Group portrait, testimonial dinner for Dr. Paul Klemperer's 10th anniversary at Mt. Sinai Hospital given by his students; Cavanagh's, New York City Portraits Groups

Digital Image

An improved real-time PCR assay for the detection of Old World screwworm flies

The Old World screwworm (OWS) fly, Chrysomya bezziana, is a serious pest of livestock, wildlife and humans in tropical Africa, parts of the Middle East, the Indian subcontinent, south-east Asia and Papua New Guinea. Although to date Australia remains free of OWS flies, an incursion would have serious economic and animal welfare implications. For these reasons Australia has an OWS fly preparedness plan including OWS fly surveillance with fly traps. The recent development of an improved OWS fly trap and synthetic attractant and a specific and sensitive real-time PCR molecular assay for the detection of OWS flies in trap catches has improved Australia's OWS fly surveillance capabilities. Because all Australian trap samples gave negative results in the PCR assay, it was deemed necessary to include a positive control mechanism to ensure that fly DNA was being successfully extracted and amplified and to guard against false negative results. A new non-competitive internal amplification control (IAC) has been developed that can be used in conjunction with the OWS fly PCR assay in a multiplexed single-tube reaction. The multiplexed assay provides an indicator of the performance of DNA extraction and amplification without greatly increasing labour or reagent costs. The fly IAC targets a region of the ribosomal 16S mitochondrial DNA which is conserved across at least six genera of commonly trapped flies. Compared to the OWS fly assay alone, the multiplexed OWS fly and fly IAC assay displayed no loss in sensitivity or specificity for OWS fly detection. The multiplexed OWS fly and fly IAC assay provides greater confidence for trap catch samples returning negative OWS fly results. © 2014 International Atomic Energy Agency.

Development of a Rapid Multiplex PCR Assay To Genotype Pasteurella multocida Strains by Use of the Lipopolysaccharide Outer Core Biosynthesis Locus

Pasteurella multocida is a Gram-negative bacterial pathogen that is the causative agent of a wide range of diseases in many animal species, including humans. A widely used method for differentiation of P. multocida strains involves the Heddleston serotyping scheme. This scheme was developed in the early 1970s and classifies P. multocida strains into 16 somatic or lipopolysaccharide (LPS) serovars using an agar gel diffusion precipitin test. However, this gel diffusion assay is problematic, with difficulties reported in accuracy, reproducibility, and the sourcing of quality serovar-specific antisera. Using our knowledge of the genetics of LPS biosynthesis in P. multocida, we have developed a multiplex PCR (mPCR) that is able to differentiate strains based on the genetic organization of the LPS outer core biosynthesis loci. The accuracy of the LPS-mPCR was compared with classical Heddleston serotyping using LPS compositional data as the "gold standard." The LPS-mPCR correctly typed 57 of 58 isolates; Heddleston serotyping was able to correctly and unambiguously type only 20 of the 58 isolates. We conclude that our LPS-mPCR is a highly accurate LPS genotyping method that should replace the Heddleston serotyping scheme for the classification of P. multocida strains.

Evaluation of a multiplex PCR to identify and serotypeActinobacillus pleuropneumoniaeserovars 1, 5, 7, 12 and 15

The aim of this study was to validate a multiplex PCR for the species identification and serotyping of Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15. All 15 reference strains and 411 field isolates (394 from Australia, 11 from Indonesia, five from Mexico and one from New Zealand) of A. pleuropneumoniae were tested with the multiplex PCR. The specificity of this multiplex PCR was validated on 26 non-A. pleuropneumoniae species. The multiplex PCR gave the expected results with all 15 serovar reference strains and agreed with conventional serotyping for all field isolates from serovars 1 (n = 46), 5 (n = 81), 7 (n = 80), 12 (n = 16) and serovar 15 (n = 117). In addition, a species-specific product was amplified in the multiplex PCR with all 411 A. pleuropneumoniae field isolates. Of 25 nontypeable field isolates only two did not yield a serovar-specific band in the multiplex PCR. This multiplex PCR for serovars 1, 5, 7, 12 and 15 is species specific and capable of serotyping isolates from diverse locations. Significance and Impact of the Study A multiplex PCR that can recognize serovars 1, 5, 7, 12 and 15 of A. pleuropneumoniae was developed and validated. This novel diagnostic tool will enable frontline laboratories to provide key information (the serovar) to guide targeted prevention and control programmes for porcine pleuropneumonia, a serious economic disease of pigs. The previous technology, traditional serotyping, is typically provided by specialized reference laboratories, limiting the capacity to respond to this key disease.

First report of Cotton leafroll dwarf virus in Thailand using a species-specific PCR validated with isolates from Brazil

Partial virus genome sequence with high nucleotide identity to Cotton leafroll dwarf virus (CLRDV) was identified from two cotton (Gossypium hirsutum) samples from Thailand displaying typical cotton leaf roll disease symptoms. We developed and validated a PCR assay for the detection of CLRDV isolates from Thailand and Brazil.

Der Prediger vom weißen Berg : Zur Rezeption der besseren Gerechtigkeit aus Mt 5 in Martin Luthers Predigtüberlieferung 1522 1546.

The purpose of this study is to examine the reception of Matthew 5 in Martin Luther s sermons; in other words to investigate how Luther interprets and applies Jesus teaching of the better righteousness and the law in Mt 5. The study applies the reception-historical approach and contributes to the history of effects and the history of interpretation in New Testament exegesis. The study shows that Luther understands the better righteousness of Mt 5 as good works and fulfillment of the law. Luther s interpretation coheres with the intention of the Evangelist, even if Luther s overall concept of righteousness is foreign to Matthew. In Luther s view righteousness is twofold: The greater righteousness of Mt 5 is the second and the actual righteousness (iustitia activa), which follows the first and the foreign righteousness (iustitia passiva). The first righteousness (faith) is for Luther the work of God, while the second righteousness (good works) is co-operation between a Christian and God. In this co-operation the law, as it is taught by Jesus, is not the opposite of the gospel, but the gospel itself in the sense of Christ as an example . The task of the law is to show the dependence of a Christian on God and to help one to love and to serve one s neighbour (brothers as well as enemies) properly. The study underlines a feature in Luther s thinking that has received little attention in Lutheran theology: Luther insists on preaching the law to Christians. In his view Mt 5 is directed to all Christians and particularly to pastors, for whom Jesus here gives an example of how to preach the law. Luther believes similarly to Matthew that Jesus reveals the real meaning of Mosaic Law and confirms its validity for Christians in Mt 5. Like Matthew, Luther insists on the practicability of the commandments of Mt 5 in his view Christians fulfil the law also with joy yet his interpretation of Mt 5 attenuates the radical nature of its commandments. Luther s reception of the individual pericopes of Mt 5 is considerably generative and occasionally contradictory, which is explained by the following factors, among others: Luther receives many ideas from tradition and reads them and his own theological concepts into Matthew s Gospel. He interprets Mt 5 through his understanding of some Old Testament passages as well as Paul. Most of all, Luther s reception of Mt 5 is shaped by his own experience as a preacher, by his relation to his religious enemies, rulers and to the congregation of Wittenberg. Here Luther shares with Matthew the experience of being opposed and concern about the upright living of the believers, which in both cases also explains the polemical tone of the paraenesis.

Development of a Mixing Device for Gas-Solid Systems and Its Application to MT Reactors

A new arrangement to achieve adequate mixing between gas and solid is described. Residence time distribution studies ensured that the behavior of this device actually approaches that of a completely mixed system. The applicability of this device in MT reactors was verified by studying the vapor phase catalytic oxidation of anthracene over vanadium pentoxide.

Gene expression profiling during Forskolin induced differentiation of BeWo cells by differential display RT-PCR

The differentiation of cytotrophoblasts into syncytiotrophoblasts in the placenta has been employed as a model to investigate stage specific expression as well as regulation of genes during this process. While the cytotrophoblasts are highly invasive and proliferative with relatively less capacity to synthesize pregnancy related proteins, the multinucleated syncytiotrophoblasts are non-proliferative and non-invasive. However, syncytiotrophoblasts are the site of synthesis of a variety of protein, peptide and steroid hormones as well as several growth factors. Both the freshly isolated cytotrophoblasts from human placenta as well as the BeWo cell, a choriocarcinoma cell line model which retain several characteristic of cytotrophoblasts has been employed by us to study regulation of differentiation. In the present study, we have employed the differential display RT-PCR analysis (DD-RT-PCR) to evaluate gene expression changes during Forskolin induced in vitro differentiation of BeWo cells. We have identified several genes which are differentially expressed during differentiation and the differential expression of 10 transcripts was confirmed by Northern blot analysis. Based on the identity of the transcripts an attempt has been made to relate the known function of the gene products, to changes observed during differentiation. Of the several transcripts, one of the transcripts, namely Secretory Leukocyte Protease Inhibitor (SLPI) which is known to have multiple functions was found to increase 15-fold in the syntiotrophoblast.

Novel fluorescence detection technique for non-contact temperature sensing in microchip PCR

DNA amplification using Polymerase Chain Reaction (PCR) in a small volume is used in Lab-on-a-chip systems involving DNA manipulation. For few microliters of volume of liquid, it becomes difficult to measure and monitor the thermal profile accurately and reproducibly, which is an essential requirement for successful amplification. Conventional temperature sensors are either not biocompatible or too large and hence positioned away from the liquid leading to calibration errors. In this work we present a fluorescence based detection technique that is completely biocompatible and measures directly the liquid temperature. PCR is demonstrated in a 3 ILL silicon-glass microfabricated device using non-contact induction heating whose temperature is controlled using fluorescence feedback from SYBR green I dye molecules intercalated within sensor DNA. The performance is compared with temperature feedback using a thermocouple sensor. Melting curve followed by gel electrophoresis is used to confirm product specificity after the PCR cycles. (c) 2007 Elsevier B.V. All rights reserved.

Performance of RT-PCR-ELISA for the detection of peste des petits ruminants virus

A highly sensitive and specific reverse transcription polymerase chain reaction enzyme linked immunosorbent assay (RT-PCR-ELISA) was developed for the objective detection of nucleoprotein (N) gene of peste des petits ruminants (PPR) virus from field outbreaks or experimentally infected sheep. Two primers (IndF and Np4) and one probe (Sp3) available or designed for the amplification/probing of the 'N' gene of PPR virus, were chosen for labeling and use in RT-PCR-ELISA based on highest analytical sensitivity of detection of infective virus or N-gene containing recombinant plasmid, higher nucleotide homology at the primer binding sites of the 'N' gene sequences available and the ability to amplify PPR viral genome from different sources of samples. RT-PCR was performed with unlabeled IndF and Np4 digoxigenin labeled primers followed by a microplate hybridization probe reaction with biotin labeled Sp3 probe. RT-PCR-ELISA was found to be 10-fold more sensitive than the conventional RT-PCR followed by agarose gel based detection of PCR product. Based on the Mean (mean +/- 3S.D.) optical density (OD) values of 47 RT-PCR negative samples, OD values above 0.306 were considered positive in RT-PCR-ELISA. A total of 82 oculo-nasal swabs and tissue samples from suspected PPR cases were analyzed by RT-PCR and RT-PCR-ELISA, which revealed 54.87 and 58.54% positivity, respectively. From an experimentally infected sheep, both RT-PCR and RT-PCR-ELISA could detect the virus from 6 days post-infection up to 9 days in oculo-nasal swabs. On post-mortem, PPR viral genome was detected in spleen, lymph node, lung, heart and liver. The correlation co-efficient between RT-PCR-ELISA OD values and either TCID50 of virus or molecules of DNA was 0.622 and 0.657, respectively. The advantages of RT-PCR-ELISA over the conventional agarose gel based detection of RT-PCR products are discussed. (c) 2006 Elsevier B.V. All rights reserved.

Cloning by genomic PCR and production of peanut agglutinin in Escherichia coli

Using the polymerase chain reaction, the coding sequence for peanut agglutinin (PNA) was cloned and expressed in Escherichia coli. Amplified PNA is identical to previously reported cDNA, suggesting the absence of any introns in PNA gene. Recombinant (re-) PNA forms inclusion bodies in E. coli. Production of PNA was confirmed by probing Western blots with polyclonal anti-PNA immunoglobulin G. Inclusion bodies were solubilized with 6 M guanidine-HCl and renatured by rapid dilution in the presence of metal ions. The renatured lectin was then purified by affinity chromatography. The re-lectin shows carbohydrate-binding properties similar to the natural PNA. This expression system provides a model for future mutagenesis studies of the carbohydrate-binding site and thus facilitates ongoing efforts to explore the molecular basis for the specificity of lectin-carbohydrate interaction.

Real-Time PCR : A Molecular Approach to Investigate the Role of Intestinal Microbiota in the Pathophysiology of Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is a common multifactorial functional intestinal disorder, the pathogenesis of which is not completely understood. Increasing scientific evidence suggests that microbes are involved in the onset and maintenance of IBS symptoms. The microbiota of the human gastrointestinal (GI) tract constitutes a massive and complex ecosystem consisting mainly of obligate anaerobic microorganisms making the use of culture-based methods demanding and prone to misinterpretation. To overcome these drawbacks, an extensive panel of species- and group-specific assays for an accurate quantification of bacteria from fecal samples with real-time PCR was developed, optimized, and validated. As a result, the target bacteria were detectable at a minimum concentration range of approximately 10 000 bacterial genomes per gram of fecal sample, which corresponds to the sensitivity to detect 0.000001% subpopulations of the total fecal microbiota. The real-time PCR panel covering both commensal and pathogenic microorganisms was assessed to compare the intestinal microbiota of patients suffering from IBS with a healthy control group devoid of GI symptoms. Both the IBS and control groups showed considerable individual variation in gut microbiota composition. Sorting of the IBS patients according to the symptom subtypes (diarrhea, constipation, and alternating predominant type) revealed that lower amounts of Lactobacillus spp. were present in the samples of diarrhea predominant IBS patients, whereas constipation predominant IBS patients carried increased amounts of Veillonella spp. In the screening of intestinal pathogens, 17% of IBS samples tested positive for Staphylococcus aureus, whereas no positive cases were discovered among healthy controls. Furthermore, the methodology was applied to monitor the effects of a multispecies probiotic supplementation on GI microbiota of IBS sufferers. In the placebo-controlled double-blind probiotic intervention trial of IBS patients, each supplemented probiotic strain was detected in fecal samples. Intestinal microbiota remained stable during the trial, except for Bifidobacterium spp., which increased in the placebo group and decreased in the probiotic group. The combination of assays developed and applied in this thesis has an overall coverage of 300-400 known bacterial species, along with the number of yet unknown phylotypes. Hence, it provides good means for studying the intestinal microbiota, irrespective of the intestinal condition and health status. In particular, it allows screening and identification of microbes putatively associated with IBS. The alterations in the gut microbiota discovered here support the hypothesis that microbes are likely to contribute to the pathophysiology of IBS. The central question is whether the microbiota changes described represent the cause for, rather than the effect of, disturbed gut physiology. Therefore, more studies are needed to determine the role and importance of individual microbial species or groups in IBS. In addition, it is essential that the microbial alterations observed in this study will be confirmed using a larger set of IBS samples of different subtypes, preferably from various geographical locations.

Excision of uracil from the ends of double stranded DNA by uracil DNA glycosylase and its use in high efficiency cloning of PCR products

We show that uracil DNA glycosylase from E. coli excises uracil residues from the ends of double stranded oligos. This information has allowed us to develop an efficient method of cloning PCR amplified DNA. In this report, we describe use of this method in cloning of E. coli genes for lysyl- and methionyl-tRNA synthetases. Efficiency of cloning by this method was found to be the same as that of subcloning of DNA restriction fragments from one vector to the other vector. Possibilities of using other DNA glycosylases for such applications are discussed.

Isolation of transforming growth factor-beta 2 cDNA from a fish, Cyprinus carpio by RT-PCR

Transforming Growth Factors-beta (TGF-beta s) have been described in many vertebrate species of amphibians, aves and mammals. In this report we demonstrate the presence of TGF-beta 2 in pisces. TGF-beta 2 has been cloned from a fish, Cyrinus carpio, by RT-PCR using degenerate oligonucleotide primers. Sequence analysis of the amplified product and alignment of the deduced amino acid sequence with the human TGF-beta 2 amino acid sequence revealed 81% and 93% identity in the precursor and the mature regions, respectively. The northern blot analysis of fish heart RNA shows a major messenger RNA species of about 8.0 kb and two messages of very low abundance of about 5.0 kb and 4.0 kb. The identification of TGF-beta 2 isoform in Pisces and it's high degree of homology with the mammalian isoform suggests that among all TGF-beta isoforms, TGF-beta 2 is the most conserved during evolution. (C) 1997 Elsevier Science B.V.