The entry inhibitor DS003 (BMS-599793): a BMS-806 analogue, provides superior activity as a pre-exposure prophylaxis candidate.

OBJECTIVE: Small molecule inhibitors able to bind to gp120 and prevent CD4+-induced HIV-1 envelope conformational change provide an important class of inhibitors. Currently, only Fostemsavir is approved for HAART, which makes this class of inhibitors attractive candidates for prevention. We assessed the activity of DS003 (BMS-599793), an analogue of BMS-378806, in different mucosal tissues and elucidated its mechanism of action. DESIGN: Preclinical analysis was performed with human mucosal tissue models as surrogates of in-vivo activity. METHODS: Antiviral efficacy of DS003 was assessed in mucosal tissue explants (ecto-cervical, penile and colorectal) and in trans-infection models (co-cultures of dendritic or mucosal migratory cells with CD4+ T cells) with several dosing times (2, 24 h and sustained) and in combination with a fusion inhibitor. Binding of DS003 to gp120 was assessed by flow cytometry and bio-layer interferometry and further probed in competitive studies using soluble CD4+ (sCD4+) and an anti-CD4+ induced antibody, 17b. RESULTS: In all models, the inhibitory activity of DS003 was increased with longer periods of exposure and by combination with a fusion inhibitor. Pre-exposure to sCD4+ impeded DS003 binding to viral envelope. In contrast, DS003 did not impact subsequent binding of sCD4+. Furthermore, sCD4+-induced epitope exposure as assessed by 17b binding was significantly reduced in the presence of DS003. CONCLUSION: DS003 inhibits HIV-1 infection by binding to or near the CD4+ binding site of gp120, preventing CD4+-induced conformational change essential for viral fusion. These data highlight the potential of DS003 for development as a pre-exposure prophylaxis candidate.

High-frequency phosphorus monitoring of the River Kennet, UK: are ecological problems due to intermittent sewage treatment works failures?

The River Kennet in southern England has exhibited excessive benthic algal growth and associated ecological problems, such as loss of macrophytes and invertebrates, since the 1980s. These ecological problems were attributed to regular peaks in phosphorus concentration, which were widely attributed to intermittent failures of the Marlborough sewage treatment works (STW). This study deployed high-frequency phosphorus auto-analysers to monitor the total reactive phosphorus (TRP) concentrations of Marlborough STW final effluent and the downstream River Kennet at hourly and 30 minute resolution respectively, between 2008 and 2009. This monitoring confirmed that the Marlborough STW was operating well within its 1000 µg l⁻¹ annual mean total phosphorus consent limit, with mean total P and soluble reactive P concentrations of 675 and 345 µg l⁻¹ respectively. There were two occasions where effluent TRP concentration exceeded 1000 µg l⁻¹, and only one of these resulted in a peak in TRP concentration of over 100 µg l⁻¹ in the River Kennet at Mildenhall. The other nine peaks of over 100 µg l⁻¹ in the River Kennet during the monitoring period were associated with storm events, indicating that diffuse-source inputs and remobilisation of stored within-channel phosphorus were the cause of the peaks in river concentration, rather than Marlborough STW. The value of high-frequency environmental monitoring and the problems associated with using nutrient auto-analysers in the field are discussed. Seasonal phosphorus consents for STWs could provide a useful and cost effective means to improve both water quality and river ecology in the upper River Kennet.

Recovery of sulfate saturated soils in the Plynlimon catchments, mid-Wales following reductions in atmospheric S inputs from the 1980s to 2011.

Sulfate adsorption capacity of B-horizons of base-poor, predominantly stagnopodzol, soils from the Plynlimon catchments, mid-Wales was determined by combination of laboratory adsorption and desorption isotherms. Results show that sulfate adsorption capacity of a range of stagnopodzol (Histic-stagno-podzol (Leptic), WRB), brown podzolic soil (Histic-umbrisol (Leptic), WRB) and stagnohumic gley (Histic-stagno-gleysol, WRB) B-horizons was positively related to the amounts of extractable (pyrophosphate and oxalate) Fe + Al, with the stagnopodzol and brown podzolic soil Bs horizon having the largest adsorption capacity and stagnohumic gley Bg horizon the smallest adsorption capacity. Results show that dissolved organic carbon (DOC) has a negative but limited effect on sulfate adsorption in these soils. Results obtained from a set of historical soil samples revealed that the grassland brown podzolic soil Bs horizon and afforested stagnopodzol Bs horizon were highly saturated with sulfate in the 1980s, at 63% and 89% respectively, whereas data from some recently sampled soil from two sites revisited in 2010-11 indicates that percentage sulfate adsorption saturation has since fallen substantially, to 41% and 50% respectively. Between 1984 and 2009 the annual rainfall-weighted mean excess SO(4)-S concentration in bulk precipitation declined linearly from 0.37 mg S l(-1) to 0.17 mg S l(-1). Over the same period, flow weighted annual mean stream water SO(4)-S concentrations decreased approximately linearly from 1.47 mg S l(-1) to 0.97 mg S l(-1) in the plantation afforested Hafren catchment compared to a drop from 1.25 to 0.69 mg S l(-1) in the adjacent moorland catchment of the Afon Gwy. In flux terms, the mean decrease in annual stream water SO(4)-S flux has been approximately 0.4 kg S ha(-1) yr(-1), whilst the recovery in stream water quality in the Afon Cyff grassland catchment has been partly offset by loss of SO(4)-S by desorption from the soil sulfur pool of approximately 0.2 kg S ha(-1) yr(-1).

Preclinical evaluation of the HIV-1 fusion inhibitor L'644 as a potential candidate microbicide.

Topical blockade of the gp41 fusogenic protein of HIV-1 is one possible strategy by which microbicides could prevent HIV transmission, working early against infection, by inhibiting viral entry into host cells. In this study, we examined the potential of gp41 fusion inhibitors (FIs) as candidate anti-HIV microbicides. Preclinical evaluation of four FIs, C34, T20, T1249, and L'644, was performed using cellular and ex vivo genital and colorectal tissue explant models. Increased and sustained activity was detected for L'644, a cholesterol-derivatized version of C34, relative to the other FIs. The higher potency of L'644 was further increased with sustained exposure of cells or tissue to the compound. The activity of L'644 was not affected by biological fluids, and the compound was still active when tissue explants were treated after viral exposure. L'644 was also more active than other FIs against a viral escape mutant resistant to reverse transcriptase inhibitors (RTIs), demonstrating the potential of L'644 to be included as part of a multiactive antiretroviral (ARV) combination-based microbicide. These data support the further development of L'644 for microbicide application.

High-frequency water quality time series in precipitation and streamflow: from fragmentary signals to scientific challenge.

Eighteen months of 7-hourly analyses of rainfall and stream water chemistry are presented, spanning a wide range of chemical determinands and building on over 20 years of weekly records for the moorland headwaters of the river Severn. The high-frequency time series data show that hydrochemical responses to major hydrological and biological drivers of short-term variability in rainfall and rivers are not captured by conventional low-frequency monitoring programmes. A wealth of flow related, flow independent, diurnal, seasonal and annual fluctuations indicate a cacophony of interactions within the catchment and stream. The complexity of the chemical dynamics is visually obvious, although there appears to be no clear way of translating this complexity into a simple algorithm. The work provides a proof of concept for the complex structure of catchment functioning revealed by extensive high-frequency measurements coupled with high analytical sensitivity and reproducibility. It provides new insights into hydrogeochemical functioning and a novel resource for catchment modelling.

Sewage effluent clean-up reduces phosphorus but not phytoplankton in lowland chalk stream (River Kennet, UK) impacted by water mixing from adjacent canal.

Information is provided on phosphorus in the River Kennet and the adjacent Kennet and Avon Canal in southern England to assess their interactions and the changes following phosphorus reductions in sewage treatment work (STW) effluent inputs. A step reduction in soluble reactive phosphorus (SRP) concentration within the effluent (5 to 13 fold) was observed from several STWs discharging to the river in the mid-2000s. This translated to over halving of SRP concentrations within the lower Kennet. Lower Kennet SRP concentrations change from being highest under base-flow to highest under storm-flow conditions. This represented a major shift from direct effluent inputs to a within-catchment source dominated system characteristic of the upper part to the catchment. Average SRP concentrations in the lower Kennet reduced over time towards the target for good water quality. Critically, there was no corresponding reduction in chlorophyll-a concentration, the waters remaining eutrophic when set against standards for lakes. Following the up gradient input of the main water and SRP source (Wilton Water), SRP concentrations in the canal reduced down gradient to below detection limits at times near its junction with the Kennet downstream. However, chlorophyll concentrations in the canal were in an order of magnitude higher than in the river. This probably resulted from long water residence times and higher temperatures promoting progressive algal and suspended sediment generations that consumed SRP. The canal acted as a point source for sediment, algae and total phosphorus to the river especially during the summer months when boat traffic disturbed the canal's bottom sediments and the locks were being regularly opened. The short-term dynamics of this transfer was complex. For the canal and the supply source at Wilton Water, conditions remained hypertrophic when set against standards for lakes even when SRP concentrations were extremely low.

Decreasing boron concentrations in UK rivers: insights into reductions in detergent formulations since the 1990s and within-catchment storage issues.

The changing patterns of riverine boron concentration are examined for the Thames catchment in southern/southeastern England using data from 1997 to 2007. Boron concentrations are related to an independent marker for sewage effluent, sodium. The results show that boron concentrations in the main river channels have declined with time especially under baseflow conditions when sewage effluent dilution potential is at its lowest. While boron concentrations have reduced, especially under low-flow conditions, this does not fully translate to a corresponding reduction in boron flux and it seems that the "within-catchment" supplies of boron to the river are contaminated by urban sources. The estimated boron reduction in the effluent input to the river based on the changes in river chemistry is typically around 60% and this figure matches with an initial survey of more limited data for the industrial north of England. Data for effluent concentrations at eight sewage treatment works within the Kennet also indicate substantial reductions in boron concentrations: 80% reduction occurred between 2001 and 2008. For the more contaminated rivers there are issues of localised rather than catchment-wide sources and uncertainties over the extent and nature of water/boron stores. Atmospheric sources average around 32 to 61% for the cleaner and 4 to 14% for the more polluted parts. The substantial decreases in the boron concentrations correspond extremely well with the timing and extent of European wide trends for reductions in the industrial and domestic usage of boron-bearing compounds. It clearly indicates that such reductions have translated into lower average and peak concentrations of boron in the river although the full extent of these reductions has probably not yet occurred due to localised stores that are still to deplete.

Declines in phosphorus concentration in the upper River Thames (UK): links to sewage effluent cleanup and extended end-member mixing analysis.

Phosphorus concentrations in the upper River Thames Basin (southeastern England) are described and linked to sewage effluent sources. Weekly surveys between 1997 and 2007 of the Thames and two of its major tributaries, the Thame and the Kennet indicated that phosphorus was mainly in soluble reactive (SRP) form. Baseflow concentrations in the Thames reduced from 1584microg/l in 1998 to 376microg/l in 2006 and from 2655 to 715microg/l for the Thame. Flow response, flux and endmember mixing analysis indicated that these declines resulted from SRP reductions in sewage treatment works (STW) effluent following phosphorus stripping for the major STWs in the region. This was confirmed by comparing our analysis with direct measurements of SRP in the effluents based on Environment Agency data. A within-river loss under baseflow of approximately 64% (range 56-78%) of the SRP-effluent input was estimated for the Thames, with a near balance for the Thame. SRP concentrations in the Kennet were an order of magnitude lower than the Thames/Thame: non-point sources dominated and were important for all the rivers at high flows. It was concluded that removal of SRP from effluents would be insufficient SRP in the Thames and Thame to meet annual average environmental targets of 50 to 120microg/l. The paper flags the value of combining hydrological/chemical tracing and concentration/flux approaches to data interrogation and the bonus of having actual measurements of the effluent. It highlights the need for fuller assessment of water storage/sediment/biota interactions for phosphorus and for caution in using boron as a long-term tracer for effluent inputs, its concentrations having declined markedly in response to reduced usage in washing powders: the value of using sodium as a tracer for examining SRP changes is shown.

Design, expression, and characterization of a multivalent, combination HIV microbicide.

Many promising microbicide candidates are proteins or peptides, including neutralizing monoclonal antibodies (mAbs). Here, the expression of the HIV-neutralizing mAb b12 in transgenic plants is described. The plant-derived mAb b12 was shown to have gp120 binding activity and HIV-neutralizing activity in vitro. However, it is likely that a protein-based microbicide will need to comprise a combination of two or more products, in order to provide long-lasting and cross-clade protection. Building on the expression of mAb b12 and to address the need for a combinational agent, the expression of a fusion protein of mAb b12 with cyanovirin-N, another protein microbicide, has been explored. This fusion protein molecule is predicted to have four binding sites for HIV gp120 with two different specificities. The fusion protein was assembled and expressed in planta, and functionality was confirmed by gp120 binding and HIV neutralization in vitro. Each moiety of the fusion protein retained its binding ability to gp120. In addition, this fusion protein demonstrated increased anti-HIV potency compared to b12 or CV-N alone. This fusion protein addresses the requirement to combine microbicide products, and the production in plants is a step toward resolving the issues of manufacturing scalability and cost for developing countries.

Preclinical evaluation of lime juice as a topical microbicide candidate.

BACKGROUND: The continued growth of the global HIV epidemic highlights the urgent need to develop novel prevention strategies to reduce HIV transmission. The development of topical microbicides is likely to take a number of years before such a product would be widely available. This has resulted in a call for the rapid introduction of simpler vaginal intervention strategies in the interim period. One suggested practice would be vaginal douching with natural products including lime or lemon juice. Here we present a comprehensive preclinical evaluation of lime juice (LiJ) as a potential intervention strategy against HIV. RESULTS: Pre-treatment of HIV with LiJ demonstrated direct virucidal activity, with 10% juice inactivating the virus within 5 minutes. However, this activity was significantly reduced in the presence of seminal plasma, where inactivation required maintaining a 1:1 mixture of neat LiJ and seminal plasma for more than 5 minutes. Additionally, LiJ demonstrated both time and dose-dependent toxicity towards cervicovaginal epithelium, where exposure to 50% juice caused 75-90% toxicity within 5 minutes increasing to 95% by 30 minutes. Cervicovaginal epithelial cell monolayers were more susceptible to the effects of LiJ with 8.8% juice causing 50% toxicity after 5 minutes. Reconstructed stratified cervicovaginal epithelium appeared more resilient to LiJ toxicity with 30 minutes exposure to 50% LiJ having little effect on viability. However viability was reduced by 75% and 90% following 60 and 120 minutes exposure. Furthermore, repeat application (several times daily) of 25% LiJ caused 80-90% reduction in viability. CONCLUSION: These data demonstrate that the virucidal activity of LiJ is severely compromised in the presence of seminal plasma. Potentially, to be effective against HIV in vivo, women would need to apply a volume of neat LiJ equal to that of an ejaculate, and maintain this ratio vaginally for 5-30 minutes after ejaculation. Data presented here suggest that this would have significant adverse effects on the genital mucosa. These data raise serious questions about the plausibility and safety of such a prevention approach.

Transgenic plant production of Cyanovirin-N, an HIV microbicide.

Cyanovirin-N (CV-N) is a microbicide candidate that inactivates a wide range of HIV strains by binding to gp120. Production of CV-N, or any protein microbicide, needs to be at extremely high levels and low cost to have an impact on global health. Thus, it is unlikely that fermentor-based systems will be suitable, including recombinant E. coli, where CV-N aggregates and dimers have consistently been found. Transgenic plants may provide a suitable expression system for protein microbicides, as production can be easily and economically scaled up. Here, Nicotiana tabacum was transformed with a gene encoding CV-N to explore proof of concept for the production of CV-N in transgenic plants. Plant-derived rCV-N was recoverable at levels of 130 ng/mg of fresh leaf tissue, or at least 0.85% of total soluble plant protein. Western blot analysis demonstrated that virtually all of the rCV-N was expressed in the desired monomeric form. Functionality was demonstrated by specific binding to gp120 and protection of T-cells from in vitro HIV infection. Hydroponic culturing of transgenic plants demonstrated CV-N rhizosecretion at levels of 0.64 mug/ml hydroponic media after 24 days. Therefore, we suggest that transgenic plants have the potential to provide strategies for large-scale protein microbicide production.