Nanopore DNA Sequencing for Metagenomic Soil Analysis.

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA sequences identified using computer software. Nanopore DNA sequencing is a flexible technique that allows for rapid microbial genome sequencing to identify bacterial and viral species, to characterize bacterial strains, and to detect genetic mutations that confer resistance to antibiotics. The advantages of nanopore sequencing (NS) for life sciences include its low complexity, reduced cost, and rapid real-time sequencing of purified genomic DNA, PCR amplicons, cDNA samples, or RNA. NS is an example of "strand sequencing" which involves sequencing DNA by guiding a single stranded DNA molecule through a nanopore that is inserted into a synthetic polymer membrane. The membrane has an electrical current applied across it, so as the individual bases pass through the nanopore the electrical current is disrupted to varying degrees by the four nucleotide bases. The identification of each nucleotide occurs by detecting the characteristic modulation of the electrical current by the different bases as they pass through the nanopore. The NS system consists of a handheld, USB powered portable device and a disposable flow cell that contains a nanopore array. The portable device plugs into a standard laptop computer that reads and records the DNA sequence using computer software.

Low molecular mass protein patterns in mycobacterial culture filtrates and purified protein derivatives.

Mycobacterial polypeptides from 2 kDa to 14 kDa may be involved in host response to infection and be useful for new diagnostics and vaccines. Tris-tricine SDS-polyacrylamide gel electrophoresis separation of proteins in tuberculin purified protein derivative (PPD) and in 6-8-week culture filtrates of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and other Mycobacterium species demonstrated as many as 10 low molecular mass bands. Common and distinct bands were observed among different species and PPD. These low molecular mass culture filtrate proteins may represent potential diagnostic reagents and vaccines for Mycobacterium tuberculosis.

Generation of a recombinant bacteriophage antibody library to mycobacterium tuberculosis.

Monoclonal antibodies to Mycobacterium tuberculosis (Mtb) may be useful for laboratory diagnosis, antigenic characterization, and studying the immune response to infection and vaccination. To investigate the potential of the recombinant phage antibody technique for the isolation of single-chain fragments (ScFv) reactive with Mtb culture proteins, we generated a bacteriophage antibody library from splenic tissue of mice immunized with heat-killed Mtb. Heavy- and light-chain immunoglobulin genes were isolated and amplified by the polymerase chain reaction (PCR), and the products were assembled into ScFv gene fragments and cloned into the pCANTAB5-E phagemid. Phagemids were introduced into E. coli TG1 by electrotransformation, followed by rescue of antibody-expressing phage using M13K07 helper-phage superinfection. Immunoselection of the Mtb phage library against Mtb cell wall extract or culture filtrate proteins selected antigen-specific and cross-reactive phage antibodies, none of which demonstrated reactivity to the immunodominant 65-kDa Mtb antigen. These results suggest that the phage antibody system has the potential to generate a diverse population of the reactive phage that may prove useful for investigating the immune response to Mtb infection and vaccination.

Production and characterization of monoclonal antibodies to human carbonic anhydrase III.

Carbonic anhydrase III (CAIII) is a cytosolic protein found almost exclusively in slow-oxidative skeletal muscle fibers. Upon excessive skeletal muscle activity or damage, CAIII is rapidly released into serum. CAIII is not found in cardiac muscle, whereas the muscle protein myoglobin (Myo) is found in skeletal and cardiac muscle. Because CAIII and Myo are released from injured muscle in a constant ratio, an increase in the Myo/CAIII ratio may be useful as an early diagnostic indicator of acute myocardial damage. Although several reliable Myo immunoassays have been established, no similar CAIII immunoassay is commercially available. We produced murine monoclonal antibodies (MAbs) to human CAIII using standard immunization and cell fusion procedures. Using an enzyme-linked immunoadsorbent assay (ELISA), three MAbs showed strong immunoreactivity with CAIII, but low to moderate levels of cross-reactivity with closely related isoenzymes CAI and CAII. The three MAbs demonstrated unique patterns of reactivity toward CAI, CAII, and CAIII, suggesting that different CAIII epitopes are recognized by the three MAbs. Specificity was further examined by Western blot analysis. These MAbs demonstrated potential for use in the development of an immunoassay for CAIII, and for investigating the biology of skeletal muscle injury in vivo.

Coevolution of virulent virus and resistant cells as a mechanism of persistence of herpes simplex virus type 1 in a human T lymphoblastoid cell line.

Infection of the lymphoblastoid CEM cell line with herpes simplex virus (HSV) type 1 results in a persistent infection with production of infectious virus. Evidence suggests that the persistent infection was not maintained by interferon or non-interferon-soluble antiviral inhibitors. Treatment of persistently infected cells with anti-HSV serum (termed CEMACR cells) or elevated temperature (39 degrees C) for 14 days (termed CEMTCR cells) resulted in loss of evidence of virus. HSV DNA was not detected in CEMACR or CEMTCR cells by Southern blot or in situ hybridization. The CEMACR or CEMTCR cells, however, were resistant to reinfection with homologous, parental virus (HSV0), but were susceptible to heterologous virus (vesicular stomatitis virus). Resistance to reinfection with HSV was not absolute; CEMACR or CEMTCR cells were less permissive to virus isolated from persistently infected cultures at times early in the course of infection, but were more permissive for HSV isolated at later times. Virus isolated later during persistent infection also displayed progressively increased virulence for the parental CEM cells. These results suggest that persistent infection of a human T lymphoblastoid cell line, CEM, with HSV-1 is maintained by a genetically determined cell-virus equilibrium, in which the resistance of cells and virulence of virus increase during persistence.

Transforming growth factor-beta regulation of IL-6 production by unstimulated and IL-1-stimulated human fibroblasts.

We characterized the ability of transforming growth factor-beta 1 (TGF-beta 1) and transforming growth factor-beta 2 (TGF-beta 2) to regulate IL-6 production by unstimulated and rIL-1-stimulated lung fibroblasts. rTGF-beta 1-, purified TGF-beta 1-, and purified TGF-beta 2-stimulated fibroblasts produced IL-6 bioactivity as assessed with the B9 hybridoma proliferation assay. These TGF-beta moieties also bidirectionally regulated the IL-6 production of rIL-1-stimulated fibroblasts. The addition of TGF-beta to cultures in which fibroblasts were vigorously stimulated with rIL-1 resulted in an inhibition of fibroblast IL-6 production and mRNA accumulation. In contrast, the addition of TGF-beta to cultures in which fibroblasts were incubated with suboptimal concentrations of rIL-1 resulted in a synergistic increase in IL-6 production and mRNA accumulation [corrected]. Nuclear run-on analysis demonstrated that IL-6 gene [corrected] transcription was synergistically augmented when rTGF-beta 1 was combined with suboptimal concentrations of rIL-1. These studies demonstrate that TGF-beta stimulates fibroblast IL-6 production. They also show that TGF-beta can augment or inhibit the IL-6 production of IL-1-stimulated fibroblasts. Lastly, [corrected] they demonstrate that the stimulatory effects of TGF-beta are, at least partially, mediated by alterations in IL-6 gene transcription. TGF-beta may be an important regulator of IL-6 production. stimulated fibroblasts. Last, they demonstrate that the stimulatory effects of TGF-beta are, at least partially, mediated by alterations in IL-6 gene transcription. TGF-beta may be an important regulator of IL-6 production.

Characterization of herpes simplex virus persistence in a human T lymphoblastoid cell line.

Persistent, dynamic-state infection with herpes simplex virus (HSV) type 1 has been maintained in human T lymphoblastoid (CEM) cells for many months after initial infection with the wild-type virus (HSV0) (input virus/cell multiplicity of 1.0). Persistently infected cells grew as well as uninfected cells, except during occasional periods of crisis (increased viral replication and cytopathic effect). Cells could survive the crisis when they were maintained for twice the usual time interval (8 to 10 rather than 4 to 5 days) before subculture. Interferon was not detectable in the cultures. HSV0 was compared with HSVp1, a small plaque-forming isolate from persistently infected CEM cells. Primary infection of CEM cells with HSV0 at a low input multiplicity (0.01) led to abortive replication, whereas infection with HSVp1 at the same multiplicity resulted in either rapidly lytic or persistent infection depending upon the time interval of subculture. Approximately 55% of plaque-purified clones of HSVp1, as compared with only 5% of HSV0 clones, displayed temperature-sensitive growth in Vero cells. Defective interfering virus was not detectable in uncloned HSVp1 by interference assay. Persistently infected cultures "cured" by treatment with HSV antiserum or incubation at 39 degrees C were resistant to reinfection with HSV but permissive for vesicular stomatitis virus replication, suggesting that these treatments modulated a shift from the dynamic-state of the static-state, latent infection. These studies provide a model for characterization of HSV persistence and latency in a highly differentiated human cell line.

Differential expression of a Mycobacterium tuberculosis protein recognized by a mouse monoclonal antibody.

Murine monoclonal antibodies were produced against Mycobacterium tuberculosis (Mtb) using standard hybridoma procedures. By a whole cell enzyme-linked immunosorbent assay (ELISA), one monoclonal antibody (mAb), HB28, demonstrated high level specific reactivity to Mtb. Western blot analysis demonstrated reactivity to a single 65 kDa Mtb protein in the cell wall extract and culture filtrate. HB28 mAb appears to be recognizing a 65 kDa Mtb protein that is over-expressed by Mtb but not other species under certain culture conditions. Differential expression and detection of this protein by HB28 mAb may have potential for diagnostic applications.

A comparison of intra-arterial and intravenous tissue-type plasminogen activator on autologous arterial emboli in the cerebral circulation of rabbits.

BACKGROUND AND PURPOSE: The efficacy of thrombolytic therapy for treatment of embolic stroke has been a subject of both experimental and clinical examination. The aim of this study was to compare the efficacy, in regard to reduction of volume of ischemic brain, of two different modes of administration (ie, intra-arterial and intravenous) of tissue-type plasminogen activator (TPA) given 30 minutes after experimental embolic stroke in rabbits. METHODS: A randomized, blinded, controlled experimental trial was undertaken. Embolic stroke was simulated in rabbits by injecting fragments of autologous arterial thrombus into one internal carotid artery. Thirty minutes after embolization, the rabbits were blindly treated with 2 mg/kg intra-arterial TPA, 2 mg/kg intravenous TPA, or saline (all n = 10). Six hours after embolization the rabbits were killed. The brains were perfused with triphenyltetrazolium chloride and cut into 0.5-cm-thick coronal sections, and the areas of ischemia were measured. RESULTS: Administration of TPA resulted in a significant reduction in the volume of ischemic cerebral injury (P < .0001): control animals sustained ischemic injury to 20.1 +/- 4.6% (mean +/- SD) of total brain compared with 4.6 +/- 4.1% for animals treated with intra-arterial TPA and 3.4 +/- 2.6% for those treated with intravenous TPA. The difference between intra-arterial and intravenous TPA treatment was not significant (P = .786). CONCLUSIONS: In this rabbit model of embolic stroke, administration of TPA within 30 minutes resulted in a dramatic reduction in the amount of ischemic injury, with equal efficacy for the two modes of administration. These results favor the treatment of acute embolic stroke with intravenous TPA, given the rapidity with which intravenous therapy can be established in the clinical setting.