Environmental bio-monitoring with high-throughput sequencing.

There is much interest in using high-throughput DNA sequencing methodology to monitor microorganisms, complex plant and animal communities. However, there are experimental and analytical issues to consider before applying a sequencing technology, which was originally developed for genome projects, to ecological projects. Many of these issues have been highlighted by recent microbial studies. Understanding how high-throughput sequencing is best implemented is important for the interpretation of recent results and the success of future applications. Addressing complex biological questions with metagenomics requires the interaction of researchers who bring different skill sets to problem solving. Educators can help by nurturing a collaborative interdisciplinary approach to genome science, which is essential for effective problem solving. Educators are in a position to help students, teachers, the public and policy makers interpret the new knowledge that metagenomics brings. To do this, they need to understand, not only the excitement of the science but also the pitfalls and shortcomings of methodology and research designs. We review these issues and some of the research directions that are helping to move the field forward.

A truncated precursor form of prostate-specific antigen is a more specific serum marker of prostate cancer.

Prostate-specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa) but has limited specificity for distinguishing early PCa from benign prostatic hyperplasia, because both PCa and benign prostatic hyperplasia release PSA into the serum. We have identified previously a truncated form of precursor PSA (pPSA) in prostate tumor extracts consisting of PSA with a serine-arginine pro leader peptide ([-2]pPSA) instead of the normally expressed 7 amino acid pro leader peptide. In the current study we developed monoclonal antibodies to detect [-2]pPSA and other isoforms of pPSA for Western blot analysis. PSA was immunoaffinity purified from 100 to 200 ml of serum from each of five men with biopsy-proven cancer and three biopsy-negative men, all with total PSA levels in the diagnostically relevant range near 10 ng/ml. The truncated [-2]pPSA was estimated to range from 25 to 95% of the free PSA in the five PCa samples but only 6-19% of the free PSA in the biopsy-negative men. Immunohistochemical studies showed positive staining for [-2]pPSA in PCa epithelium and that [-2]pPSA was enriched in cancer cell secretions. In vitro activation studies showed that human kallikrein 2 and trypsin readily activated full-length pPSA but were unable to activate [-2]pPSA to mature PSA. Thus, [-2]pPSA, once formed, is a stable but inactive isoform of PSA. Truncated [-2]pPSA may represent an important new diagnostic marker for the early detection of PCa.

Generation of PSA-ACT-specific monoclonal antibodies and their application in a sandwich immunoassay.

The prostate-specific antigen (PSA) immunoassay is an important tool for the detection and monitoring of prostate cancer. PSA exists in serum mainly as complexes with serine protease inhibitors including alpha1 antichymotrypsin (ACT) and alpha2 macroglobulin (MG). PSA-MG complex is not detected by the existing PSA immunoassays since MG (720 kDa) sequesters PSA and masks the antibody binding sites. Existing immunoassays for quantitation of total serum PSA measure PSA-ACT (CPSA) and free PSA (FPSA), which comprise the major and minor components of total PSA, respectively. Monoclonal antibodies (MAb) specific for CPSA alone were generated using a unique immunogen prepared by blocking the major antigenic determinants on FPSA and ACT. The blocked immunogen greatly enhanced the frequency of hybridomas reactive against the CPSA complex. CPSA prototype immunoassays were established using anti-CPSA (PX1G359) or anti-ACT (AC1A212) MAb as tracer MAb and anti-PSA (PSA399) MAb as capture MAb. The complex-selective MAbs demonstrated minimal cross-reactivity with Cathepsin-G (CG) ACT (CG-ACT), ACT or FPSA. CG-ACT complex interfered with the accuracy of initial prototype assays specific for CPSA measurement and caused over-recovery (1 to 3 ng/mL, with 40 to 180 ng/mL range of CG-ACT in serum) of apparent CPSA values. Addition of 0.4% NP-40 and 0.1% 0.088 micron microparticles in clinical specimens eliminated this interference. Specimens from 39 prostate cancer (PCa) patients and 44 benign prostatic hyperplasia (BPH) patients were analyzed with the PX1G359 CPSA assay. In this study, the area under the curve (AUC) values for ROC analysis of total PSA (CPSA+FPSA), FPSA to total PSA ratio (f/t), and FPSA to CPSA ratio (f/c) were 0.357, 0.634, and 0.624, respectively. In a second study using AC1A212 CPSA assay, where specimens from 16 PCa patients and 48 BPH patients were tested, the AUC values for total PSA, f/t and f/c ratios were 0.62, 0.785, and 0.732, respectively. Using the CPSA assay with minimal interference our studies are consistent with previous CPSA data showing that the f/t PSA ratio remains superior to the f/c PSA ratio in differentiating PCa and BPH diseases. Complex PSA by itself or as ratio with free or total PSA does not provide any advantage over the established method of FPSA to total PSA ratio.

Western blotting analysis of antibodies to prostate-specific antigen: cross-reactivity with human kallikrein-2.

Immunoassays for prostate-specific antigen (PSA) using monoclonal or polyclonal antibodies have clinical applications, such as monitoring and early detection of prostate cancer. However, PSA shares 80% sequence homology with human glandular kallikrein (hK2). In the present study, we have used SDS-PAGE and Western blotting of a recombinant form of hK2 to determine the degree of cross-reactivity in a panel of 83 antibodies submitted to the ISOBM TD-3 PSA Workshop. From this panel, 24 of the 83 antibodies showed cross-reactivity with hK2. The majority of these antibodies showed binding to conformationally independent or linear epitopes. Fourteen of these antibodies appear to map to the same epitope group, indicating the existence of a dominant and linear immunogenic domain shared by PSA and hK2.

Western blotting analysis of antibodies to prostate-specific antigen: specificities for prostate-specific antigen and prostate-specific antigen fragments.

Approximately 40% of the prostate-specific antigen (PSA) purified from seminal fluid comprises cleaved or fragmented forms of PSA. These fragments are observed by reduced SDS-PAGE and have been identified in preparations of purified seminal plasma. The comparative analysis of 53 antibodies, submitted to an international PSA Workshop, with different PSA variants was studied using SDS-PAGE and Western blotting. Six different patterns of reactivity were observed which may reflect different epitopes recognized by this panel of antibodies. Additional antibody studies to nonreduced intact PSA suggest the epitopes are conformation-dependent.

Summary report of the TD-3 workshop: characterization of 83 antibodies against prostate-specific antigen.

Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86-91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158-163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3-11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.

Growth and metabolic responses in preterm infants fed fortified human milk or a preterm formula.

Preterm infants fed human milk have been shown to grow poorly and develop mineral deficiencies that may lead to osteopenia. This study has investigated the efficacy of a human milk fortifier made up of glucose polymers, a mixture of bovine milk protein fractions and free amino acids, minerals and vitamins designed to improve these nutritional deficiencies. Growth and bone mineral deficiencies were compared in 38 preterm infants fed fortified mother's milk and 21 preterm infants fed a preterm formula until they reached 1800 g; all had a birthweight below 1600 g. Weight gain was similar in each group with a mean (SD) increase of 19.6 (3.5) g/kg/day in the fortified group and 19.9 (4.1) g/kg/day in the preterm formula group. There were also no significant differences in linear growth, head circumference, skin fold thickness or mid-arm circumference. Serum phosphate, alkaline phosphatase and plasma urea concentrations were similar and there was no clinical evidence of osteopenia. These results indicate that the growth and metabolic disadvantages associated with feeding human milk to preterm infants are ameliorated by the addition of a milk fortifier that increases the calorific, protein and mineral content of breast milk.

The effect of ephedrine isomers and their oxazolidines on locomotor activity in rats.

1. The four stereoisomeric ephedrines and four oxazolidines formed by reaction of each ephedrine isomer with salicylaldehyde were tested for their ability to increase locomotor activity in rats. 2. All ephedrine isomers except (-)pseudoephedrine increased locomotor activity; the order of potency was (-)ephedrine > (+) pseudoephedrine > (+) ephedrine. 3. All oxazolidine derivatives increased locomotor activity except (-)pseudoephedrine oxazolidine. 4. The oxazolidines derived from (-) and (+) ephedrine were slightly more active than their parent drugs. (+) Pseudoephedrine oxazolidine was less active than its parent. 5. Half-lives of hydrolysis were measured for the oxazolidines in aqueous buffer, ephedrine oxazolidines hydrolyze faster than pseudoephedrine oxazolidines.

Value of the Hybritech ICON Candida Assay in the diagnosis of invasive candidiasis in high-risk patients.

A total of 314 sera from 114 patients at risk for invasive candidiasis were analyzed for the presence of antigenemia using the Hybritech enzyme immunoassay (EIA) for detection of Candida mannan in serum (ICON Candida Assay, Hybritech Inc., San Diego, CA). Fourteen patients (12%) had invasive candidiasis documented by positive blood cultures, deep biopsy culture, and histopathology or autopsy, and five patients had probable invasive candidiasis based on a single positive blood culture and no additional signs of candidiasis. Nine patients had candiduria, 43 patients had mucous membrane colonization, 25 patients were not colonized but received empiric amphotericin B, and 18 patients were not colonized and not treated with amphotericin B. All sera were enzymatically extracted, heat treated, and reacted in a solid-phase sandwich EIA. Test results were read visually and with the ICON reader. The sensitivity and specificity of the mannan EIA in detection of documented invasive candidiasis was 86% and 92%, respectively. The positive predictive value was 60% and the negative predictive value was 98%. Among all patients with invasive candidiasis (documented plus probable), the sensitivity was 68%, the positive predictive value 62%, and the negative predictive value 94%. Specimens were positive within 3 days of the first positive culture in 11 (79%) of 14 patients with documented invasive candidiasis.

Enzyme-linked immunosorbent assay (ELISA) for metallothionein.

A heterogeneous, double-antibody, fluorometric enzyme-linked immunosorbent assay (ELISA) is described for the detection and quantitation of metallothionein(MT) The protocol uses the same immunological reagents as the radioimmunoassay(RIA) developed previously in this laboratory; fluorescence replaces radioactivity for detection of the reference antigen in the specific binding reaction. Present results indicate that the developed ELISA has approximately the same range of capability in detecting and quantitating MT as is characteristic of the RIA. The ELISA has the advantage that the time required to perform a typical assay is significantly less than that required for the RIA.

Fluorometric ELISA for the detection and quantitation of metallothionein.

The development of a heterogeneous fluorometric ELISA for the detection and quantitation of metallothionein (MT) is described. A radioimmunoassay (RIA) previously developed in our laboratory is used as a reference assay to characterize the performance of the ELISA. The standard curves (logit-log regressions) that are typical of either assay have similar ranges (customarily from 20 000 to 100 pg of competing antigen); both assays are capable of quantitating MT in unknowns with 5-10% accuracy. Aspects of MT measurement in cytosols and physiological fluids are discussed.

Significance of urinary metallothionein in workers exposed to cadmium.

Cadmium in blood (Cd-B) and in urine (Cd-U) and metallothionein (Mt-U) and beta 2-microglobulin in urine (beta 2m-U) were measured in 94 male Cd workers. The results were examined according to the workers' current exposure to cadmium (group C, n = 73, workers currently exposed to Cd; group R, n = 21, Cd workers removed from exposure or retired) and according to their renal status (group N, n = 66, normal beta 2m-U; group I, n = 28, beta 2m-U greater than 200 micrograms/g creatinine). The interrelationships between Mt-U, Cd-U, Cd-B and years of cadmium exposure were examined in the various subgroups. The study of the correlations between these variables demonstrates that Mt-U is directly correlated with Cd-U but not with Cd-B or years of Cd exposure. The association between Cd-U and Mt-U is independent of the status of renal function and the intensity of current exposure to cadmium. Under moderate chronic exposure to cadmium, the fraction of Cd-U which is directly influenced by recent exposure (Cd-B) is small in comparison with that influenced by the cadmium body burden.