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Individuals with the apolipoprotein E e4 genetic risk factor for Alzheimer's disease (AD) show deficits in olfactory function. The purpose of the present study was to examine longitudinally odor identification (odor ID), odor threshold, picture identification, and global cognitive status in allele positive (e4+) and negative (e4-) persons. Participants were initially given the San Diego Odor Identification test, an odor threshold test, and the Dementia Rating Scale (DRS). Participants were re-tested approximately four years later. The results indicate: (1) odor ID declined more rapidly in e4+ than in e4- normal elderly adults; (2) neither group exhibited a significant decline in odor threshold, picture identification or DRS scores. These results suggest that declines in odor identification occur before declines in other measures of dementia in persons at risk for AD because of their APOE allele genetic status.

The article discusses the study which examines the differences in storm water quality measured during sampling techniques at several locations in stream cross-sections. The researchers have observed different degrees of concentration variability within stream cross-sections for dissolved and particulate constituents. The study indicates some substantial temporal concentration variability for dissolved and particulate constituents, but fails to account for potential variability in cross-sectional concentrations.

A new soil extractant (H3A-1) with the ability to extract ammonium (NH4) nitrogen (N), nitrate (NO3) N, and phosphorus (P) from soil was originally developed and tested against 32 soils (Haney et al. 2006). H3A-1 is composed of organic acid root exudates, which are naturally occurring, and lithium citrate. In this study, H3A-1 was modified to reduce the extractable iron and aluminum and improve the nutrient extracting relationships with other well-known soil extractants. Correlations show improved relationships with NO3, NH4, PO4, P, potassium, calcium, and zinc when compared to the original H3A-1 as well as standard soil-test methods [Olsen, potassium chloride (KCl), water, Mehlich 3, Bray 1, ammonium acetate (NH4 OAc), and diethylenetriaminepentaacetic acid (DTPA)]. The modifications were tested against the results from the North American Proficiency Testing (NAPT) database, which included 60 soils, varying in pH, organic carbon, and clay content. [ABSTRACT FROM AUTHOR]

Soil microbial activity is greatly affected by soil water content. Determining the appropriate moisture content to rewet soils that have been dried in preparation for laboratory incubations to determine microbial activity can be laborious and time-consuming. The most common methods used achieve sufficient moisture content for peak microbial respiration are gravimetric water content, soil matric potential, or percentage of water-filled pore space (WFPS). Alternatively, a fast, simple, and accurate way to ensure that a given soil receives the appropriate amount of water for peak soil microbial respiration is to rely on natural capillary action for rewetting the dry soil. The capillary method is related to the gravimetric method for water uptake and has a strong correlation with WFPS. A microbial respiration test was conducted to compare rewetting methods. The 24-h carbon dioxide (CO2) / carbon (C) results were very similar and strongly correlated using the gravimetric method and the capillary method for rewetting dried soil. [ABSTRACT FROM AUTHOR]

Improving access to care, health outcomes, and patient satisfaction are primary objectives for healthcare practices. This article outlines benefits, concerns, and possible challenges of shared medical appointments (SMAs) for patients and providers. The SMA model was designed to support providers' demanding schedules by allowing patients with the same chronic condition to be seen in a group setting. By concentrating on patient education and disease management, interactive meetings provide an opportunity for patients to share both successes and struggles with others experiencing similar challenges. Studies demonstrated that SMAs improved patient access, enhanced outcomes, and promoted patient satisfaction. This article describes the potential benefits of SMAs for patients with chronic heart disease, which consumes a large number of healthcare dollars related to hospital admissions, acute exacerbations, and symptom management Education for self-management of chronic disease can become repetitive and time consuming. The SMA model introduces a fresh and unique of healthcare visits, allowing providers to devote more time and attention to patients and improve productivity. The SMA model provides an outstanding method for nurse practitioners to demonstrate their role as a primary care provider, by leading patients in group discussions and evaluating their current health status. Patient selection, preparation, and facilitation of an SMA are discussed to demonstrate the complementary nature of an SMA approach in a healthcare practice. [ABSTRACT FROM AUTHOR]

The coastal South American species Cyprinodon dearborni contains two lineages distinct at both mitochondrial and nuclear loci. One appears to be a long-term South American endemic, whereas the other is a more recent colonizer related to the widespread Cyprinodon variegatus. [ABSTRACT FROM AUTHOR]

The measurement of soil carbon dioxide (CO2) respiration is a means to gauge biological soil fertility. Test methods for respiration employed in the laboratory vary somewhat, and to date the equipment and labor required have limited more widespread adoption of such methodologies. A new method to measure soil respiration was tested along with the traditional alkali trap and titration method. The new method involves the Solvita gel system, which was originally designed for CO2 respiration from compost but has been applied in this research to soils with treatments of increasing dairy manure compost. The objectives of this research are to (1) examine the relationship between the CO2 release after 1 day of incubation from soils amended with dairy manure compost that have been dried and rewetted as determined using the titration method and the Solvita gel system, and (2) compare water-soluble organic nitrogen (N), as well as carbon (C), N, and phosphorus (P) mineralization after 28 days of incubation with 1-day CO2 release from the titration method and Solvita gel system. One-day CO2 from both titration and the Solvita gel system were highly correlated with cumulative 28-day CO2 as well as the basal rate from 7-28 days of incubation. Both methods were also highly correlated with 28-day N and P mineralization as well as the initial water-extractable organic N and C concentration. The data suggest that the Solvita gel system for soil CO2 analysis could be a simple and easily used method to quantify soil microbial activity and possibly provide an estimate of potential mineralizable N and P. Once standardized soil sampling and laboratory analysis protocols are established, the Solvita method could be easily adapted to commercial soil testing laboratories as an index of soil microbial activity. [ABSTRACT FROM AUTHOR]

An evaluation of soil quality, which integrates biological, chemical and physical processes, would be beneficial to regulators as well as mining companies when making reclamation decisions. Coal mining regulations require that industry perform soil sampling and submit laboratory results in order to determine pre-mine and reclaimed soil quality. A rapid and accurate biological soil quality method, such as one-day CO2 (1-day CO2) analysis, can be used to determine soil microbial activity which is related to the soils' ability to sustain nutrient cycling. Our objective in this study was to compare native and reclaimed soils from surface-mine operations in order to assess the effectiveness of the 1-day CO2 method as a tool for determining biological soil quality. Soil samples were taken from sites that visually had poor and well-vegetated reclaimed areas of a surface-mine operation as well as an undisturbed native site. Chemical, physical and biological indicators were compared to 1-day CO2 analysis for microbial activity. Results indicate that the biological soil quality indicators as summarized by 1-day CO2 analysis are a more sensitive indicator of soil health on the reclaimed soils tested than chemical analysis alone. One-day CO2 analysis can be a useful additional tool for regulators and mining companies when assessing the soils ability to sustain plant growth and evaluate reclamation success. [ABSTRACT FROM AUTHOR]

The pelagic larval stage of most coral reef fishes might allow extensive dispersal or, alternatively, some level of local recruitment might be important. Molecular markers can be used to obtain indirect estimates of dispersal to evaluate these alternatives, yet the extent of gene flow among populations is known for only a small number of species. The use of such markers must take into account the properties of the markers and the demographic history of the population when making inferences about current gene flow. In the Caribbean bluehead wrasse, Thalassoma bifasciatum, previous studies have found both substantial levels of local recruitment, in studies interpreting otolith microchemistry and, conversely, a lack of genetic differentiation inferred from mitochondrial DNA (mtDNA) restriction-fragment length polymorphism (RFLP) data and allozymes. However, if subtle differentiation exists, larger sample sizes and highly variable markers may be required to discern it. Here we present results from mitochondrial control region sequence and microsatellite data that indicate a lack of genetic differentiation at both small and large spatial scales. However, historical processes, such as changes in population size, may have affected the current distribution of genetic variation.Heredity (2007) 98, 294–302. doi:10.1038/sj.hdy.6800940; published online 31 January 2007 [ABSTRACT FROM AUTHOR]

A new soil extractant (H 3 A) with the ability to extract NH 4 , NO 3 , and P from soil was developed and tested against 32 soils, which varied greatly in clay content, organic carbon (C), and soil pH. The extractant (H 3 A) eliminates the need for separate phosphorus (P) extractants for acid and calcareous soils and maintains the extract pH, on average, within one unit of the soil pH. The extractant is composed of organic root exudates, lithium citrate, and two synthetic chelators (DTPA, EDTA). The new soil extractant was tested against Mehlich 3, Olsen, and water for extractable P, and 1 M KCl and water‐extractable NH 4 and NO 2 /NO 3 . The pH of the extractant after adding soil, shaking, and filtration was measured for each soil sample (5 extractants×2 reps×32 soils=320 samples) and was shown to be highly influential on extractable P but has no effect on extractable NH 4 or NO 2 /NO 3 . H 3 A was highly correlated with soil‐extractable inorganic N (NH 4 , NO 2 /NO 3 ) from both water (r=0.98) and 1 M KCl (r=0.97), as well as being significantly correlated with water (r=0.71), Mehlich 3 (r=0.83), and Olsen (r=0.84) for extractable P. [ABSTRACT FROM AUTHOR]

The article focuses on neutralization potential (NP) determination of siderite (FeCO3) with the use of selected oxidants. The determination of NP can be interfered by siderite. Neutrolize acid has an effect on siderite . The reaction of the siderite has an effect on the NP. An examination was conducted on the effects of hydrogen peroxide, oxygen, and potassium permanganate on the NP of siderate samples. Oxidation treatments on siderite samples have different results. Oxidation with oxygen is the most accurate in examining NP.

Reports that a hybrid poultry litter and inorganic N fertilization program can supply necessary crop nutrients without detrimental impacts on runoff water quality, but only if recommended best management practices are followed. Demonstration that a properly managed annual litter application of 1 to 2 tons per acre provides a sustainable alternative to disposal techniques and produces little or no negative water quality impact.

On-farm adoption of agricultural conservation practices or management alternatives depends on conservation ethic, social pressure, regulatory attention, and perceived impact on yield and economic return. Although agro-economic and environmental impacts are assumed to conflict, little research has been conducted to address potential tradeoffs and provide a scientific basis for decision-making. Thus, this 16-year evaluation of adaptive nutrient management was conducted in the Texas Blackland Prairies ecoregion on six fields with structural conservation practices already in place. Each field was randomly selected to receive either commercial fertilizer or poultry litter at rates of 4.5 to 13.4 Mg ha-1. Two major nutrient management adaptations were made (i.e., soil test nitrogen [N] rate recommendations in 2009, and reduction of fallow period length and cover cropping during prolonged fallow periods in 2013). Important results included (1) soil test recommendations that consider historical crop yields reduced N application 25% to 38% for low rates of poultry litter, but did not reduce profits; (2) interannual variability of economic and weather conditions contributed to the lack of statistically significant differences in profit, although profit reduction for high nutrient rate treatments was clear; and (3) litter application, especially at rates in excess of crop phosphorus (P) needs, also increased runoff P losses by 1 to 1.4 kg ha-1 indicating the need for careful management of organic nutrient sources. Results of this long-term study showed that maintaining or increasing economic return does not have to be sacrificed to improve environmental impacts, which is an important consideration as producers make on-farm management decisions. [ABSTRACT FROM AUTHOR]

Water quality experts have suggested that no-till induces phosphorus (P) stratification, which may exacerbate soluble P (SP) runoff from agricultural fields, contributing to eutrophication. Conservationists have been concerned about increased SP loading to Lake Erie, which has been partially blamed on adoption of no-till and the concomitant P stratification of no-till soils. This study was conducted to provide better insight into the potential link between P stratification from no-till soils and P losses via runoff with the objective of exploring P fertilization strategies on P stratification and P runoff from a corn (Zea mays L.)–soybean (Glycine max L.) rotation. Plots were established with nine treatments, including unfertilized, diammonium phosphate (DAP) applications, monoammonium phosphate (MAP) applications, surface applied, injecting fertilizer or tilling fertilizer in, and the use of cover crops. Fertilizer applications were made at 24.4 kg P ha−1 (21.8 lb P ac−1) every other year or at 9.6 kg P ha−1 (8.7 lb P ac−1) every year. Disking, which was intended to minimize P stratification, resulted in the greatest stratification, with significantly higher water SP and Mehlich 3 P in the 0 to 5 cm (0 to 2 in) soil layer compared to the other treatments. There were no differences in SP or total P (TP) runoff from rainfall simulations between fertilizer source (MAP versus DAP) or fertilizer rate (annual versus biennial). The highest SP concentrations observed were from DAP applied to cover crops at the high application rate (24.4 kg P ha−1 applied every other year). This may suggest cover crops are not the ideal practice to decrease SP losses from agricultural fields. Incorporation of fertilizer reduced SP but increased erosion and could potentially increase TP loss. Injecting liquid fertilizer (polyphosphate [Poly]) at the time of planting resulted in lower SP and TP loads than surface applied fertilizers. We encourage other researchers to confirm these results at the field-to-watershed scale to ensure there are not unintended consequences of adopting this fertilization strategy. Further, fertilizer dealers, crop consultants, and farmers should be encouraged to consider liquid fertilizer applications as one option to minimize P losses. [ABSTRACT FROM AUTHOR]

Conservation planners need simple yet accurate tools to predict sediment and nutrient losses from agricultural fields to guide conservation practice implementation and increase cost-effectiveness. The Texas Best Management Practice Evaluation Tool (TBET), which serves as an input/output interpreter and vastly simplified interface for the Soil and Water Assessment Tool (SWAT), was developed to predict mean annual runoff, sediment, nitrogen (N), and phosphorus (P) losses from agricultural fields in Texas under a variety of management scenarios and conservation practices. The Texas Best Management Practice Evaluation Tool was developed to provide a scientific basis for decision making, evaluation, and reporting in state and federal conservation programs. A rigorous, data-intensive calibration and validation process was employed in TBET development. First, hydrology outputs were calibrated with basin scale streamflow data from 20 US Geological Survey sites. Then, runoff, sediment, total N, and total P outputs were calibrated and validated with measured field scale data (260+ site years). Even without calibration,TBET was able to make "good" to "very good" predictions of mean annual runoff and total P losses according to commonly applied model evaluation methods. Calibrated TBET predictions of sediment and total N losses were not as accurate but were still "satisfactory." These much-improved results relative to those of often applied tools such as P Indices are very encouraging since the design goals of simple operation and accurate predictions for diverse Texas conditions with only readily available inputs were certainly met. Thus, TBET meets an important need by providing accurate, science-based estimates of conservation practice benefits at the field scale. [ABSTRACT FROM AUTHOR]

Application of litter and other organic by-products to agricultural land off site of animal production facilities has created both environmental concerns and agro-economic opportunities, but limited long-term, field-scale data are available to guide management decisions. Thus, the objective of this study was to determine the water quality effects of repeated annual poultry litter application as a cropland and pasture fertilizer. Eight years of data collected on ten field-scale watersheds indicated several significant water quality differences based on litter rate (0.0 to 13.4 Mg ha-1 [0 to 6 ton ac-1]) and land use (cropland and pasture). On cropland fields, increasing litter rates (with corresponding decreases in supplemental inorganic nitrogen [Ni) increased runoff orthophosphate phosphorus (PO4-P) concentrations but reduced extreme high nitrate nitrogen (NO3-N) concentrations. Whereas run off PO4-P concentrations were somewhat similar between land uses, NO3-N concentrations were much lower in pasture runoff because of supplemental inorganic N application, reduced nutrient uptake potential, and faster litter mineralization on cropland. Although considerable variability was observed, intra-annual runoff NO3-N and PO4-P concentrations generally exhibited curvilinear decay based on time since fertilizer application. In spite of repeated annual litter application and buildup of soil phosphorus (P) at high litter rates, few long-term trends in N and P runoff were evident due to the dynamic interaction between transport and source factors. These results support several practical implications, specifically: (1) combining organic and inorganic nutrient sources can be environmentally friendly and economically sound if application rates are carefully managed; (2) high runoff N and P concentrations can occur from well-managed fields, which presents difficulty in regulating edge-of-field water quality; and (3) change in the animal industry mindset to view by-products as marketable resources could mitigate environmental problems, provide alternative fertilizer sources, and enhance animal industry revenue opportunities. [ABSTRACT FROM AUTHOR]

The size of the harmful algal blooms in western Lake Erie is strongly correlated with agricultural phosphorus (P) loading from tributaries. Despite farmers' efforts to reduce sediment-bound P loadings and fertilize using current guidance, the media and public have singled them out as the culprit in Lake Erie re-eutrophication. In this paper, two farmer surveys were used to evaluate if farmers in the Lake Erie region follow P fertilizer recommendations, and we also review historic and current P management guidance provided by the scientific community and agricultural industry. The majority (56% to 80%) of farmers apply P fertilizers at or below the current fertility recommendations. Wholesale agronomic changes (e.g., no-tillage adoption, crop cultivar advances, and fertilizer application and formulation) have occurred since current fertilizer recommendations were developed. Although crop P uptake mechanisms have not changed, these agronomic changes have altered P cycling in soil and water. Based on these results, it is time that the scientific community and agricultural industry acknowledge that our current guidance may be contributing to eutrophication. We must ask whether or not we have (1) developed appropriate fertility guidance, (2) developed and recommended appropriate practices to protect water quality, (3) adequately considered "the law of unintended consequences" in conservation recommendations, and (4) focused too much on short-term economic outcomes while disregarding environmental quality. Improved understanding, reconsideration of traditional recommendations, and wider farmer adoption of the most effective practices are needed to develop a sustainable agricultural system in the Western Lake Erie Basin that produces needed commodities while preserving ecosystem integrity. [ABSTRACT FROM AUTHOR]

Excess nutrient loading from numerous sources (e.g., agricultural and urban runoff, treatment plant discharge, and streambank erosion) continues to adversely impact water resources, and determination of the cause(s) of accelerated nutrient enrichment has become a contentious and litigious issue in several US regions. This paper addresses one fundamental question: What are acceptable levels of nutrients in runoff from agricultural fields? It focuses on the field scale where farmers and ranchers make management decisions. Not answering this question limits the effectiveness of on-farm management and policy alternatives to address agriculture's contribution. To answer the question, some might suggest "direct comparison"with reference site data, existing criteria/standards, or measured data compilations. Alternatively, "indirect assessments"using soil test phosphorus (P) levels, P indices, field-scale models, or certainty programs might be suggested. Thus, to provide a scientific basis for policy debate and management decisions related to nutrient runoff from agricultural fields, we evaluated "direct comparisons"with measured data from case studies and evaluated "indirect assessment"alternatives. While acknowledging that scientific challenges and practical realities exist for each alternative, we concluded that certainty programs offer the most promise for ensuring acceptable nutrient runoff, and that field-scale models linked with watershed decision support tools are the most promising for assessing impacts on downstream water quality. Recognizing the reality that some nutrient loss is unavoidable from natural and anthropogenic sources, agriculture, industry, and municipalities are each encouraged to commit to implementing enhanced management where needed to minimize their sector's contribution to excess nutrients in our nation's waters. [ABSTRACT FROM AUTHOR]

The application of poultry litter to agricultural land has become a topic of interest for policy makers due to public concern about its effects on water quality. The Soil and Water Assessment Tool (SWAT) version 2005 is designed to assess nonpoint and point sources of pollution. In this study, six subwatersheds in Texas (HUC-8; 12070101) are used to evaluate the model's ability to simulate water quality at a small scale. Each of these subwatersheds randomly received poultry litter rates of 0.0 to 13.4 Mg ha-1. Monthly and daily data from 2002 were used for calibration purposes, while 2000, 2001, 2003, and 2004 were used for validation. The SCS runoff curve number for moisture condition II (CN2) and the soil evaporation compensation factor (ESCO) parameters were found to be more sensitive than the surface runoff lag time (SURLAG) and initial soil water content expressed as a fraction of field capacity (FFCB). The monthly and daily runoff model simulations for the six subwatersheds resulted in calibration Nash-Sutcliffe efficiency (NSE) values of 0.59 and 0.53 and validation NSE values 0.82 and 0.80, respectively. The monthly and daily R² runoff values for the six subwatersheds resulted in calibration values of at least 0.60 and 0.53 and validation R² values of 0.86 and 0.81, respectively. The observed trends included SWAT's overestimation of runoff in the dry periods and underestimation in the wet periods. The monthly NSE and R² values for sediment and nutrient losses were generally above 0.4 and 0.5, respectively. Paired t-tests for the monthly manually adjusted parameter simulation of sediment, organic N and P, NO3-IV, and soluble P for the 2000-2004 period losses showed that their respective SWAT means were not significantly different from the measured values (α = 0.05), except for NO3-N losses for the Y10 subwatershed (p-value 0.042). The control subwatershed's measured and simulated water quality results were significantly different (α = 0.05) from the treated subwatersheds, most likely due to the amount of inorganic N present. Almost all of the subwatersheds that had poultry litter applied resulted in higher sediment, organic N, organic P, and soluble P losses than the control subwatershed upon averaging thee monthly validation values. High NO3-N losses may have been a function of poultry litter and commercial fertilizers being applied before a large rainfall event occurred. The subwatersheds that received smaller amounts of commercial fertilizer and/or poultry litter lost more sediment, organic N, and organic P than the subwatersheds that received the higher litter and/or fertilizer treatments. Overall, the SWAT simulated the hydrology and the water quality constituents at the subwatershed scale more adequately when all of the data were used to simulate the model, as evidenced by statistical measures. [ABSTRACT FROM AUTHOR]

The scientific community has not established an adequate understanding of the uncertainty inherent in measured water quality data, which is introduced by four procedural categories: streamflow measurement, sample collection, sample preservation/storage, and laboratory analysis. Although previous research has produced valuable information on relative differences in procedures within these categories, little information is available that compares the procedural categories or presents the cumulative uncertainty in resulting water quality data. As a result, quality control emphasis is often misdirected, and data uncertainty is typically either ignored or accounted for with an arbitrary margin of safety. Faced with tile need for scientifically defensible estimates of data uncertainty to support water resource management, the objectives of this research were to: (1) compile selected published information on uncertainty related to measured stream, flow and water quality data for small watersheds, (2) use a root mean square error propagation method to compare the uncertainty introduced by each procedural category, and (3) use the error propagation method to determine the cumulative probable uncertainty in measured streamflow, sediment, and nutrient data. Best case, typical, and worst case "data quality" scenarios were examined. Averaged across all constituents, the calculated cumulative probable uncertainty (±%) contributed under typical scenarios ranged from 6% to 19% for streamflow measurement, from 4% to 48% for sample collection, from 2% to 16% for sample preservation/storage, and from 5% to 21% for laboratory analysis. Under typical conditions, errors in storm loads ranged from 8% to 104% for dissolved nutrients, from 8% to 110% for total N and P, and from 7% to 53% for TSS. Results indicated that uncertainty can increase substantially under poor measurement conditions and limited quality control effort. This research provides introductory scientific estimates of uncertainty in measured water quality data. The results and procedures presented should also assist modelers in quantifying tile "quality" of calibration and evaluation data sets, determining model accuracy goals, and evaluating model performance. [ABSTRACT FROM AUTHOR]

Phosphorus (P) indices were developed to address nonpoint source P losses from agricultural fields; however, only limited information on P index performance at the field- and watershed-scale is available. Evaluation of P indices is necessary to provide the basis of modification and improvement of their usefulness as P management tools. In this study, the ability of the Texas P index to estimate P loss potential was evaluated by comparison with measured annual P loads over three years on four new pasture and six new cultivated litter application sites in the Texas Blackland Prairie. The Arkansas and Iowa P indices were also evaluated. The Texas and Iowa versions were able to provide reasonable estimates of P loss potential as illustrated with significant linear relationships (p <0.01) between P index values and measured annual P loads. In general, the P index values, Mehlich 3 soil test P, and poultry litter application rate were better correlated with dissolved P concentrations and loads (r² ranged from 0.12 to 0.91) than with total P and particulate P loads (r² ranged from 0.00-0.31). A major source of error in P index load estimations was their inability to capture variability in annual soil erosion. This source of error was dramatically reduced by using measured erosion instead of estimated annual average erosion (average r² values increased from 0.24 to 0.58). While these results illustrate a potential for the P Indices to make relative P loss assessments, research on incentives to prevent buildup of soil P levels, linkages between P levels in soils and receiving waters, and other important issues related to the use of P indices is warranted. [ABSTRACT FROM AUTHOR]

Lenacapavir (LEN) is a highly potent, long-acting antiretroviral medication for treating people infected with muti-drug-resistant HIV-1 phenotypes. The inhibitor targets multifaceted functions of the viral capsid protein (CA) during HIV-1 replication. Previous studies have mainly focused on elucidating LEN's mode of action during viral ingress. Additionally, the inhibitor has been shown to interfere with mature capsid assembly during viral egress. However, the mechanism for how LEN affects HIV-1 maturation is unknown. Here, we show that pharmacologically relevant LEN concentrations do not impair proteolytic processing of Gag in virions. Instead, we have elucidated the primary mechanism for highly potent inhibition of HIV-1 maturation by sub-stoichiometric LEN:CA ratios. The inhibitor exerts opposing effects on formation of CA pentamers versus hexamers, the key capsomere intermediates in mature capsid assembly. LEN impairs formation of pentamers, whereas it induces assembly of hexameric lattices by imposing an opened CA conformation and stabilizing a dimeric form of CA. Consequently, LEN treatment results in morphologically atypical virus particles containing malformed, hyper-stable CA assemblies, which fail to infect target cells. Moreover, we have uncovered an inverse correlation between inhibitor potency and CA levels in cell culture assays, which accounts for LEN's ability to potently (with pM EC 50 values) inhibit HIV-1 maturation at clinically relevant drug concentrations., Author Summary: Lenacapavir (LEN) is the first-in-class HIV-1 capsid targeting antiretroviral that exhibits multimodal modality to inhibit both early and late steps of viral replication. Our studies here have elucidated previously undescribed structural and mechanistic bases for a highly potent antiviral activity of LEN during viral egress. These findings will inform clinical applications of LEN as a potent HIV-1 maturation inhibitor and aid the development of second-generation inhibitors targeting assembly of the mature viral capsid.

Lenacapavir (LEN) is the first in class viral capsid protein (CA) targeting antiretroviral for treating multi-drug-resistant HIV-1 infection. Clinical trials and cell culture experiments have identified resistance associated mutations (RAMs) in the vicinity of the hydrophobic CA pocket targeted by LEN. The M66I substitution conferred by far the highest level of resistance to the inhibitor compared to other RAMs. Here we investigated structural and mechanistic bases for how the M66I change affects LEN binding to CA and viral replication. The high-resolution X-ray structure of the CA(M66I) hexamer revealed that the β-branched side chain of Ile66 induces steric hindrance specifically to LEN thereby markedly reducing the inhibitor binding affinity. By contrast, the M66I substitution did not affect binding of Phe-Gly (FG)-motif-containing cellular cofactors CPSF6, NUP153, or SEC24C, which engage the same hydrophobic pocket of CA. In cell culture the M66I variant did not acquire compensatory mutations or replicate in the presence of LEN. Analysis of viral replication intermediates revealed that HIV-1 (M66I CA) predominantly formed correctly matured viral cores, which were more stable than their wildtype counterparts. The mutant cores stably bound to the nuclear envelope but failed to penetrate inside the nucleus. Furthermore, the M66I substitution markedly altered HIV-1 integration targeting. Taken together, our findings elucidate mechanistic insights for how the M66I change confers remarkable resistance to LEN and affects HIV-1 replication. Moreover, our structural findings provide powerful means for future medicinal chemistry efforts to rationally develop second generation inhibitors with a higher barrier to resistance., Importance: Lenacapavir (LEN) is a highly potent and long-acting antiretroviral that works by a unique mechanism of targeting the viral capsid protein. The inhibitor is used in combination with other antiretrovirals to treat multi-drug-resistant HIV-1 infection in heavily treatment-experienced adults. Furthermore, LEN is in clinical trials for preexposure prophylaxis (PrEP) with interim results indicating 100 % efficacy to prevent HIV-1 infections. However, one notable shortcoming is a relatively low barrier of viral resistance to LEN. Clinical trials and cell culture experiments identified emergent resistance mutations near the inhibitor binding site on capsid. The M66I variant was the most prevalent capsid substitution identified in patients receiving LEN to treat muti-drug resistant HIV-1 infections. The studies described here elucidate the underlying mechanism by which the M66I substitution confers a marked resistance to the inhibitor. Furthermore, our structural findings will aid future efforts to develop the next generation of capsid inhibitors with enhanced barriers to resistance.

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are investigational antiretroviral agents that potently impair virion maturation by inducing hyper-multimerization of IN and inhibiting its interaction with viral genomic RNA. The pyrrolopyridine-based ALLINI pirmitegravir (PIR) has recently advanced into phase 2a clinical trials. Previous cell culture-based viral breakthrough assays identified the HIV-1 (Y99H/A128T IN) variant that confers substantial resistance to this inhibitor. Here, we have elucidated the unexpected mechanism of viral resistance to PIR. Although both Tyr99 and Ala128 are positioned within the inhibitor binding V-shaped cavity at the IN catalytic core domain (CCD) dimer interface, the Y99H/A128T IN mutations did not substantially affect the direct binding of PIR to the CCD dimer or functional oligomerization of full-length IN. Instead, the drug-resistant mutations introduced a steric hindrance at the inhibitor-mediated interface between CCD and C-terminal domain (CTD) and compromised CTD binding to the CCD Y99H/A128T + PIR complex. Consequently, full-length IN Y99H/A128T was substantially less susceptible to the PIR-induced hyper-multimerization than the WT protein, and HIV-1 (Y99H/A128T IN) conferred >150-fold resistance to the inhibitor compared with the WT virus. By rationally modifying PIR, we have developed its analog EKC110, which readily induced hyper-multimerization of IN Y99H/A128T in vitro and was ~14-fold more potent against HIV-1 (Y99H/A128T IN) than the parent inhibitor. These findings suggest a path for developing improved PIR chemotypes with a higher barrier to resistance for their potential clinical use.IMPORTANCEAntiretroviral therapies save the lives of millions of people living with HIV (PLWH). However, the evolution of multi-drug-resistant viral phenotypes is a major clinical problem, and there are limited or no treatment options for heavily treatment-experienced PLWH. Allosteric HIV-1 integrase inhibitors (ALLINIs) are a novel class of antiretroviral compounds that work by a unique mechanism of binding to the non-catalytic site on the viral protein and inducing aberrant integrase multimerization. Accordingly, ALLINIs potently inhibit both wild-type HIV-1 and all drug-resistant viral phenotypes that have so far emerged against currently used therapies. Pirmitegravir, a highly potent and safe investigational ALLINI, is currently advancing through clinical trials. Here, we have elucidated the structural and mechanistic bases behind the emergence of HIV-1 integrase mutations in infected cells that confer resistance to pirmitegravir. In turn, our findings allowed us to rationally develop an improved ALLINI with substantially enhanced potency against the pirmitegravir-resistant virus., Competing Interests: Kyungjin Kim was the chief executive officer of ST Pharm Co. Ltd. No other authors declare a potential conflict of interest.