of lepromatous leprosy patients on short term chemotherapy and in 8 of 13 tuberculoid leprosy patients (62%). Sera from patients with tuberculosis or atypical mycobacterial infections were devoid of anti-phenolic glycolipid I activity. The structurally related phenolic glycolipids from Mycobacterium kansasii and Mycobacterium bovis and the aglycone segments of the M. leprae product showed no significant activity. Thus, the trisaccharide determinant of phenolic glycolipid I is specific in its structure, serological activity, and, to a lesser extent, the antibody class it evokes.     

Adrenocortical adenoma and carcinoma: histopathological and molecular comparative analysis.

We compared histomorphological features and molecular expression profiles of adrenocortical adenomas (ACAd) and carcinomas (ACCa). A critical histopathological review (mean, 11 slides per patient) was conducted of 37 ACAd and 67 ACCa. Paraffin-embedded tissue cores of ACAd (n = 33) and ACCa (n = 38) were arrayed in triplicate on tissue microarrays. Expression profiles of p53, mdm-2, p21, Bcl-2, cyclin D1, p27, and Ki-67 were investigated by immunohistochemistry and correlated with histopathology and patient outcome using standard statistical methodology. Median follow-up period was 5 years. Tumor necrosis, atypical mitoses, and >1 mitosis per 50 high-power fields were factors that were highly specific for ACCa (P <.001). Number (0 to 4) of unfavorable markers [Ki-67 (+), p21 (+), p27 (+), mdm-2(-)] expressed was significantly associated with mitotic activity and morphologic index (i.e., number of adverse morphologic features) and highly predictive of malignancy (P <.001). Ki-67 overexpression occurred in 0 ACAd and 36% ACCa (P <.001) and was significantly associated with mitotic rate and unfavorable morphologic index (P <.001). Tumor necrosis, atypical mitoses, >5 mitoses per 50 high-power fields, sinusoidal invasion, histologic index of >5, and presence of more than two unfavorable molecular markers were associated significantly with metastasis in ACCa. Well-established histopathologic criteria and Ki-67 can specifically distinguish ACCAd from ACCa. Tumor cell proliferation (Ki-67) correlates with mitotic activity and morphologic index. Tumor morphology is a better predictor of metastatic risk in ACCa than current immunohistochemistry-detected cell cycle regulatory and proliferation-associated proteins.     

Altered expression of the retinoblastoma gene product in human sarcomas

BACKGROUND. The retinoblastoma-susceptibility (Rb) gene is a prototype tumor-suppressor gene originally isolated from patients with heritable retinoblastoma. This gene encodes a nuclear phosphoprotein whose expression is altered in several types of human tumors. METHODS. We studied the expression of the Rb protein in 44 primary and 12 metastatic high-grade human sarcomas by means of immunohistochemical methods and Western blotting. Computerized image analysis was used to quantify the level of Rb gene product in individual tumor cells. The expression of the Rb gene was then correlated with clinical outcome in the patients with primary tumors. RESULTS. Of the 44 patients with primary sarcomas, 13 (30 percent) had tumors with normal, homogeneous expression of the Rb protein in essentially all tumor cells. Thirty-one patients with primary tumors (70 percent) had altered Rb expression; in 18 (40 percent) the Rb protein was heterogeneously expressed, and in 13 (30 percent) it was detected in fewer than 20 percent of the tumor cells. All 12 of the patients with metastatic sarcomas had altered expression of the Rb protein. When the findings in the patients with primary tumors were correlated with clinical outcome, survival was found to be significantly increased in the patients whose tumors had homogeneous Rb expression, as compared with those with either heterogeneous expression (P = 0.026) or no expression (P = 0.012). CONCLUSIONS. Tumors in which the expression of Rb gene product was decreased were more aggressive than tumors in which this protein was expressed by nearly all cells. The Rb gene product may be an important prognostic variable in patients with these tumors.     

Maxillary artery embolisation in the management of epistaxis

Serious consideration needs to be given to the importance of early embolisation of the maxillary artery in severe and refractory epistaxis. This is particularly true in the young fit person with traumatic epistaxis and in the elderly person who is unfit for general anaesthesia. Embolisation is now a safe and reliable technique. Six patients were embolised. In the two institutions from 1999 - 2000. All responded well to embolisation without complication. This paper describes the technique used in our hospitals and suggests that with the advent of platinum coils and pre embolisation arteriography, the chances of cerebrovascular accident is very small.     

Health-related quality of life following percutaneous coronary intervention during the COVID-19 pandemic

Quality of Life Research - During the COVID-19 pandemic, widespread public health measures were implemented to control community transmission. The association between these measures and...