Biogeochemistry and community ecology in a spring-fed urban river following a major earthquake.

In February 2011 a MW 6.3 earthquake in Christchurch, New Zealand inundated urban waterways with sediment from liquefaction and triggered sewage spills. The impacts of, and recovery from, this natural disaster on the stream biogeochemistry and biology were assessed over six months along a longitudinal impact gradient in an urban river. The impact of liquefaction was masked by earthquake triggered sewage spills (~20,000 m(3) day(-1) entering the river for one month). Within 10 days of the earthquake dissolved oxygen in the lowest reaches was <1 mg l(-1), in-stream denitrification accelerated (attenuating 40-80% of sewage nitrogen), microbial biofilm communities changed, and several benthic invertebrate taxa disappeared. Following sewage system repairs, the river recovered in a reverse cascade, and within six months there were no differences in water chemistry, nutrient cycling, or benthic communities between severely and minimally impacted reaches. This study highlights the importance of assessing environmental impact following urban natural disasters.

Evaluation of methodology for detection of human adenoviruses in wastewater, drinking water, stream water and recreational waters.

AIMS: This study evaluates dialysis filtration and a range of PCR detection methods for identification and quantification of human adenoviruses in a range of environmental waters. METHODS AND RESULTS: Adenovirus was concentrated from large volumes (50-200 l) of environmental and potable water by hollow fibre microfiltration using commercial dialysis filters. By this method, an acceptable recovery of a seeded control bacteriophage MS2 from seawater (median 95.5%, range 36-98%, n=5), stream water (median 84.7%, range 23-94%, n=5) and storm water (median 59.5%, range 6.3-112%, n=5) was achieved. Adenovirus detection using integrated cell culture PCR (ICC-PCR), direct PCR, nested PCR, real-time quantitative PCR (qPCR) and adenovirus group F-specific direct PCR was tested with PCR products sequenced for confirmation. Adenovirus was routinely detected from all water types by most methods, with ICC-PCR more sensitive than direct-nested PCR or qPCR. Group F adenovirus dominated in wastewater samples but was detected very infrequently in environmental waters. CONCLUSIONS AND IMPLICATIONS: Human adenoviruses (HAdv) proved relatively common in environmental and potable waters when assessed using an efficient concentration method and sensitive detection method. ICC-PCR proved most sensitive, could be used semiquantitatively and demonstrated virus infectivity but was time consuming and expensive. qPCR provided quantitative results but was c. ten-fold less sensitive than the best methods.

The role of resuspension in enterococci distribution in water at an urban beach.

This study investigated the process and effects of bacterial resuspension on microbiological water quality in a small urban embayment. Water and sediments were sampled for enterococci at a small urban bay, on both irregular and intensive time scales, with a focus on the potential sources of faecal contamination to the system. Distribution of enterococci in sediments was influenced by the location and microbiological quality of major sources of enterococci to the embayment. Stream and storm water contributed the greatest numbers of enterococci and, consequently, high numbers of enterococci were found in both water and sediments surrounding discharge points for these sources. To investigate bacterial resuspension, water samples were collected from within the surf zone (at water depths of 1-1.5 m) as a wave crest passed. Two samples were collected simultaneously at each sampling location at 10 cm above the seabed and 10 cm below the water surface. Samples were analysed for enterococci and data compared with bacterial numbers in adjacent sediments as well as in stream and storm water sources. Vertical distribution data for enterococci in the water column revealed evidence of spatial and temporal variability in bacterial resuspension and the role of wave action was demonstrated. Bacterial resuspension under waves was directly related to weather and wave conditions. The resuspension of enterococci was not detected beyond the surf zone suggesting that wave action was the main cause of resuspension at the study site.

Evaluation of integrated cell culture-PCR (C-PCR) for virological analysis of environmental samples.

AIMS: The aims of this study were to establish an integrated culture-polymerase chain reaction (C-PCR) method for detection of enteric viruses in environmental samples, and to evaluate it for sensitivity, speed and provision of virus infectivity data. METHODS AND RESULTS: C-PCR, direct reverse transcription (RT)-PCR, PCR and plaque assay methods were used to detect enteroviruses and adenoviruses in seeded and naturally contaminated environmental samples. Using C-PCR, infectious enterovirus presence was confirmed in 3 d and adenovirus presence in 5 d, compared with up to 10 d required by conventional cell culture methods. CONCLUSIONS: C-PCR was the preferred method for detection of enteric viruses in environmental samples containing high viral concentrations. It was less successful for samples with low viral concentrations or containing toxic materials or inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: C-PCR provides sensitive, specific results within 2-5 d and is useful as a rapid screen for environmental samples of low toxicity.

Influence of environmental factors on virus detection by RT-PCR and cell culture.

The effects of clay, humic acid, u.v. light and shellfish tissue residues on the detection of poliovirus type 2 from environmental samples by culture and RT-PCR were investigated. RT-PCR showed 10-100 times greater sensitivity for PV2 detection in the absence of sample contaminants than did culture by plaque assay in BGM cell monolayers. Bentonite clay (100-1000 mg l-1) and shellfish tissue residues reduced virus detection by plaque assay, but the effect of bentonite was mitigated by simple elution procedures. Bentonite clay, humic acid (5-150 mg l-1) and mussel tissue reduced virus detection by RT-PCR by between 1 and 8 logs, although this was mitigated in part by elution and Sephadex filtration of extracts. Sephadex filtration of samples reduced culturable PV2 by 32-50%. Exposure of PV2 in water to u.v. light reduced culturability of PV2 but not detection by RT-PCR. This study demonstrates that virus detection in environmental samples is strongly influenced by naturally occurring substances and disinfection approaches. The accuracy of results of viral analyses of this nature should be carefully scrutinized with respect to sample constituents.

Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin).

HER2 is a ligand-less member of the human epidermal growth factor receptor or ErbB family of tyrosine kinases. In normal biological systems, HER2 functions as a co-receptor for a multitude of epidermal growth factor-like ligands that bind and activate other HER family members. HER2 overexpression is observed in a number of human adenocarcinomas and results in constitutive HER2 activation. Specific targeting of these tumors can be accomplished with antibodies directed against the extracellular domain of the HER2 protein. One of these antibodies, 4D5, has been fully humanized and is termed trastuzumab (Herceptin; Genentech, San Francisco, CA). Treatment of HER2-overexpressing breast cancer cell lines with trastuzumab results in induction of p27KIP1 and the Rb-related protein, p130, which in turn significantly reduces the number of cells undergoing S-phase. A number of other phenotypic changes are observed in vitro as a consequence of trastuzumab binding to HER2-overexpressing cells. These phenotypic changes include downmodulation of the HER2 receptor, inhibition of tumor cell growth, reversed cytokine resistance, restored E-cadherin expression levels, and reduced vascular endothelial growth factor production. Interaction of trastuzumab with the human immune system via its human immunoglobulin G1 Fc domain may potentiate its antitumor activities. In vitro studies demonstrate that trastuzumab is very effective in mediating antibody-dependent cell-mediated cytotoxicity against HER2-overexpressing tumor targets. Trastuzumab treatment of mouse xenograft models results in marked suppression of tumor growth. When given in combination with standard cytotoxic chemotherapeutic agents, trastuzumab treatment generally results in statistically superior antitumor efficacy compared with either agent given alone. Taken together, these studies suggest that the mechanism of action of trastuzumab includes antagonizing the constitutive growth-signaling properties of the HER2 system, enlisting immune cells to attack and kill the tumor target, and augmenting chemotherapy-induced cytotoxicity.

Receptor binding and biological activity of mammalian expressed sensory and motor neuron-derived factor (SMDF).

Sensory and motor neuron-derived factor (SMDF) is a member of the neuregulin family of proteins. SMDF is structurally characterized by a novel N-terminal domain. Using the signal sequence and N-terminal 28 amino acids (the "epitope") of herpes simplex virus type 1 glycoprotein D (gD), we have expressed SMDF as an epitope-tagged protein (gD-SMDF) in 293 cells, and purified it to > 98% homogeneity on a monoclonal anti-gD column. gD-SMDF stimulates human Schwann cell growth and 3H-thymidine incorporation in MCF-7 and T47D human breast tumor cells in vitro. The biological activity of gD-SMDF is consistent with its ability to compete with 125I-labeled heregulinbeta1 peptide (rHRGbeta1(177-244)) to bind to soluble dimeric ErbB receptor-IgG fusion proteins. gD-SMDF binds with low affinity to homodimeric ErbB3-IgG and ErbB4-IgG but with higher affinity to heterodimeric ErbB2/ErbB3-IgG and ErbB2/ErbB4-IgG. Using a SMDF-IgG(Fc) fusion protein we generated a monoclonal antibody (3G11) which binds SMDF, crossreacts with rHRGbeta1(177-244), and neutralizes the in vitro activities of gD-SMDF and rHRGbeta1(177-244) in human Schwann cells.

RT-PCR and chemiluminescent ELISA for detection of enteroviruses.

Reverse transcription followed by polymerase chain reaction amplification (RT-PCR) is now used commonly to detect the presence of enteric RNA viruses in environmental samples. A sensitive, non-isotopic microtitre plate hybridisation assay was developed and applied for detection of enteroviruses in environmental samples. Following reverse transcription, viral cDNA was labelled with digoxigenin (DIG)-dUTP during the PCR amplification step. The labelled PCR products were then hybridised with enterovirus-specific biotinylated oligonucleotide probe and captured in streptavidin-coated microtitre wells. Hybridised enteroviral PCR products were detected by an anti-digoxigenin peroxidase conjugate using either a colourimetric or a chemiluminescent substrate and automated measurement. Standard curves were established for poliovirus and other enteroviruses. The chemiluminescent assay was more sensitive than the colourimetric assay for detection of poliovirus, and was specific for enteroviruses. The chemiluminescent ELISA assay was used to confirm the presence of enteroviruses in environmental water samples.

Backscattering target detection in a turbid medium by polarization discrimination.

We describe a method for increasing target contrast within a turbid medium by means of the polarization state of the scattered light. The backscattered Mueller matrices for various concentrations of 0.1-microm spherical scatterers were measured with and without a painted metal target. Simple discrimination based on detecting cross-polarized intensities is shown to be more effective than the use of total intensity information. As a result, the choice of polarization state is dictated primarily by the requirement to maximize depolarization at the target. This in general means that circularly polarized light is the optimum choice.

The effect of pH on the inhibition of bacterial growth by physiological concentrations of butyric acid: implications for neonates fed on suckled milk.

Butyric acid is released from milk by pre-intestinal lipases during suckling. It is also known to inhibit bacterial growth. To investigate whether butyric acid may be a significant factor in controlling bacterial growth in the stomach of pre-weaned animals, the ability of butyric acid to inhibit growth of selected bacteria was tested over physiological ranges of pH and butyric acid concentrations. Six enteric and environmental strains of bacteria were used: two strains of Escherichia coli, Klebsiella pneumoniae, Enterococcus faecium, Enterococcus faecalis, and Enterococcus casseliflavus. At pH 4.5 and 5.0, the growth of all organisms was significantly inhibited in the presence of butyrate, and in some cases growth was completely arrested. At pH 6.0, butyric acid did not affect bacterial growth until the concentration reached 40 mM. The maximum concentration of butyric acid available in cow's milk after incubation with pre-gastric lipase is approximately 16 mM, which would be sufficient to prevent growth of the organisms tested at pH values occurring in the stomach. Therefore, butyric acid inhibition of bacterial growth may explain in part, the role of pre-intestinal lipases in young animals' natural defenses against bacteria in ingested food prior to weaning.

Backscatter linear and circular polarization analysis of roughened aluminum.

A study of cross-polarized and copolarized intensities backscattered from roughened aluminum surfaces is presented for both linear and circular incident polarization states. The angular variation of measured Mueller matrices is shown to contain only diagonal elements, as predicted by the reciprocity theorem. The ratio of cross-depolarized to copolarized scattered intensities is significantly larger for circular than for linear input polarization states. In the linear case the ratio saturates beyond 50 degrees , whereas in the circular case the ratio continues to increase monotonically with angle. A phenomenological model for copolarization and cross-polarization intensities is shown to predict the observed behavior of both linear and circular input polarization states up to incident angles of 70 degrees .

A genomic polymorphism located downstream of the gcvP gene of Escherichia coli that correlates with ecological niche.

Current evolutionary theory proposes that niche-adapted microbial populations might evolve through selection for favoured genotypes followed by clonal expansion (Maynard-Smith, 1991). Possible correlations between genomic variation and ecological niche in Escherichia coli isolates derived from human and animal sources were investigated by randomly amplified polymorphic DNA (RAPD) analysis. A 1.6-kb polymorphic marker was identified which was present in 60% of isolates from human clinical specimens but was present in less than 5% of isolates derived from ovine and bovine faeces. The marker maps to a region of the chromosome located immediately downstream from the gene encoding the glycine decarboxylase P-protein (gcvP). DNA sequences from marker-positive and marker-negative isolates exhibit an abrupt loss of homology immediately downstream from the transcription termination point of the gene which extends for at least 130-base pairs beyond the gcvP transcription terminator. Sequences spanning this region in marker-negative isolates exhibit similarity to the cognate sequence from E. coli K-12, while the corresponding region in marker-positive isolates bears no similarity to any other published sequence. The utility of the marker for investigating the occurrence of human-derived E. coli in the environment was studied in a rural stream. Although the stream carried a high background of animal-derived E. coli, the marker could only be detected in isolates obtained downstream of the human faecal input. The polymorphism therefore shows promise for identification of human-derived E. coli within environments containing isolates from multiple and diverse sources. The methods described here could be used to generate further markers suitable for investigating microbial population ecology.

Growth regulation of human breast and ovarian tumor cells by heregulin: Evidence for the requirement of ErbB2 as a critical component in mediating heregulin responsiveness.

Alterations in the expression of the epidermal growth factor (EGF) receptor ErbB family are frequently encountered in a number of human cancers. Two of these receptors, ErbB3 and ErbB4, are known to bind a family of related proteins termed heregulins (HRGs) or neu differentiation factors. In biologically relevant systems, interaction of HRG with ErbB3 or ErbB4 results in the transactivation of ErbB2. In this report, we show that ErbB2 is a critical component in mediating HRG responsiveness in a panel of human breast and ovarian tumor cell lines. Because HRGs have been reported to elicit diverse biological effects on cultured cells, including growth stimulation, growth inhibition, and induction of differentiation, we systematically examined the effect of rHRG beta 1 on tumor cell proliferation. HRG binding studies were performed with a panel of breast and ovarian tumor cell lines expressing a range of levels of ErbB2. The biological responses to HRG were also compared to EGF and to the growth-inhibitory anti-ErbB2 antibody, 4D5. In most cases, HRG stimulation of DNA synthesis correlated with positive effects on cell cycle progression and cell number and with enhancement of colony formation in soft agar. On each cell line tested, the HRG effects were distinguishable from EGF and 4D5. Our findings indicate that HRG induces cell proliferation in a number of tumor cell lines. In addition, we show that methods for measuring cell proliferation, as well as experimental conditions, are critical for determining HRGs effect on tumor cell growth in vitro.

Emission polarization of roughened glass and aluminum surfaces.

Ellipsometer measurements of the effective complex refractive index at a wavelength of 10.6 µm are made on a series of glass and aluminum surfaces of increasing surface roughness. The measured values are then used to calculate the degree of emission polarization and are shown to be in agreement with the experimentally determined values when depolarization is small. Comparisons are also made with calculations based on the Kirchhoff scattering theory. Both the theory and the experimental results indicate that it is the local surface slope and not the roughness magnitude that is the prime factor in determining the degree of emission polarization from the samples studied.

Engineering high affinity humanized anti-p185HER2/anti-CD3 bispecific F(ab')2 for efficient lysis of p185HER2 overexpressing tumor cells.

We previously constructed a humanized anti-p185HER2/anti-CD3 bispecific antibody variant, BsF(ab')2 v1 which retargets the cytotoxic activity of human T cells in vitro against human breast tumor cells which overexpress the p185HER2 product of the HER2/neu (c-erbB-2) protooncogene. Subsequently we identified an improved anti-CD3 variant, v9, which binds to T cells with approx. 100-fold higher affinity than the original variant, v1. Here we demonstrate that BsF(ab')2 v9 is more potent than BsF(ab')2 v1 in stimulating the proliferation of both resting peripheral blood lymphocytes (PBL) and IL-2-activated, long-term cultured T lymphocytes (ATL). In addition, at low concentrations (0.01-1 ng/ml) BsF(ab')2 v9 is much more efficient than BsF(ab')2 v1 in directing lysis of p185HER2-overexpressing tumor cells by IL-2 activated PBL. In contrast, at higher concentration BsF(ab')2 v9 and BsF(ab')2 v1 have similar potency in retargeted cytotoxicity. At BsF(ab')2 v9 concentrations of > or = 1 ng/ml the susceptibility of p185HER2-expressing tumor cells to lysis is apparently independent of the level of p185HER2 expression. At lower concentrations of BsF(ab')2 v9 and/or lower ratios of effector to target cells the extent of lysis is reduced, in some cases improving the selectivity of lysis of high p185HER2 expressors over low expressors. Thus selection of a high affinity anti-CD3 arm is likely important in the design of BsF(ab')2 for retargeting the cytotoxicity of T cells to tumors. The dose of BsF(ab')2 v9 in any future clinical evaluation will require optimization to maximize anti-tumor efficacy whilst minimizing potential toxicity against normal tissue expressing p185HER2.