Simultaneous monitoring of faecal indicators and harmful algae using an in-situ autonomous sensor.

UNLABELLED: Faecal indicator bacteria (FIB) and harmful algal blooms (HABs) threaten the health and the economy of coastal communities worldwide. Emerging automated sampling technologies combined with molecular analytical techniques could enable rapid detection of micro-organisms in-situ, thereby improving resource management and public health decision-making. We evaluated this concept using a robotic device, the Environmental Sample Processor (ESP). The ESP automates in-situ sample collection, nucleic acid extraction and molecular analyses. Here, the ESP measured and reported concentrations of FIB (Enterococcus spp.), a microbial source-tracking marker (human-specific Bacteriodales) and a HAB species (Psuedo-nitzschia spp.) over a 45-day deployment on the Santa Cruz Municipal Wharf (Santa Cruz, CA, USA). Both FIB and HABs were enumerated from single in-situ collected water samples. The in-situ qPCR efficiencies ranged from 86% to 105%, while the limit of quantifications during the deployment was 10 copies reaction(-1) . No differences were observed in the concentrations of enterococci, the human-specific marker in Bacteroidales spp., and P. australis between in-situ collected sample and traditional hand sampling methods (P > 0·05). Analytical results were Internet-accessible within hours of sample collection, demonstrating the feasibility of same-day public notification of current water quality conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents the first report of in-situ qPCR enumeration of both faecal indicators and harmful algal species in coastal marine waters. We utilize a robotic device for in-situ quantification of enterococci, the human-specific marker in Bacteriodales and Pseudo-nitzschia spp. from the same water samples collected and processed in-situ. The results demonstrate that rapid, in-situ monitoring can be utilized to identify and quantify multiple health-relevant micro-organisms important in water quality monitoring and that this monitoring can be used to inform same-day notifications.

Leaching of nitrogen and phenolics from wood waste and co-composts used for road rehabilitation.

Rehabilitation and reforestation of disused forest roads and landings can be facilitated by the incorporation of organic matter. The British Columbia forest industry creates residual woody materials, but they are nutrient poor and may leach phenolic compounds. We assessed the potential for wood wastes (chipped cedar wood waste, sort-yard waste, hogfuel) and co-composts with shellfish waste or municipal biosolids to provide inorganic N and release phenolics and condensed tannins, compared with natural forest floor and mineral soil. Initial concentrations of tannins and phenolics were low, and 13C cross-polarization and magic-angle spinning nuclear magnetic resonance spectroscopy showed that composts were still dominated by wood. During a 426-d laboratory leaching experiment, release of phenolics from woody amendments (other than cedar wood) was lower than from native forest floor. The pH levels of woody amendments and their leachates were also within the range of native forest floor and soil (except cedar wood, which was the most acidic material). Co-composts had higher total N and available P, greatly reduced tannins and phenolics, and negligible leaching of polyphenols. Uncomposted materials released very little N during the incubation. Hogfuel-biosolids compost released a large amount of nitrate, but only during the first 100 d. Shrimp-wood compost released moderate amounts of ammonium and nitrate throughout the incubation, had high available P and low tannin content, and released less polyphenols than did native forest floors. Our results indicate that appropriate use of these amendments does not pose an environmental risk with regard to the parameters measured in this study.

Sequestration of soil nitrogen as tannin-protein complexes may improve the competitive ability of sheep laurel (Kalmia angustifolia) relative to black spruce (Picea mariana).

The role of litter tannins in controlling soil nitrogen (N) cycling may explain the competitive ability of Kalmia relative to black spruce (Picea mariana), although this has not been demonstrated experimentally. Here, the protein-precipitation capacities of purified tannins and leaf extracts from Kalmia and black spruce were compared. The resistance to degradation of tannin-protein precipitates from both species were compared by monitoring carbon (C) and N dynamics in humus amended with protein, purified tannins or protein-tannin precipitates. The purity of the precipitates was verified using solid-state (13)C nuclear magnetic resonance (NMR) spectra. The ability of mycorrhizal fungi associated with both species to grow on media amended with tannin-protein complexes as the principal N source was also compared. The protein precipitation capacity of Kalmia tannins was superior to those of black spruce. Humus amended with protein increased both mineral and microbial N, whereas humus amended with tannin-protein precipitates increased dissolved organic N. Mycorrhizal fungi associated with Kalmia showed better growth than those associated with black spruce when N was provided as tannin-protein precipitates. These data suggest that Kalmia litter increases the amount of soil N sequestered as tannin-protein complexes, which may improve the competitive ability of Kalmia relative to black spruce by favouring N uptake by mycorrhizas associated with the former.

Soil enzyme inhibition by condensed litter tannins may drive ecosystem structure and processes: the case of Kalmia angustifolia.

Kalmia angustifolia is an ericaceous shrub that can rapidly spread on recently harvested boreal forest sites, causing a slow-down in soil nutrient cycling and reduced growth of spruce seedlings. It has been hypothesized that tannins released from Kalmia litter suppress soil enzyme activity, and are thus important in controlling ecosystem structure and processes. Here the effects of different concentrations of tannins extracted from both Kalmia and black spruce (Picea mariana) foliage were tested on enzyme activities of soil extracts. Then the effects of various Kalmia-black spruce litter mixtures on soil enzyme activity were investigated. Lastly, the correlation between Kalmia cover in the field and soil enzyme activity was measured. Both tannin types suppressed beta-glucosidase and acid phosphatase activities, and the magnitude of these effects was concentration-dependent. beta-glucosidase and amidase activity decreased linearly with an increasing Kalmia : spruce litter ratio added to soil. A field survey of 24 sites revealed a negative relationship between percentage Kalmia cover and beta-glucosidase activity. Collectively, results of the three experiments converge to support the claim that enzyme inhibition by litter tannins has evolved as an important mechanism controlling ecosystem processes and structure following Kalmia invasion on recently disturbed forest sites.

Towards an understanding of the molecular basis of herpes simplex virus latency.

The survival strategy of herpes simplex virus centres on the establishment of latency in sensory neurons innervating the site of primary infection followed by periodic reactivation to facilitate transmission. This is a highly evolved and efficient survival mechanism, which despite being the subject of intense research, has proven remarkably difficult to dissect at a molecular level. This review will focus on data, emerging from both in vitro and in vivo model systems, which provide a framework for a mechanistic understanding of latency and the existence and possible significance of non-uniform latent states.

Chemical and carbon-13 cross-polarization magic-angle spinning nuclear magnetic resonance characterization of logyard fines from British Columbia.

Phasing out beehive burners and rising costs for landfilling have increased the need to widen options for utilization of the smaller size fractions of woody wastes generated during log handling and sawmilling in British Columbia. We characterized several size classes of logyard fines up to 16 mm sampled from coastal and interior operations. Total C, total N, ash, and condensed tannin concentrations were consistent with properties derived largely from wood, with varying proportions of bark and mixing with mineral soil. Especially for < 3-mm fractions, the latter resulted in high ash contents that would make them unsuitable for fuel. Solid-state 13C cross-polarization magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectra were consistent with the chemical data, with high O-alkyl intensity and similarity to naturally occurring woody forest floor; no samples were high in aromatic or phenolic C. Aqueous extracts of two < 16-mm fines, which accounted for only a small proportion of the total C, were enriched in alkyl C and had low or undetectable tannins. Application to forest sites might cause short-term immobilization of N, but also might include possible longer-term benefits from reduction of N loss after harvesting and restoration of soil organic matter in degraded sites.

Phylogenetic screening of ribosomal RNA gene-containing clones in Bacterial Artificial Chromosome (BAC) libraries from different depths in Monterey Bay.

Marine picoplankton are central mediators of many oceanic biogeochemical processes, but much of their biology and ecology remains ill defined. One approach to better defining these environmentally significant microbes involves the acquisition of genomic data that can provide information about genome content, metabolic capabilities, and population variability in picoplankton assemblages. Previously, we constructed and phylogenetically screened a Bacterial Artificial Chromosome (BAC) library from surface water picoplankton of Monterey Bay. To further describe niche partitioning, metabolic variability, and population structure in coastal picoplankton populations, we constructed and compared several picoplankton BAC libraries recovered from different depths in Monterey Bay. To facilitate library screening, a rapid technique was developed (ITS-LH-PCR) to identify and quantify ribosomal RNA (rRNA) gene-containing BAC clones in BAC libraries. The approach exploited natural length variations in the internal transcribed spacer (ITS) located between SSU and LSU rRNA genes, as well as the presence and location of tRNA-alanine coding genes within the ITS. The correspondence between ITS-LH-PCR fragment sizes and 16S rRNA gene phylogenies facilitated rapid identification of rRNA genes in BAC clones without requiring direct DNA sequencing. Using this approach, 35 phylogenetic groups (previously identified by cultivation or PCR-based rRNA gene surveys) were detected and quantified among the BAC clones. Since the probability of recovering chimeric rRNA gene sequences in large insert BAC clones was low, we used these sequences to identify potentially chimeric sequences from previous PCR amplified clones deposited in public databases. Full-length SSU rRNA gene sequences from picoplankton BAC libraries, cultivated bacterioplankton, and nonchimeric RNA genes were then used to refine phylogenetic analyses of planktonic marine gamma Proteobacteria, Roseobacter, and Rhodospirillales species.

Linking chemical reactivity and protein precipitation to structural characteristics of foliar tannins.

Tannins influence ecosystem function by affecting decomposition rates, nutrient cycling, and herbivory. To determine the role of tannins in ecological processes, it is important to quantify their abundance and understand how structural properties affect reactivity. In this study, purified tannins from the foliage of nine species growing in the pygmy forest of the northern California coast were examined for chemical reactivity, protein precipitation capacity (PPC), and structural characteristics (13C NMR). Reactivity of purified tannins varied among species 1.5-fold for the Folin total phenol assay, and 7-fold and 3-fold, respectively, for the acid butanol and vanillin condensed tannin assays. There was about a 5-fold difference in PPC. Variation in chemical reactivity and PPC can be largely explained by differences in structural characteristics of the tannins determined by 13C NMR. In particular, the condensed versus hydrolyzable tannin content, as well as the hydroxylation pattern of the B-ring and stereochemistry at the C-2-C-3 position appear to influence reactivity. Due to the large differences in chemical reactivity among species, it is necessary to use a well-characterized purified tannin from the species of interest to convert assay values to concentrations. Our results suggest that structural characteristics of tannins play an important role in regulating their reactivity in ecological processes.

Effects of wheat variety and specific weight on dietary apparent metabolisable energy concentration and performance of broiler chicks.

1. This study used 4 wheat cultivars (Brigadier, Chaucer, Consort, Reaper) from three locations (Crossnacreevy, C; Downpatrick, D; Limavady, L), which had given rise to differences in wheat specific weight (SW), to examine the relationships between apparent metabolisable energy (AME) concentration, broiler performance and wheat SW. 2. The diets contained (g/kg): wheat 744, casein 142, blended vegetable fat 50, dicalcium phosphate 22, potassium bicarbonate 10.8, sodium bicarbonate 7.5, arginine 5, methionine 2, binder 8, trace minerals/vitamins 7.2, titanium dioxide 1.5. The diets were heat-treated (80 degrees C for 2 min) prior to pelleting (3 mm die). 3. SW ranged from 63 to 77 kg/hectolitre (hl), averaging 66, 69 and 76 kg/hl at D, C and L, respectively. In vitro viscosity of the wheat samples ranged from 5.2 to 17.5 cps and thousand grain weight (TGW) from 33.4 to 47.3 g. Mean TGW was similar at C and D (38.7, 37.0 g) but higher at L (43.1 g). In vitro viscosity was similar at C and L (11.2, 10.2 cps) but somewhat higher at D (14.4 cps). Crude protein (6.25 N) ranged from 116 to 147 g/kg and tended to be higher at D. Starch, which ranged from 612 to 656 g/kg, was least at D (617 g/kg) and greatest at L (641 g/kg). 4. Crude protein, crude fibre and total non-starch polysaccharide (NSP) were negatively correlated with SW, the R2 being respectively 0.38 (P<0.05), 0.16 (NS) and 0.45 (P<0.05). TGW and starch concentration were positively correlated with SW (R2=0.70, 0.44, respectively). There was a weak (NS) negative relationship (R2=0.19) between in vitro viscosity and SW. For both TGW and in vitro viscosity, correlations improved when variety was taken into account (R2=0.95, 0.92, respectively). 5. There were no significant effects of variety on dry matter (DM) intake or live weight gain (LWG). Gain: food was significantly higher (P<0.05) for Consort than for the other three varieties and the metabolisable energy ratio (ME:GE) just failed to attain significance (P=0.062). Calculated wheat AME (MJ/kg DM) was significantly (P<0.05) higher for Consort than for the other three wheats. There was a good correlation (R2=0.49) for the total data set between gain:food and ME:GE. In vivo viscosity varied from 13.6 to 28.6 cps for individual treatments and was significantly affected by variety (P<0001). 6. Although there were no significant differences in DM intake or LWG due to site the values for L (SW 76) were 6 and 5% lower, respectively, than for D (SW 66). Gain:food was lower (P<0.05) for C (SW 69) than for D. ME:GE, wheat AME and ME:gain were not significantly different between sites. 7. There was a weak (R2=0.18) positive relationship between ME:GE and SW corresponding to a 2.5% increase in energy value for a 10 kg/hl increase in SW and no relationship between gain:food and SW. When variety was taken into the regression the slope was similar but R2 increased to 0.82. 8. ME:GE and wheat AME concentration were negatively correlated with wheat in vitro viscosity (R2=0.64, 0.55, respectively). 9. It was concluded that in vitro viscosity appears to provide a better basis than SW for prediction of the nutritive value of wheats of unknown variety. If the variety is known then SW could be used to predict energy value. However, the effect of quite a large change in SW (10 kg/hl) was relatively small.

Harvesting and climate effects on organic matter characteristics in British Columbia coastal forests.

As part of investigations into the effects of harvesting old-growth forest, we characterized carbon in five organic matter pools in eight forest chronosequences of coastal British Columbia. Each chronosequence comprised stands in four seral stages from regeneration (3-8 yr) to old-growth (>250 yr), with second-growth stands mostly of harvest origin. Stands were located in two biogeoclimatic subzones with contrasting climate (wetter, slightly cooler conditions on the west coast of Vancouver Island than on the east). Carbon concentrations in fine woody debris (FWD), forest floor (LFH), fine roots from LFH, and two water-floatable fractions from 10 to 30 cm mineral soil (MIN-ROOT, 2-8 mm and MIN-FLOAT, <2 mm) showed no significant effects due to climate, seral stage, or site. There were some significant differences in N concentrations, but none related to seral stage. Carbon-13 cross-polarization with magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectra with principal component analysis of relative areas also showed little harvesting effect, but greater variation related to input of coarse woody debris (CWD) vs. roots high in tannin. Overall, there tended to be more spectral features associated with wood and lignin in the west; whereas some MIN-ROOT samples from the drier east side had aromatic intensity attributed to charcoal. The minimal effects of one harvest on organic matter are most likely due to the large legacy effect; however, more intensive management will probably result in less CWD retention, less charcoal input, and less microsite variability in these pools of poorly decomposed organic matter.

Activation of interferon response factor-3 in human cells infected with herpes simplex virus type 1 or human cytomegalovirus.

Activation of cellular interferon-stimulated genes (ISGs) after infection with herpes simplex virus type 1 (HSV-1) or human cytomegalovirus (HCMV) was investigated. The level of ISG54-specific RNA in human fetal lung (HFL) or human foreskin (BJ) fibroblasts increased substantially after infection with either virus in the presence of cycloheximide. HSV-1 particles lacking glycoprotein D or glycoprotein H failed to induce ISG54-specific RNA synthesis, demonstrating that entry of virus particles rather than binding of virions to the cell surface was required for the effect. A DNA-binding complex that recognized an interferon-responsive sequence motif was induced upon infection with HSV-1 or HCMV in the presence of cycloheximide, and the complex was shown to contain the cell proteins interferon response factor 3 (IRF-3) and CREB-binding protein. IRF-3 was modified after infection with HSV-1 or HCMV to a form of lower electrophoretic mobility, consistent with phosphorylation. De novo transcription of viral or cellular genes was not required for the activation of IRF-3, since the effect was not sensitive to inhibition by actinomycin D. Infection of HFL fibroblasts with HSV-1 under conditions in which viral replication proceeded normally resulted in severely reduced levels of the IRF-3-containing complex, defining the activation of IRF-3 as a target for viral interference with ISG induction. In BJ fibroblasts, however, significant activation of IRF-3 was detected even when the viral gene expression program progressed to later stages, demonstrating that the degree of inhibition of the response was dependent on host cell type. As a consequence of IRF-3 activation, endogenous interferon was released from BJ cells and was capable of triggering the appropriate signal transduction pathway in both infected and uninfected cells. Activation of ISG54-specific RNA synthesis was not detected after infection of human U-373MG glioblastoma cells, showing that the induction of the response by infection is cell type dependent.

Latency associated promoter transgene expression in the central nervous system after stereotaxic delivery of replication-defective HSV-1-based vectors.

The herpes simplex virus type 1 (HSV-1) latency associated promoter (LAP) has been shown to sustain long-term reporter gene expression within sensory neurones. Its activity within the CNS is, however, less well understood. In this study we characterise the activity of the LAP after stereotaxic delivery of recombinant HSV-1-based vectors to the brain. Two classes of vectors were utilised in these studies: (1) a replication-defective vector lacking the glycoprotein H and thymidine kinase genes, designated CS1, and (2) a virus mutant severely impaired for immediate-early (IE) gene expression which lacks functional VP16, ICP4 and ICP0 genes, designated in1388. Both vectors contain the LacZ gene under the control of the LAP. Following delivery of either vector to the striatum, beta-gal expression was detected within anatomically related CNS regions distal to the site of injection. At these sites the number of beta-gal-positive cells increased with time and remained stable up to 4 weeks p.i. beta-Gal expression could not be detected at the site of injection after delivery of CS1 but beta-gal expression within neurones located at this site was observed after delivery of in1388, indicating reduced toxicity of this severely disabled virus. Transgene expression decreased dramatically with both vectors at later time-points (>4 weeks after delivery), but PCR analysis demonstrated that viral genomes were stably maintained for up to 180 days following delivery, indicating that the loss of beta-gal-positive neurones was not likely to be due to a loss of vector-transduced cells. Moreover, after delivery of an equivalent virus to the rat striatum in situ hybridisation analysis showed a similar decrease in the number of neurones expressing the endogenous LATs with time. These data indicate that although the HSV-1 LAP can drive the expression of foreign genes in a variety of CNS neurones, in these cells there is a slow down-regulation of the viral promoter which eventually results in the loss of detectable transgene expression.

Decomposition and nitrogen mineralization from biosolids and other organic materials: relationship with initial chemistry.

Biosolids are effective forest fertilizers. In order to facilitate their use it is important that one be able to predict the amount and rate of mineralization of nutrients, particularly nitrogen, and the relationship between substrate chemistry and N release. We examined the relationships between substrate quality and nitrogen release in a variety of organic materials. Rates of decomposition and net N mineralization from four biosolids, wheat straw, paper fines, and Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] needle litter were measured during 391-d incubations in a greenhouse, and at two field sites in wet coastal and dry interior forests. Decomposition rates were best predicted by a model incorporating the ratio of carbon to organic matter. The decomposition model extrapolated well to the field when site-specific correction factors were applied. There was a weak relationship between rates of decomposition and net N mineralization. Rates of net N mineralization were best predicted by a model incorporating the initial organic N concentration and the proportion of phenolic C determined from solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. The mineralization model extrapolated less well to the field, but the effect of substrate chemistry was still apparent. Among the four biosolids there was a strong correlation between organic N concentration and indices or protein determined from 13C NMR, suggesting that these protein indices may be useful for predicting N mineralization from biosolids. There was some evidence that the protein content and N mineralization in biosolids may be predictable from the sewage treatment process employed.

Herpes simplex virus type 1 blocks the apoptotic host cell defense mechanisms that target Bcl-2 and manipulates activation of p38 mitogen-activated protein kinase to improve viral replication.

Wild-type (wt) herpes simplex virus type 1 (HSV-1) suppresses cell death. We investigated the apoptotic pathways triggered during infection with mutant viruses tsk and 27lacZ (which lack functional ICP4 and ICP27 viral proteins, respectively) and examined the mechanisms used by wt HSV-1 to protect against programmed cell death induced by the DNA-damaging compound cisplatin. In our studies, we used BHK and HeLa cells, with similar results. We suggest that a decrease in the levels of Bcl-2 protein is a key event during apoptosis induced by the mutant viruses and that Bcl-2 levels are targeted by (i) a decrease of bcl-2 RNA, (ii) caspase-related proteolysis, and (iii) p38 mitogen-activated protein kinase (p38MAPK)-dependent destabilization of Bcl-2 protein. We show that wt HSV-1, but not the mutant viruses, maintains bcl-2 RNA and protein levels during infection and protects from the cisplatin-induced decrease in bcl-2 RNA; our data suggest that both ICP27 and ICP4 are required for this function. Additionally, wt HSV-1 evades but does not actively block activation of caspases. Although wt HSV-1 induces p38MAPK activation during infection, it prevents p38MAPK-dependent destabilization of Bcl-2 and exploits p38MAPK stimulation to enhance transcription of specific viral gene promoters to increase viral yields.

Effect of wheat content, fat source and enzyme supplementation on diet metabolisability and broiler performance.

1. A 3x3x2 factorial experiment studied the interactions of fat source (tallow, soya, tallow:soya [2:1] blend), wheat level (700, 350, 0 g/kg) and enzyme inclusion (Avizyme 1300, absent, present) in diets for broilers fed ad libitum in individual cages from 7 to 35d. Bird performance, fat digestibility, viscosity of ileal contents and diet metabolisability (AME) were measured. 2. There were no significant effects of fat source on bird performance. However, there was a significant effect on fat digestibility, which was highest for soya and lowest for tallow. Diet AME content was also significantly affected by fat source and reflected differences in fat digestibility. 3. Dry matter (DM) intake, liveweight gain (LWG) and gain:food were all reduced at 700 g wheat/kg. Viscosity of ileal contents increased with increasing wheat inclusion. 4. There were no significant effects of enzyme on DM intake or LWG but gain:food was improved by 2%. Diet AME content was increased with enzyme addition, the effect being greatest (9%) with tallow at 700 g wheat/kg. 5. Viscosity of ileal contents was reduced and fat digestibility increased with enzyme addition and there were significant wheat enzyme interactions attributable to no differences with zero wheat but marked responses to enzyme at 700 g wheat/kg. 6. The results confirm important interactions between wheat content and fat composition in relation to fat digestibility, AME content and food efficiency.

Effect of diet form and enzyme supplementation on growth, efficiency and energy utilisation of wheat-based diets for broilers.

1. The effect of diet form (mash, cold-pelleted, steam-conditioned/pelleted, wet mash, whole wheat with balancer pellet, restricted pellet) and enzyme inclusion (Avizyme 1300, absent, present) was studied in 2 trials using individually caged, male broilers from 14 to 42 d. Bird performance, viscosity of ileal contents and diet metabolisability (AME) were measured. 2. The performance of mash-fed birds was significantly poorer than for the other treatments in relation to dry matter intake, liveweight gain and gain:food. This was not due to reduced diet AME content. 3. There was no significant effect of heat treatment on any of the variables measured, although viscosity of ileal contents was increased by 30% as compared to the cold-pelleted diet. 4. Gain:food was improved with wet-mash feeding in comparison to the dry mash treatment but it was concluded that this was not due to any intrinsic improvement in diet quality, but rather to voluntary food restriction on introduction of the wet food. 5. Whole wheat feeding improved gain:food and diet AME content by 3% as compared to the complete diets and caused approximately a 50% increase in gizzard weight as compared with the pelleted diets. 6. Food enzyme inclusion did not improve performance although a significant improvement in diet AME content was observed with enzyme inclusion in trial 1.

Subcellular post-transcriptional targeting: delivery of an intracellular protein to the extracellular leaflet of the plasma membrane using a glycosyl-phosphatidylinositol (GPI) membrane anchor in neurons and polarised epithelial cells.

The effectiveness of viral vector-mediated gene transfer depends on the expression of therapeutic transgenes in the correct target cell types. So far, however, little attention has been given to targeted subcellular distribution of expressed transgenes. Targeting individual transgenes to particular subcellular compartments will provide various advantages in increasing the safety, efficacy, and specificity of viral vector-mediated gene delivery. Viruses normally hijack the cellular protein synthesis machinery for their own advantages. It is thus unknown whether cells infected with viral vectors will be able to target proteins to the correct subcellular organelles, or whether the subcellular targeting machinery would be selectively disrupted by viral infection. In this article we explored whether a herpes simplex virus type 1-derived vector could be used to deliver a transgene engineered to be targeted to the extracellular membrane of target cells. To do so we constructed a temperature-sensitive mutant HSV-1 vector, tsK-TT21 expressing a recombinant marker protein, tissue inhibitor of metalloproteinases (TIMP), linked to sequence encoding a signal for the addition of a glycosyl-phosphatidylinositol (GPI)-anchor within the endoplasmic reticulum. Our results demonstrate that HSV1-derived viral vectors can be used to target transgenes as GPI anchored proteins to the outside leaflet of plasma membranes, without disrupting the targeting machinery of host epithelial cells or neurons. This approach could then be used to target specific proteins to the cell membrane to modify cell-cell interactions, the function of specific plasma membrane proteins, or their interactions with other membrane proteins, and also to target a prodrug converting enzyme to the plasma membrane of target cells, therefore enhancing its cell killing effects.

Long-term transgene expression in mice infected with a herpes simplex virus type 1 mutant severely impaired for immediate-early gene expression.

The role of viral immediate-early (IE) gene expression in herpes simplex virus type 1 (HSV-1) latency was investigated. The HSV-1 multiple mutant in1312, defective for the expression of the virion transactivator VP16 and the IE proteins ICP0 and ICP4, was used as the parent for these studies. The coding sequences of the Escherichia coli lacZ gene, preceded by the encephalomyocarditis virus internal ribosome entry site, were inserted into the region of in1312 that encodes the latency-associated transcripts (LATs) such that transcription of the transgene was controlled by the LAT promoter. This insert has previously been shown to direct long-term latent-phase expression of beta-galactosidase in a wild-type HSV-1 genome (R. H. Lachmann and S. Efstathiou, J. Virol. 71, 3197-3207, 1997). The resulting recombinant, in1388, was apathogenic after inoculation into mice via the footpad and did not detectably replicate in dorsal root ganglia (DRG) or footpads. Mutant in1388 established latency in DRG, and beta-galactosidase was expressed in increasing numbers of neurons over the first 25 days of infection. During latency, more than 1% of neurons in ganglia that innervate the footpad expressed beta-galactosidase, with the number of positive cells remaining constant for at least 5 months. Rescue of the VP16, ICP0, or ICP4 mutations of in1388 did not affect the number of beta-galactosidase-expressing neurons detected during latency. The results demonstrate that HSV-1 mutants severely impaired for IE gene expression are capable of establishing latency and efficiently expressing a foreign gene product under control of the LAT promoter.

Visualization and enumeration of marine planktonic archaea and bacteria by using polyribonucleotide probes and fluorescent in situ hybridization.

Fluorescent in situ hybridization (FISH) using rRNA-specific oligonucleotide probes has emerged as a popular technique for identifying individual microbial cells. In natural samples, however, the signal derived from fluor-labeled oligonucleotide probes often is undetectable above background fluorescence in many cells. To circumvent this difficulty, we applied fluorochrome-labeled polyribonucleotide probes to identify and enumerate marine planktonic archaea and bacteria. The approach greatly enhanced the sensitivity and applicability of FISH with seawater samples, allowing confident identification and enumeration of planktonic cells to ocean depths of 3,400 m. Quantitative whole-cell hybridization experiments using these probes accounted for 90 to 100% of the total 4',6-diamidino-2-phenylindole (DAPI)-stained cells in most samples. As predicted in a previous study (R. Massana, A. E. Murray, C. M. Preston, and E. F. DeLong, Appl. Environ. Microbiol. 63:50-56, 1997), group I and II marine archaea predominate in different zones in the water column, with maximal cell densities of 10(5)/ml. The high cell densities of archaea, extending from surface waters to abyssal depths, suggest that they represent a large and significant fraction of the total picoplankton biomass in coastal ocean waters. The data also show that the vast majority of planktonic prokaryotes contain significant numbers of ribosomes, rendering them easily detectable with polyribonucleotide probes. These results imply that the majority of planktonic cells visualized by DAPI do not represent lysed cells or "ghosts," as was suggested in a previous report.