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To aid in understanding black carbon (BC) formation during smoldering combustion in forest fires, we characterized charring of a softwood and hardwood. Charring (150, 340, 480°C) caused mass loss (7-84%), enrichment of organic carbon (OC) (0-32%), and 13C depletion (> 150°C). As determined by 13C MAS NMR, the OC composition of the woods was dominated by (di)-O-alkyl structures, and the chars by alkyl and aromatic structures. With increasing temperature, aromatic structures increased and the chars became more similar, although initial differences in OC concentration and δ 13C of woods persisted. The BC cluster sizes apparently remained small, pointing towards a low resistance against oxidation. Crown Copyright © 2002 Published by Elsevier Science Ltd. All rights reserved.

Identifying mechanisms by which bacterial species evolve and maintain genomic diversity is particularly challenging for the uncultured lineages that dominate the surface ocean. A longitudinal analysis of bacterial genes, genomes, and transcripts during a coastal phytoplankton bloom revealed two co-occurring, highly related Rhodobacteraceae species from the deeply branching and uncultured NAC11-7 lineage. These have identical 16S rRNA gene amplicon sequences, yet their genome contents assembled from metagenomes and single cells indicate species-level divergence. Moreover, shifts in relative dominance of the species during dynamic bloom conditions over 7 weeks confirmed the syntopic species' divergent responses to the same microenvironment at the same time. Genes unique to each species and genes shared but divergent in per-cell inventories of mRNAs accounted for 5% of the species' pangenome content. These analyses uncover physiological and ecological features that differentiate the species, including capacities for organic carbon utilization, attributes of the cell surface, metal requirements, and vitamin biosynthesis. Such insights into the coexistence of highly related and ecologically similar bacterial species in their shared natural habitat are rare., (© 2023. The Author(s).)

Importance: Ultra-widefield (UWF) imaging improves the ability to identify peripheral diabetic retinopathy (DR) lesions compared with standard imaging. Whether detection of predominantly peripheral lesions (PPLs) better predicts rates of disease worsening over time is unknown., Objective: To determine whether PPLs identified on UWF imaging are associated with increased disease worsening beyond the risk associated with baseline Early Treatment Diabetic Retinopathy Study (ETDRS) Diabetic Retinopathy Severity Scale (DRSS) score., Design, Setting, and Participants: This cohort study was a prospective, multicenter, longitudinal observational study conducted at 37 US and Canadian sites with 388 participants enrolled between February and December 2015. At baseline and annually through 4 years, 200° UWF-color images were obtained and graded for DRSS at a reading center. Baseline UWF-color and UWF-fluorescein angiography (FA) images were evaluated for the presence of PPL. Data were analyzed from May 2020 to June 2022., Interventions: Treatment of DR or diabetic macular edema was at investigator discretion., Main Outcomes and Measures: Predominantly peripheral lesions were defined as DR lesions with a greater extent outside vs inside the 7 standard ETDRS fields. Primary outcome was disease worsening defined as worsening 2 steps or more on the DRSS or receipt of DR treatment. Analyses were adjusted for baseline DRSS score and correlation between 2 study eyes of the same participant., Results: Data for 544 study eyes with nonproliferative DR (NPDR) were analyzed (182 [50%] female participants; median age, 62 years; 68% White). The 4-year disease worsening rates were 45% for eyes with baseline mild NPDR, 40% for moderate NPDR, 26% for moderately severe NPDR, and 43% for severe NPDR. Disease worsening was not associated with color PPL at baseline (present vs absent: 38% vs 43%; HR, 0.78; 95% CI, 0.57-1.08; P = .13) but was associated with FA PPL at baseline (present vs absent: 50% vs 31%; HR, 1.72; 95% CI, 1.25-2.36; P < .001)., Conclusions and Relevance: Although no association was identified with color PPL, presence of FA PPL was associated with greater risk of disease worsening over 4 years, independent of baseline DRSS score. These results suggest that use of UWF-FA to evaluate retinas peripheral to standard ETDRS fields may improve the ability to predict disease worsening in NPDR eyes. These findings support use of UWF-FA for future DR staging systems and clinical care to more accurately determine prognosis in NPDR eyes.

Importance: Presence of predominantly peripheral diabetic retinopathy (DR) lesions on ultra-widefield fluorescein angiography (UWF-FA) was associated with greater risk of DR worsening or treatment over 4 years. Whether baseline retinal nonperfusion assessment is additionally predictive of DR disease worsening is unclear., Objective: To assess whether the extent and location of retinal nonperfusion identified on UWF-FA are associated with worsening in Diabetic Retinopathy Severity Scale (DRSS) score or DR treatment over time., Design, Setting, and Participants: This cohort study was a prospective, multicenter, longitudinal observational study with data for 508 eyes with nonproliferative DR and gradable nonperfusion on UWF-FA at baseline. All images were graded at a centralized reading center; 200° ultra-widefield (UWF) color images were graded for DR at baseline and annually for 4 years. Baseline 200° UWF-FA images were graded for nonperfused area, nonperfusion index (NPI), and presence of predominantly peripheral lesions on UWF-FA (FA PPL)., Interventions: Treatment of DR or diabetic macular edema was at investigator discretion., Main Outcomes and Measures: Association of baseline UWF-FA nonperfusion extent with disease worsening, defined as either 2 or more steps of DRSS worsening within Early Treatment Diabetic Retinopathy Study fields on UWF-color images or receipt of DR treatment., Results: After adjusting for baseline DRSS, the risk of disease worsening over 4 years was higher in eyes with greater overall NPI (hazard ratio [HR] for 0.1-unit increase, 1.11; 95% CI, 1.02-1.21; P = .02) and NPI within the posterior pole (HR for 0.1-unit increase, 1.35; 95% CI, 1.17-1.56; P < .001) and midperiphery (HR for 0.1-unit increase, 1.08; 95% CI, 1.00-1.16; P = .04). In a multivariable analysis adjusting for baseline DRSS score and baseline systemic risk factors, greater NPI (HR, 1.11; 95% CI, 1.02-1.22; P = .02) and presence of FA PPL (HR, 1.89; 95% CI, 1.35-2.65; P < .001) remained associated with disease worsening., Conclusions and Relevance: This 4-year longitudinal study has demonstrated that both greater baseline retinal nonperfusion and FA PPL on UWF-FA are associated with higher risk of disease worsening, even after adjusting for baseline DRSS score and known systemic risk. These associations between disease worsening and retinal nonperfusion and FA PPL support the increased use of UWF-FA to complement color fundus photography in future efforts for DR prognosis, clinical care, and research.

Background: In eyes with diabetic macular edema, the relative efficacy of administering aflibercept monotherapy as compared with bevacizumab first with a switch to aflibercept if the eye condition does not improve sufficiently (a form of step therapy) is unclear., Methods: At 54 clinical sites, we randomly assigned eyes in adults who had diabetic macular edema involving the macular center and a visual-acuity letter score of 24 to 69 (on a scale from 0 to 100, with higher scores indicating better visual acuity; Snellen equivalent, 20/320 to 20/50) to receive either 2.0 mg of intravitreous aflibercept or 1.25 mg of intravitreous bevacizumab. The drug was administered at randomization and thereafter according to the prespecified retreatment protocol. Beginning at 12 weeks, eyes in the bevacizumab-first group were switched to aflibercept therapy if protocol-specified criteria were met. The primary outcome was the mean change in visual acuity over the 2-year trial period. Retinal central subfield thickness and visual acuity at 2 years and safety were also assessed., Results: A total of 312 eyes (in 270 adults) underwent randomization; 158 eyes were assigned to receive aflibercept monotherapy and 154 to receive bevacizumab first. Over the 2-year period, 70% of the eyes in the bevacizumab-first group were switched to aflibercept therapy. The mean improvement in visual acuity was 15.0 letters in the aflibercept-monotherapy group and 14.0 letters in the bevacizumab-first group (adjusted difference, 0.8 letters; 95% confidence interval, -0.9 to 2.5; P = 0.37). At 2 years, the mean changes in visual acuity and retinal central subfield thickness were similar in the two groups. Serious adverse events (in 52% of the patients in the aflibercept-monotherapy group and in 36% of those in the bevacizumab-first group) and hospitalizations for adverse events (in 48% and 32%, respectively) were more common in the aflibercept-monotherapy group., Conclusions: In this trial of treatment of moderate vision loss due to diabetic macular edema involving the center of the macula, we found no evidence of a significant difference in visual outcomes over a 2-year period between aflibercept monotherapy and treatment with bevacizumab first with a switch to aflibercept in the case of suboptimal response. (Funded by the National Institutes of Health; Protocol AC ClinicalTrials.gov number, NCT03321513.)., (Copyright © 2022 Massachusetts Medical Society.)

The deep chlorophyll maximum (DCM) layer is an ecologically important feature of the open ocean. The DCM cannot be observed using aerial or satellite remote sensing; thus, in situ observations are essential. Further, understanding the responses of microbes to the environmental processes driving their metabolism and interactions requires observing in a reference frame that moves with a plankton population drifting in ocean currents, i.e., Lagrangian. Here, we report the development and application of a system of coordinated robots for studying planktonic biological communities drifting within the ocean. The presented Lagrangian system uses three coordinated autonomous robotic platforms. The focal platform consists of an autonomous underwater vehicle (AUV) fitted with a robotic water sampler. This platform localizes and drifts within a DCM community, periodically acquiring samples while continuously monitoring the local environment. The second platform is an AUV equipped with environmental sensing and acoustic tracking capabilities. This platform characterizes environmental conditions by tracking the focal platform and vertically profiling in its vicinity. The third platform is an autonomous surface vehicle equipped with satellite communications and subsea acoustic tracking capabilities. While also acoustically tracking the focal platform, this vehicle serves as a communication relay that connects the subsea robot to human operators, thereby providing situational awareness and enabling intervention if needed. Deployed in the North Pacific Ocean within the core of a cyclonic eddy, this coordinated system autonomously captured fundamental characteristics of the in situ DCM microbial community in a manner not possible previously., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Environmental DNA (eDNA) is increasingly used for monitoring marine organisms; however, offshore sampling and time lag from sampling to results remain problematic. In order to overcome these challenges a robotic sampler, a 2nd generation Environmental Sample Processor (ESP), was tested for autonomous analysis of eDNA from four commercial fish species in a 4.5 million liter mesocosm. The ESP enabled in situ analysis, consisting of water collection, filtration, DNA extraction and qPCR analysis, which allowed for real-time remote reporting and archival sample collection, consisting of water collection, filtration and chemical preservation followed by post-deployment laboratory analysis. The results demonstrate that the 2G ESP was able to consistently detect and quantify target molecules from the most abundant species (Atlantic mackerel) both in real-time and from the archived samples. In contrast, detection of low abundant species was challenged by both biological and technical aspects coupled to the ecology of eDNA and the 2G ESP instrumentation. Comparison of the in situ analysis and archival samples demonstrated variance, which potentially was linked to diel migration patterns of the Atlantic mackerel. The study demonstrates strong potential for remote autonomous in situ monitoring which open new possibilities for the field of eDNA and marine monitoring.

Metagenomic and metatranscriptomic time-series data covering a 52-day period in the fall of 2016 provide an inventory of bacterial and archaeal community genes, transcripts, and taxonomy during an intense dinoflagellate bloom in Monterey Bay, CA, USA. The dataset comprises 84 metagenomes (0.8 terabases), 82 metatranscriptomes (1.1 terabases), and 88 16S rRNA amplicon libraries from samples collected on 41 dates. The dataset also includes 88 18S rRNA amplicon libraries, characterizing the taxonomy of the eukaryotic community during the bloom. Accompanying the sequence data are chemical and biological measurements associated with each sample. These datasets will facilitate studies of the structure and function of marine bacterial communities during episodic phytoplankton blooms.

Dimethylsulfoniopropionate (DMSP) is an abundant organic sulfur metabolite produced by many phytoplankton species and degraded by bacteria via two distinct pathways with climate-relevant implications. We assessed the diversity and abundance of bacteria possessing these pathways in the context of phytoplankton community composition over a 3-week time period spanning September-October, 2014 in Monterey Bay, CA. The dmdA gene from the DMSP demethylation pathway dominated the DMSP gene pool and was harboured mostly by members of the alphaproteobacterial SAR11 clade and secondarily by the Roseobacter group, particularly during the second half of the study. Novel members of the DMSP-degrading community emerged from dmdA sequences recovered from metagenome assemblies and single-cell sequencing, including largely uncharacterized gammaproteobacteria and alphaproteobacteria taxa. In the DMSP cleavage pathway, the SAR11 gene dddK was the most abundant early in the study, but was supplanted by dddP over time. SAR11 members, especially those harbouring genes for both DMSP degradation pathways, had a strong positive relationship with the abundance of dinoflagellates, and DMSP-degrading gammaproteobacteria co-occurred with haptophytes. This in situ study of the drivers of DMSP fate in a coastal ecosystem demonstrates for the first time correlations between specific groups of bacterial DMSP degraders and phytoplankton taxa., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Kalmia angustifolia is a boreal ericaceous shrub that can rapidly spread on black spruce forest cutovers in eastern Canada, where CPRS (i.e. Cutting with Protection of Regeneration and Soils") is practiced. The proliferation of Kalmia often coincides with a reduction in the growth rate of regenerating black spruce seedlings. We report on a study where we compared the local effects of Kalmia and black spruce seedling patches (i.e. two types of "Vegetation") on chemical and biochemical soil properties in CPRS cutovers within mesic spruce-moss and xeric spruce-lichen ecosystems, as well as in four mature spruce-moss forests (i.e. three "Site Types"). Results from 13C-CPMAS-NMR revealed lower O-alkyl C (i.e. carbohydrates), higher aromatic C (i.e. lignin and other phenolics) and higher carbonyl-C (i.e. amide-C and carboxyl groups) in spruce-moss than in spruce-lichen forest floors (F-horizon). In spite of these distinctions, we observed only a small number of Site Type x Vegetation interactions controlling soil properties. Vegetation had a significant effect on ten forest floor properties. Most notably, Kalmia patches had higher concentrations of condensed tannins and lower mineral N cycling. On the other hand, Site Type had a relatively greater effect on the deeper podzolic-B horizons, where mineral N and microbial activity were higher in mature spruce-moss forests than in the cutovers. Green and senescent Kalmia leaves collected at these sites had higher N, tannin and phenolic concentrations than green and senescent spruce needles. A 25 month litter bag study found lower decomposition of Kalmia leaf litter in spruce patches on spruce-lichen cutovers compared to spruce patches on spruce-moss cutovers, or to Kalmia patches on spruce-lichen cutovers. Given that black spruce seedlings obtain most of their nutrients from the forest floor, our results suggest that CPRS may have long-term negative effects on black spruce forest productivity if the spread of Kalmia is left unchecked., Competing Interests: The authors have declared that no competing interests exist.

Environmental stresses not only influence production of plant metabolites but could also modify their resorption during leaf senescence. The production-resorption dynamics of polyphenolic tannins, a class of defense compound whose ecological role extends beyond tissue senescence, could amplify the influence of climate on ecosystem processes. We studied the quantity, chemical composition, and tissue-association of tannins in green and freshly-senesced leaves of Quercus rubra exposed to different temperature ( Warming and No Warming ) and precipitation treatments ( Dry, Ambient, Wet ) at the Boston-Area Climate Experiment (BACE) in Massachusetts, USA. Climate influenced not only the quantity of tannins, but also their molecular composition and cell-wall associations. Irrespective of climatic treatments, tannin composition in Q. rubra was dominated by condensed tannins (CTs, proanthocyanidins). When exposed to Dry and Ambient * Warm conditions, Q. rubra produced higher quantities of tannins that were less polymerized. In contrast, under favorable conditions ( Wet ), tannins were produced in lower quantities, but the CTs were more polymerized. Further, even as the overall tissue tannin content declined, the content of hydrolysable tannins (HTs) increased under Wet treatments. The molecular composition of tannins influenced their content in senesced litter. Compared to the green leaves, the content of HTs decreased in senesced leaves across treatments, whereas the CT content was similar between green and senesced leaves in Wet treatments that produced more polymerized tannins. The content of total tannins in senesced leaves was higher in Warming treatments under both dry and ambient precipitation treatments. Our results suggest that, though climate directly influenced the production of tannins in green tissues (and similar patterns were observed in the senesced tissue), the influence of climate on tannin content of senesced tissue was partly mediated by the effect on the chemical composition of tannins. These different climatic impacts on leaves over the course of a growing season may alter forest dynamics, not only in decomposition and nutrient cycling dynamics, but also in herbivory dynamics.

The production of pyrogenic carbon (PyC; a continuum of organic carbon (C) ranging from partially charred biomass and charcoal to soot) is a widely acknowledged C sink, with the latest estimates indicating that ~50% of the PyC produced by vegetation fires potentially sequesters C over centuries. Nevertheless, the quantitative importance of PyC in the global C balance remains contentious, and therefore, PyC is rarely considered in global C cycle and climate studies. Here we examine the robustness of existing evidence and identify the main research gaps in the production, fluxes and fate of PyC from vegetation fires. Much of the previous work on PyC production has focused on selected components of total PyC generated in vegetation fires, likely leading to underestimates. We suggest that global PyC production could be in the range of 116-385 Tg C yr(-1) , that is ~0.2-0.6% of the annual terrestrial net primary production. According to our estimations, atmospheric emissions of soot/black C might be a smaller fraction of total PyC (<2%) than previously reported. Research on the fate of PyC in the environment has mainly focused on its degradation pathways, and its accumulation and resilience either in situ (surface soils) or in ultimate sinks (marine sediments). Off-site transport, transformation and PyC storage in intermediate pools are often overlooked, which could explain the fate of a substantial fraction of the PyC mobilized annually. We propose new research directions addressing gaps in the global PyC cycle to fully understand the importance of the products of burning in global C cycle dynamics., (© 2015 The Authors. Global Change Biology Published by John Wiley & Sons Ltd.)

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., (© 2015 The Society for Applied Microbiology.)

Wildfires release substantial quantities of carbon (C) into the atmosphere but they also convert part of the burnt biomass into pyrogenic organic matter (PyOM). This is richer in C and, overall, more resistant to environmental degradation than the original biomass, and, therefore, PyOM production is an efficient mechanism for C sequestration. The magnitude of this C sink, however, remains poorly quantified, and current production estimates, which suggest that ~1-5% of the C affected by fire is converted to PyOM, are based on incomplete inventories. Here, we quantify, for the first time, the complete range of PyOM components found in-situ immediately after a typical boreal forest fire. We utilized an experimental high-intensity crown fire in a jack pine forest (Pinus banksiana) and carried out a detailed pre- and postfire inventory and quantification of all fuel components, and the PyOM (i.e., all visually charred, blackened materials) produced in each of them. Our results show that, overall, 27.6% of the C affected by fire was retained in PyOM (4.8 ± 0.8 t C ha(-1)), rather than emitted to the atmosphere (12.6 ± 4.5 t C ha(-1)). The conversion rates varied substantially between fuel components. For down wood and bark, over half of the C affected was converted to PyOM, whereas for forest floor it was only one quarter, and less than a tenth for needles. If the overall conversion rate found here were applicable to boreal wildfire in general, it would translate into a PyOM production of ~100 Tg C yr(-1) by wildfire in the global boreal regions, more than five times the amount estimated previously. Our findings suggest that PyOM production from boreal wildfires, and potentially also from other fire-prone ecosystems, may have been underestimated and that its quantitative importance as a C sink warrants its inclusion in the global C budget estimates., (© 2014 The Authors. Global Change Biology Published by John Wiley & Sons Ltd.)

To facilitate studies of herpes simplex virus 1 latency, cell culture models of quiescent or latent infection have been developed. Using deep sequencing, we analyzed the expression of viral microRNAs (miRNAs) in two models employing human fibroblasts and one using rat neurons. In all cases, the expression patterns differed from that in productively infected cells, with the rat neuron pattern most closely resembling that found in latently infected human or mouse ganglia in vivo.

Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate Ostreopsis species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of Ostreopsis spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of Ostreopsis. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.

By inhibiting soil enzymes, tannins play an important role in soil carbon (C) and nitrogen (N) mineralization. The role of tannin chemistry in this inhibitory process, in conjunction with enzyme classes and isoforms, is less well understood. Here, we compared the inhibition efficiencies of mixed tannins (MTs, mostly limited to angiosperms) and condensed tannins (CTs, produced mostly by gymnosperms) against the potential activity of β-glucosidase (BG), N-acetyl-glucosaminidase (NAG), and peroxidase in two soils that differed in their vegetation histories. Compared with CTs, MTs exhibited 50% more inhibition of almond (Prunus dulcis) BG activity and greater inhibition of the potential NAG activity in the gymnosperm-acclimatized soils. CTs exhibited lower BG inhibition in the angiosperm-acclimated soils, whereas both types of tannins exhibited higher peroxidase inhibition in the angiosperm soils than in gymnosperm soils. At all of the tested tannin concentrations, irrespective of the tannin type and site history, the potential peroxidase activity was inhibited two-fold more than the hydrolase activity and was positively associated with the redox-buffering efficiency of tannins. Our finding that the inhibitory activities and mechanisms of MTs and CTs are dependent on the vegetative history and enzyme class is novel and furthers our understanding of the role of tannins and soil isoenzymes in decomposition., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

Monterey Bay, CA is an Eastern boundary upwelling system that is nitrogen limited much of the year. In order to resolve population dynamics of microorganisms important for nutrient cycling in this region, we deployed the Environmental Sample Processor with quantitative PCR assays targeting both ribosomal RNA genes and functional genes for subclades of cyanobacteria (Synechococcus) and ammonia-oxidizing Archaea (Thaumarchaeota) populations. Results showed a strong correlation between Thaumarchaea abundances and nitrate during the spring upwelling but not the fall sampling period. In relatively stratified fall waters, the Thaumarchaeota community reached higher numbers than in the spring, and an unexpected positive correlation with chlorophyll concentration was observed. Further, we detected drops in Synechococcus abundance that occurred on short (that is, daily) time scales. Upwelling intensity and blooms of eukaryotic phytoplankton strongly influenced Synechococcus distributions in the spring and fall, revealing what appear to be the environmental limitations of Synechococcus populations in this region. Each of these findings has implications for Monterey Bay biogeochemistry. High-resolution sampling provides a better-resolved framework within which to observe changes in the plankton community. We conclude that controls on these ecosystems change on smaller scales than are routinely assessed, and that more predictable trends will be uncovered if they are evaluated within seasonal (monthly), rather than on annual or interannual scales.

Planktonic microbial activity and community structure is dynamic, and can change dramatically on time scales of hours to days. Yet for logistical reasons, this temporal scale is typically under-sampled in the marine environment. In order to facilitate higher-resolution, long-term observation of microbial diversity and activity, we developed a protocol for automated collection and fixation of marine microbes using the Environmental Sample Processor (ESP) platform. The protocol applies a preservative (RNALater) to cells collected on filters, for long-term storage and preservation of total cellular RNA. Microbial samples preserved using this protocol yielded high-quality RNA after 30 days of storage at room temperature, or onboard the ESP at in situ temperatures. Pyrosequencing of complementary DNA libraries generated from ESP-collected and preserved samples yielded transcript abundance profiles nearly indistinguishable from those derived from conventionally treated replicate samples. To demonstrate the utility of the method, we used a moored ESP to remotely and autonomously collect Monterey Bay seawater for metatranscriptomic analysis. Community RNA was extracted and pyrosequenced from samples collected at four time points over the course of a single day. In all four samples, the oxygenic photoautotrophs were predominantly eukaryotic, while the bacterial community was dominated by Polaribacter-like Flavobacteria and a Rhodobacterales bacterium sharing high similarity with Rhodobacterales sp. HTCC2255. However, each time point was associated with distinct species abundance and gene transcript profiles. These laboratory and field tests confirmed that autonomous collection and preservation is a feasible and useful approach for characterizing the expressed genes and environmental responses of marine microbial communities.

Herpes simplex virus (HSV) type-1 establishes lifelong latency in sensory neurones and it is widely assumed that latency is the consequence of a failure to initiate virus immediate-early (IE) gene expression. However, using a Cre reporter mouse system in conjunction with Cre-expressing HSV-1 recombinants we have previously shown that activation of the IE ICP0 promoter can precede latency establishment in at least 30% of latently infected cells. During productive infection of non-neuronal cells, IE promoter activation is largely dependent on the transactivator VP16 a late structural component of the virion. Of significance, VP16 has recently been shown to exhibit altered regulation in neurones; where its de novo synthesis is necessary for IE gene expression during both lytic infection and reactivation from latency. In the current study, we utilized the Cre reporter mouse model system to characterize the full extent of viral promoter activity compatible with cell survival and latency establishment. In contrast to the high frequency activation of representative IE promoters prior to latency establishment, cell marking using a virus recombinant expressing Cre under VP16 promoter control was very inefficient. Furthermore, infection of neuronal cultures with VP16 mutants reveals a strong VP16 requirement for IE promoter activity in non-neuronal cells, but not sensory neurones. We conclude that only IE promoter activation can efficiently precede latency establishment and that this activation is likely to occur through a VP16-independent mechanism.

Recent studies suggest that unidentified prokaryotes fix inorganic carbon at globally significant rates in the immense dark ocean. Using single-cell sorting and whole-genome amplification of prokaryotes from two subtropical gyres, we obtained genomic DNA from 738 cells representing most cosmopolitan lineages. Multiple cells of Deltaproteobacteria cluster SAR324, Gammaproteobacteria clusters ARCTIC96BD-19 and Agg47, and some Oceanospirillales from the lower mesopelagic contained ribulose-1,5-bisphosphate carboxylase-oxygenase and sulfur oxidation genes. These results corroborated community DNA and RNA profiling from diverse geographic regions. The SAR324 genomes also suggested C(1) metabolism and a particle-associated life-style. Microautoradiography and fluorescence in situ hybridization confirmed bicarbonate uptake and particle association of SAR324 cells. Our study suggests potential chemolithoautotrophy in several uncultured Proteobacteria lineages that are ubiquitous in the dark oxygenated ocean and provides new perspective on carbon cycling in the ocean's largest habitat.

• Climate change could increase the frequency with which plants experience abiotic stresses, leading to changes in their metabolic pathways. These stresses may induce the production of compounds that are structurally and biologically different from constitutive compounds. • We studied how warming and altered precipitation affected the composition, structure, and biological reactivity of leaf litter tannins in Acer rubrum at the Boston-Area Climate Experiment, in Massachusetts, USA. • Warmer and drier climatic conditions led to higher concentrations of protective compounds, including flavonoids and cutin. The abundance and structure of leaf tannins also responded consistently to climatic treatments. Drought and warming in combination doubled the concentration of total tannins, which reached 30% of leaf-litter DW. This treatment also produced condensed tannins with lower polymerization and a greater proportion of procyanidin units, which in turn reduced sequestration of tannins by litter fiber. Furthermore, because of the structural flexibility of these tannins, litter from this treatment exhibited five times more enzyme (β-glucosidase) complexation capacity on a per-weight basis. Warmer and wetter conditions decreased the amount of foliar condensed tannins. • Our finding that warming and drought result in the production of highly reactive tannins is novel, and highly relevant to climate change research as these tannins, by immobilizing microbial enzymes, could slow litter decomposition and thus carbon and nutrient cycling in a warmer, drier world., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

Background: Viruses have a profound influence on both the ecology and evolution of marine plankton, but the genetic diversity of viral assemblages, particularly those in deeper ocean waters, remains poorly described. Here we report on the construction and analysis of a viral metagenome prepared from below the euphotic zone in a temperate, eutrophic bay of coastal California., Methods: We purified viruses from approximately one cubic meter of seawater collected from 200 m depth in Monterey Bay, CA. DNA was extracted from the virus fraction, sheared, and cloned with no prior amplification into a plasmid vector and propagated in E. coli to produce the MBv200m library. Random clones were sequenced by the Sanger method. Sequences were assembled then compared to sequences in GenBank and to other viral metagenomic libraries using BLAST analyses., Results: Only 26% of the 881 sequences remaining after assembly had significant (E≤0.001) BLAST hits to sequences in the GenBank nr database, with most being matches to bacteria (15%) and viruses (8%). When BLAST analysis included environmental sequences, 74% of sequences in the MBv200m library had a significant match. Most of these hits (70%) were to microbial metagenome sequences and only 0.7% were to sequences from viral metagenomes. Of the 121 sequences with a significant hit to a known virus, 94% matched bacteriophages (Families Podo-, Sipho-, and Myoviridae) and 6% matched viruses of eukaryotes in the Family Phycodnaviridae (5 sequences) or the Mimivirus (2 sequences). The largest percentages of hits to viral genes of known function were to those involved in DNA modification (25%) or structural genes (17%). Based on reciprocal BLAST analyses, the MBv200m library appeared to be most similar to viral metagenomes from two other bays and least similar to a viral metagenome from the Arctic Ocean., Conclusions: Direct cloning of DNA from diverse marine viruses was feasible and resulted in a distribution of virus types and functional genes at depth that differed in detail, but were broadly similar to those found in surface marine waters. Targeted viral analyses are useful for identifying those components of the greater marine metagenome that circulate in the subcellular size fraction.

The cellular protein hypoxia-inducible factor 1 alpha (HIF-1α) was induced after infection of human fibroblasts with human cytomegalovirus (HCMV). HCMV irradiated with ultraviolet light (uv-HCMV) also elicited the effect, demonstrating that the response was provoked by interaction of the infecting virion with the cell and that viral gene expression was not required. Although induction of HIF-1α was initiated by an early event, accumulation of the protein was not detected until 9 hours post infection, with levels increasing thereafter. Infection with uv-HCMV resulted in increased abundance of HIF-1α-specific RNA, indicating stimulation of transcription. In addition, greater phosphorylation of the protein kinase Akt was observed, and the activity of this enzyme was required for induction of HIF-1α to occur. HIF-1α controls the expression of many cellular gene products; therefore the findings reveal new ways in which interaction of the HCMV particle with the host cell may cause significant alterations to cellular physiology., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The infection of human fetal foreskin fibroblasts (HFFF2) with human cytomegalovirus (HCMV) resulted in the induction of autophagy. This was demonstrated by the increased lipidation of microtubule-associated protein 1 light chain 3 (LC3), a hallmark of autophagy, and by the visualization of characteristic vesicles within infected cells. The response was detected first at 2 h postinfection and persisted for at least 3 days. De novo protein synthesis was not required for the effect, since HCMV that was irradiated with UV light also elicited the response, and furthermore the continuous presence of cycloheximide did not prevent induction. Infection with herpes simplex virus type 1 (HSV-1) under conditions that inhibited viral gene expression provoked autophagy, whereas UV-irradiated respiratory syncytial virus did not. The induction of autophagy occurred when cells were infected with HCMV or HSV-1 that was gradient purified, but HCMV dense bodies and HSV-1 light particles, each of which lack nucleocapsids and genomes, were inactive. The depletion of regulatory proteins Atg5 and Atg7, which are required for autophagy, reduced LC3 modification in response to infection but did not result in any detectable difference in viral or cellular gene expression at early times after infection. The electroporation of DNA into HFFF2 cultures induced the lipidation of LC3 but double-stranded RNA did not, even though both agents stimulated an innate immune response. The results show a novel, early cellular response to the presence of the incoming virion and additionally demonstrate that autophagy can be induced by the presence of foreign DNA within cells.

The control of human cytomegalovirus (HCMV) immediate early (IE) gene expression in infected human fibroblasts was compared with that in the U2OS human osteosarcoma cells. Viral IE expression was stimulated by the virion protein pp71 and repressed by the cell protein hDaxx in fibroblasts, as expected from published data. Neither of these events occurred in infected U2OS cells, suggesting that this cell line lacks one or more factors that repress HCMV IE expression. The chromatin remodeling factor ATRX is absent from U2OS cells, therefore the effect of introducing this protein by electroporation of plasmid DNA was investigated. Provision of ATRX inhibited HCMV IE expression, and the presence of the HCMV-specified virion phosphoprotein pp71 overcame the repression. The experiments demonstrate that ATRX can act as a cellular intrinsic antiviral defense in U2OS cells by blocking gene expression from incoming HCMV genomes. In contrast, ATRX did not affect the replication of herpes simplex virus type 1, showing that there are differences in the way U2OS cells respond to the presence of the herpesviral genomes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We examined how tannin structure influences reactivity in tannin assays and carbon and nitrogen mineralization. Condensed tannins from the foliage of ten tree and shrub species and from pecan shells (Carya illinoensis) had different proportions of: (a) epicatechin (cis) and catechin (trans) isomers, (b) procyanidin (PC) and prodelphinidin (PD) monomers, and (c) different chain lengths. The response of each tannin to several widely used tannin assays was determined. Although there was some variation in response to proanthocyanidin (butanol/HCl) and Folin Ciocalteu assays, we did not deduce any predictable relationship between tannin structure and response to either assay. There was little variation in protein precipitation among the different tannins. To assess biological activity, six of the tannins were incubated with forest humus for 22 days. We determined that, while PC-based tannins remained at least partly extractable for the duration of the incubation, tannins with a high proportion of PD subunits rapidly became unextractable from soil. There was a positive correlation between net nitrogen mineralization and cis chemical structure. Carbon mineralization was enhanced initially by the addition of tannins to humus, but after 22 days, a negative correlation between the proportion of cis subunits and respiration was determined. Overall, we were not able to demonstrate consistent effects of structure on either microbial mineralization or reactivity to chemical assays; such relationships remain elusive.

Alzheimer's disease (AD) afflicts around 20 million people worldwide and so there is an urgent need for effective treatment. Our research showing that herpes simplex virus type 1 (HSV1) is a risk factor for AD for the brains of people who possess a specific genetic factor and that the virus causes accumulation of key AD proteins (β-amyloid (Aβ) and abnormally phosphorylated tau (P-tau)), suggests that anti-HSV1 antiviral agents might slow AD progression. However, currently available antiviral agents target HSV1 DNA replication and so might be successful in AD only if Aβ and P-tau accumulation depend on viral DNA replication. Therefore, we investigated firstly the stage(s) of the virus replication cycle required for Aβ and P-tau accumulation, and secondly whether antiviral agents prevent these changes using recombinant strains of HSV1 that progress only partly through the replication cycle and antiviral agents that inhibit HSV1 DNA replication. By quantitative immunocytochemistry we demonstrated that entry, fusion and uncoating of HSV1, are insufficient to induce Aβ and P-tau production. We showed also that none of the "immediate early" viral proteins is directly responsible, and that Aβ and P-tau are produced at a subsequent stage of the HSV1 replication cycle. Importantly, the anti-HSV1 antiviral agents acyclovir, penciclovir and foscarnet reduced Aβ and P-tau accumulation, as well as HSV1, with foscarnet being less effective in each case. P-tau accumulation was found to depend on HSV1 DNA replication, whereas Aβ accumulation was not. The antiviral-induced decrease in Aβ is attributable to the reduced number of new viruses, and hence the reduction in viral spread. Since antiviral agents reduce greatly Aβ and P-tau accumulation in HSV1-infected cells, they would be suitable for treating AD with great advantage unlike current AD therapies, only the virus, not the host cell, would be targeted.

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions.

The Affordable Care Act (ACA) was signed into law on March 23, 2010 and will fundamentally alter health care in the United States for years to come. The US is currently one of the only industrialized countries without universal health insurance. The new law expands existing public insurance for the poor. It also provides financial credits to low income individuals and some small businesses to purchase health insurance. By government estimates, the law will bring insurance to 30 million people. The law also provides for a significant new investment in prevention and wellness. It appropriates an unprecedented $15 billion in a prevention and public health fund, to be disbursed over 10 years, as well as creates a national prevention council to oversee the government's prevention efforts. This paper discusses 3 major prevention provisions in the legislation: 1) the waiving of cost-sharing for clinical preventive services, 2) new funding for community preventive services, and 3) new funding for workplace wellness programs. The paper examines the scientific evidence behind these provisions as well as provides examples of some model programs. Taken together, these provisions represent a significant advancement for prevention in the US health care system, including a shift towards healthier environments. However, in this turbulent economic and political environment, there is a real threat that much of the law, including the prevention provisions, will not receive adequate funding.

Background: Human cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in situations where the immune system is immature or compromised. HCMV immediate early (IE) gene expression is stimulated by the virion phosphoprotein pp71, encoded by open reading frame (ORF) UL82, and this transactivation activity is important for the efficient initiation of viral replication. It is currently recognized that pp71 acts to overcome cellular intrinsic defences that otherwise block viral IE gene expression, and that interactions of pp71 with the cell proteins Daxx and ATRX are important for this function. A further property of pp71 is the ability to enable prolonged gene expression from quiescent herpes simplex virus type 1 (HSV-1) genomes. Non-human primate cytomegaloviruses encode homologs of pp71, but there is currently no published information that addresses their effects on gene expression and modes of action., Results: The UL82 homolog encoded by simian cytomegalovirus (SCMV), strain Colburn, was identified and cloned. This ORF, named S82, was cloned into an HSV-1 vector, as were those from baboon, rhesus monkey and chimpanzee cytomegaloviruses. The use of an HSV-1 vector enabled expression of the UL82 homologs in a range of cell types, and permitted investigation of their abilities to direct prolonged gene expression from quiescent genomes. The results show that all UL82 homologs activate gene expression, and that neither host cell type nor promoter target sequence has major effects on these activities. Surprisingly, the UL82 proteins specified by non-human primate cytomegaloviruses, unlike pp71, did not direct long term expression from quiescent HSV-1 genomes. In addition, significant differences were observed in the intranuclear localization of the UL82 homologs, and in their effects on Daxx. Strikingly, S82 mediated the release of Daxx from nuclear domain 10 substructures much more rapidly than pp71 or the other proteins tested. All UL82 homologs stimulated the early release of ATRX from nuclear domain 10., Conclusion: All of the UL82 homolog proteins analysed activated gene expression, but surprising differences in other aspects of their properties were revealed. The results provide new information on early events in infection with cytomegaloviruses.

A sandwich hybridization assay (SHA) was developed to detect 16S rRNAs indicative of phylogenetically distinct groups of marine bacterioplankton in a 96-well plate format as well as low-density arrays printed on a membrane support. The arrays were used in a field-deployable instrument, the Environmental Sample Processor (ESP). The SHA employs a chaotropic buffer for both cell homogenization and hybridization, thus target sequences are captured directly from crude homogenates. Capture probes for seven of nine different bacterioplankton clades examined reacted specifically when challenged with target and non-target 16S rRNAs derived from in vitro transcribed 16S rRNA genes cloned from natural samples. Detection limits were between 0.10-1.98 and 4.43- 12.54 fmole ml(-1) homogenate for the 96-well plate and array SHA respectively. Arrays printed with five of the bacterioplankton-specific capture probes were deployed on the ESP in Monterey Bay, CA, twice in 2006 for a total of 25 days and also utilized in a laboratory time series study. Groups detected included marine alphaproteobacteria, SAR11, marine cyanobacteria, marine group I crenarchaea, and marine group II euryarchaea. To our knowledge this represents the first report of remote in situ DNA probe-based detection of marine bacterioplankton.

The mechanism controlling the exit from herpes simplex virus latency (HSV) is of central importance to recurrent disease and transmission of infection, yet interactions between host and viral functions that govern this process remain unclear. The cascade of HSV gene transcription is initiated by the multifunctional virion protein VP16, which is expressed late in the viral replication cycle. Currently, it is widely accepted that VP16 transactivating function is not involved in the exit from latency. Utilizing the mouse ocular model of HSV pathogenesis together with genetically engineered viral mutants and assays to quantify latency and the exit from latency at the single neuron level, we show that in vivo (i) the VP16 promoter confers distinct regulation critical for viral replication in the trigeminal ganglion (TG) during the acute phase of infection and (ii) the transactivation function of VP16 (VP16TF) is uniquely required for the exit from latency. TG neurons latently infected with the VP16TF mutant in1814 do not express detectable viral proteins following stress, whereas viruses with mutations in the other major viral transcription regulators ICP0 and ICP4 do exit the latent state. Analysis of a VP16 promoter/reporter mutant in the background of in1814 demonstrates that the VP16 promoter is activated in latently infected neurons following stress in the absence of other viral proteins. These findings support the novel hypothesis that de novo expression of VP16 regulates entry into the lytic program in neurons at all phases of the viral life cycle. HSV reactivation from latency conforms to a model in which stochastic derepression of the VP16 promoter and expression of VP16 initiates entry into the lytic cycle.

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.

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.

The human cytomegalovirus (HCMV) tegument protein pp71, encoded by gene UL82, stimulates viral immediate-early (IE) transcription. pp71 interacts with the cellular protein hDaxx at nuclear domain 10 (ND10) sites, resulting in the reversal of hDaxx-mediated repression of viral transcription. We demonstrate that pp71 displaces an hDaxx-binding protein, ATRX, from ND10 prior to any detectable effects on hDaxx itself and that this event contributes to the role of pp71 in alleviating repression. Introduction of pp71 into cells by transfection, infection with a pp71-expressing herpes simplex virus type 1 vector, or by generation of transformed cell lines promoted the rapid relocation of ATRX from ND10 to the nucleoplasm without alteration of hDaxx levels or localization. A pp71 mutant protein unable to interact with hDaxx did not affect the intranuclear distribution of ATRX. Infection with HCMV at a high multiplicity of infection resulted in rapid displacement of ATRX from ND10, the effect being observed maximally by 2 h after adsorption, whereas infection with the UL82-null HCMV mutant ADsubUL82 did not affect ATRX localization even at 7 h postinfection. Cell lines depleted of ATRX by transduction with shRNA-expressing lentiviruses supported increased IE gene expression and virus replication after infection with ADsubUL82, demonstrating that ATRX has a role in repressing IE transcription. The results show that ATRX, in addition to hDaxx, is a component of cellular intrinsic defenses that limit HCMV IE transcription and that displacement of ATRX from ND10 by pp71 is important for the efficient initiation of viral gene expression.

Herpes simplex virus type 1 (HSV-1) mutants impaired in the activities of the structural protein VP16 and the immediate-early (IE) proteins ICP0 and ICP4 establish a quiescent infection in human fibroblasts, with most cells retaining an inactive, repressed viral genome for sustained periods in culture. To date, the quiescent state has been considered stable, since it has been reversed only by provision of herpesviral proteins, such as ICP0, not by alteration of the cell physiological state. We report that the interaction of HSV-1 with human fibroblasts can be altered significantly by transient treatment of cultures with sodium arsenite, an inducer of heat shock and oxidative stress, or gramicidin D, a toxin that selectively permeabilizes cell membranes, prior to infection. These regimens stimulated gene expression from IE-deficient HSV-1 mutants in a promoter sequence-independent manner and also overcame the replication defect of ICP0-null mutants. Reactivation of gene expression from quiescent HSV-1 genomes and the resumption of virus replication were observed following addition of arsenite or gramicidin D to cultures. Both agents induced reorganization of nuclear domain 10 structures, the sites of quiescent genomes, but appeared to do so through different mechanisms. The results demonstrate that the physiological state of the cell is important in determining the outcome of infection with IE-deficient HSV-1 and show novel methods for reactivating quiescent HSV-1 in fibroblasts with a high efficiency.

During an initial encounter with herpes simplex virus type 1 (HSV-1) it takes several days for an adaptive immune response to develop and for herpes-specific CD8(+) T cells to infiltrate sites of infection. By this time the virus has firmly established itself within the innervating sensory nervous system where it then persists indefinitely. Preventing the establishment of viral latency would require blocking the skin to nervous system transmission of the virus. We wished to examine if CD8(+) T cells present early during acute HSV-1 infection could block this transmission. We show that effector CD8(+) T cells failed to prevent the establishment of HSV latency even when present prior to infection. This lack of blocking likely reflects the delayed infiltration of the CD8(+) T cells into the infected skin. Examination of the kinetics of HSV-1 infection highlighted the rapidity at which the virus infects the sensory ganglia and singled out early viral replication within the skin as an important factor in determining the magnitude of the ensuing latent infection. Though unable to prevent the establishment of latency, CD8(+) T cells could reduce the average viral copy number of the residual latent infection by dampening the skin infection and thus limiting the skin-to-nerve transmission of virus.

Studies on the herpes simplex virus type 1 UL25-null mutant KUL25NS have shown that the capsid-associated UL25 protein is required at a late stage in the encapsidation of viral DNA. Our previous work on UL25 with the UL25 temperature-sensitive (ts) mutant ts1204 also implicated UL25 in a role at very early times in the virus growth cycle, possibly at the stage of penetration of the host cell. We have reexamined this mutant and discovered that it had an additional ts mutation elsewhere in the genome. The ts1204 UL25 mutation was transferred into wild-type (wt) virus DNA, and the UL25 mutant ts1249 was isolated and characterized to clarify the function of UL25 at the initial stages of virus infection. Indirect immunofluorescence assays and in situ hybridization analysis of virus-infected cells revealed that the mutant ts1249 was not impaired in penetration of the host cell but had an uncoating defect at the nonpermissive temperature. When ts1249-infected cells were incubated initially at the permissive temperature to allow uncoating of the viral genome and subsequently transferred to the restrictive temperature, a DNA-packaging defect was evident. The results suggested that ts1249, like KUL25NS, had a block at a late stage of DNA packaging and that the packaged genome was shorter than the full-length genome. Examination of ts1249 capsids produced at the nonpermissive temperature revealed that, in comparison with wt capsids, they contained reduced amounts of UL25 protein, thereby providing a possible explanation for the failure of ts1249 to package full-length viral DNA.