A new capacity of gut microbiota: Fermentation of engineered inorganic carbon nanomaterials into endogenous organic metabolites.

Carbon-based nanomaterials (CNMs) have recently been found in humans raising a great concern over their adverse roles in the hosts. However, our knowledge of the in vivo behavior and fate of CNMs, especially their biological processes elicited by the gut microbiota, remains poor. Here, we uncovered the integration of CNMs (single-walled carbon nanotubes and graphene oxide) into the endogenous carbon flow through degradation and fermentation, mediated by the gut microbiota of mice using isotope tracing and gene sequencing. As a newly available carbon source for the gut microbiota, microbial fermentation leads to the incorporation of inorganic carbon from the CNMs into organic butyrate through the pyruvate pathway. Furthermore, the butyrate-producing bacteria are identified to show a preference for the CNMs as their favorable source, and excessive butyrate derived from microbial CNMs fermentation further impacts on the function (proliferation and differentiation) of intestinal stem cells in mouse and intestinal organoid models. Collectively, our results unlock the unknown fermentation processes of CNMs in the gut of hosts and underscore an urgent need for assessing the transformation of CNMs and their health risk via the gut-centric physiological and anatomical pathways.

Quantitative assessment of translocator protein (TSPO) in the non-human primate brain and clinical translation of [18F]LW223 as a TSPO-targeted PET radioligand.

OBJECTIVES: Translocator protein 18 kDa (TSPO) positron emission tomography (PET) can be harnessed for the non-invasive detection of macrophage-driven inflammation. [18F]LW223, a newly reported TSPO PET tracer which was insensitive to rs6971 polymorphism, showed favorable performance characteristics in a recent imaging study involving a rat myocardial infarction model. To enable quantitative neuroimaging with [18F]LW223, we conducted kinetic analysis in the non-human primate (NHP) brain. Further, we sought to assess the utility of [18F]LW223-based TSPO imaging in a first-in-human study. METHODS: Radiosynthesis of [18F]LW223 was accomplished on an automated module, whereas molar activities, stability in formulation, lipophilicity and unbound free fraction (fu) of the probe were measured. Brain penetration and target specificity of [18F]LW223 in NHPs were corroborated by PET-MR imaging under baseline and pre-blocking conditions using the validated TSPO inhibitor, (R)-PK11195, at doses ranging from 5 to 10 mg/kg. Kinetic modeling was performed using one-tissue compartment model (1TCM), two-tissue compartment model (2TCM) and Logan graphical analyses, using dynamic PET data acquisition, arterial blood collection and metabolic stability testing. Clinical PET scans were performed in two healthy volunteers (HVs). Regional brain standard uptake value ratio (SUVr) was assessed for different time intervals. RESULTS: [18F]LW223 was synthesized in non-decay corrected radiochemical yields (n.d.c. RCYs) of 33.3 ± 6.5% with molar activities ranging from 1.8 ± 0.7 Ci/µmol (n = 11). [18F]LW223 was stable in formulation for up to 4 h and LogD7.4 of 2.31 ± 0.13 (n = 6) and fu of 5.80 ± 1.42% (n = 6) were determined. [18F]LW223 exhibited good brain penetration in NHPs, with a peak SUV value of ca. 1.79 in the whole brain. Pre-treatment with (R)-PK11195 substantially accelerated the washout and attenuated the area under the time-activity curve, indicating in vivo specificity of [18F]LW223 towards TSPO. Kinetic modeling demonstrated that 2TCM was the most suitable model for [18F]LW223-based neuroimaging. Global transfer rate constants (K1) and total volumes of distribution (VT) were found to be 0.10 ± 0.01 mL/cm3/min and 2.30 ± 0.17 mL/cm3, respectively. Dynamic PET data analyses across distinct time windows revealed that the VT values were relatively stable after 60 min post-injection. In a preliminary clinical study with two healthy volunteers, [18F]LW223 exhibited good brain uptake and considerable tracer retention across all analyzed brain regions. Of note, an excellent correlation between SUVr with VT was obtained when assessing the time interval from 20 to 40 min post tracer injection (SUVr(20-40 min), R2 = 0.94, p < 0.0001), suggesting this time window may be suitable to estimate specific binding to TSPO in human brain. CONCLUSION: Our findings indicate that [18F]LW223 is suitable for quantitative TSPO-targeted PET imaging in higher species. Employing state-of-the-art kinetic modeling, we found that [18F]LW223 was effective in mapping TSPO throughout the NHP brain, with best model fits obtained from 2TCM and Logan graphical analyses. Overall, our results indicate that [18F]LW223 exhibits favorable tracer performance characteristics in higher species, and this novel imaging tool may hold promise to provide effective neuroinflammation imaging in patients with neurological disease.

Discussion on "Instrumented difference-in-differences" by Ye, Ertefaie, Flory, Hennessy, Small.

Ye, Ertefaie, Flory, Hennessy, and Small (YEFHS) proposed a new method, instrumented difference-in-differences, for dealing with unmeasured confounding. In this note, I connect and compare assumptions and identifications in instrumental variable (IV) and difference-in-differences (DID) methods with those in YEFHS, derive new identification results, and discuss different choices when extending such results to adjust for covariates.

Nickel Oxide Nanoparticles Improve Soybean Yield and Enhance Nitrogen Assimilation.

Nickel (Ni) is a trace element beneficial for plant growth and development and could improve crop yield by stimulating urea decomposition and nitrogen-fixing enzyme activity. A full life cycle study was conducted to compare the long-term effects of soil-applied NiO nanoparticles (n-NiO), NiO bulk (b-NiO), and NiSO4 at 10-200 mg kg-1 on plant growth and nutritional content of soybean. n-NiO at 50 mg kg-1 significantly promoted the seed yield by 39%. Only 50 mg kg-1 n-NiO promoted total fatty acid content and starch content by 28 and 19%, respectively. The increased yield and nutrition could be attributed to the regulatory effects of n-NiO, including photosynthesis, mineral homeostasis, phytohormone, and nitrogen metabolism. Furthermore, n-NiO maintained a Ni2+ supply for more extended periods than NiSO4, reducing potential phytotoxicity concerns. Single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS) for the first time confirmed that the majority of the Ni in seeds is in ionic form, with only 28-34% as n-NiO. These findings deepen our understanding of the potential of nanoscale and non-nanoscale Ni to accumulate and translocate in soybean, as well as the long-term fate of these materials in agricultural soils as a strategy for nanoenabled agriculture.

Consistent and robust inference in hazard probability and odds models with discrete-time survival data.

For discrete-time survival data, conditional likelihood inference in Cox's hazard odds model is theoretically desirable but exact calculation is numerical intractable with a moderate to large number of tied events. Unconditional maximum likelihood estimation over both regression coefficients and baseline hazard probabilities can be problematic with a large number of time intervals. We develop new methods and theory using numerically simple estimating functions, along with model-based and model-robust variance estimation, in hazard probability and odds models. For the probability hazard model, we derive as a consistent estimator the Breslow-Peto estimator, previously known as an approximation to the conditional likelihood estimator in the hazard odds model. For the hazard odds model, we propose a weighted Mantel-Haenszel estimator, which satisfies conditional unbiasedness given the numbers of events in addition to the risk sets and covariates, similarly to the conditional likelihood estimator. Our methods are expected to perform satisfactorily in a broad range of settings, with small or large numbers of tied events corresponding to a large or small number of time intervals. The methods are implemented in the R package dSurvival.

Dissolved metal ion removal by online hollow fiber ultrafiltration for enhanced size characterization of metal-containing nanoparticles with single-particle ICP-MS.

Single particle-inductively coupled plasma mass spectrometry (SP-ICP-MS) is a powerful tool for size-characterization of metal-containing nanoparticles (MCNs) at environmentally relevant concentrations, however, coexisting dissolved metal ions greatly interfere with the accuracy of particle size analysis. The purpose of this study is to develop an online technique that couples hollow fiber ultrafiltration (HFUF) with SP-ICP-MS to improve the accuracy and size detection limit of MCNs by removing metal ions from suspensions of MCNs. Through systematic optimization of conditions including the type and concentration of surfactant and complexing agent, carrier pH, and ion cleaning time, HFUF completely removes metal ions but retains the MCNs in suspension. The optimal conditions include using a mixture of 0.05 vol.% FL-70 and 0.5 mmol/L Na2S2O3 (pH = 8.0) as the carrier and 4 min as the ion cleaning time. At these conditions, HFUF-SP-ICP-MS accurately determines the sizes of MCNs, and the results agree with the size distribution determined by transmission electron microscopy, even when metal ions also are present in the sample. In addition, reducing the ionic background through HFUF also lowers the particle size detection limit with SP-ICP-MS (e.g., from 28.3 to 14.2 nm for gold nanoparticles). This size-based ion-removal principle provided by HFUF is suitable for both cations (e.g., Ag+) and anions (e.g., AuCl4-) and thus has good versatility compared to ion exchange purification and promising prospects for the removal of salts and macromolecules before single particle analysis.

Total Organic Carbon as a Quantitative Index of Micro- and Nano-Plastic Pollution.

The global pollution of micro- and nano-plastics (MNPs) calls for monitoring methods. As diverse mixtures of various sizes, morphologies, and chemical compositions in the environment, MNPs are currently quantified based on mass or number concentrations. Here, we show total organic carbon (TOC) as an index for quantifying the pollution of total MNPs in environmental waters. Two parallel water samples are respectively filtered with a carbon-free glass fiber membrane. Then, one membrane with the collected particulate substances is treated by potassium peroxodisulfate oxidation and Fenton digestion in sequence for quantifying the sum of MNPs and particulate black carbon (PBC) as TOCMNP&PBC using a TOC analyzer, another membrane is treated by sulfonation and Fenton digestion for quantifying PBC as TOCPBC, and the TOC of MNPs is calculated by subtracting TOCPBC from TOCMNP&PBC. The feasibility of our method is demonstrated by determination of various MNPs of representative plastic types and sizes (0.5-100 µm) in tap, river, and sea water samples, with low detection limits (∼7 µg C L-1) and high spiked recoveries (83.7-114%). TOC is a powerful index for routine monitoring of MNP pollution.

Dithizone-functionalized C18 online solid-phase extraction-HPLC-ICP-MS for speciation of ultra-trace organic and inorganic mercury in cereals and environmental samples.

A simple and efficient dithizone-functionalized solid-phase extraction (SPE) procedure, online coupled with high-performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry, was developed for the first time for enrichment and determination of ultra-trace mercury (Hg) species (inorganic divalent Hg (Hg(II)), methylmercury (CH3Hg(II)) and ethylmercury (C2H5Hg(II)) in cereals and environmental samples. In the proposed method, functionalization of the commercial C18 column with dithizone, enrichment, and elution of the above Hg species can be completed online with the developed SPE device. A simple solution of 2-mercaptoethanol (1% (V/V)) could be used as an eluent for both the SPE and HPLC separation of Hg species, significantly simplifying the method and instrumentation. The online SPE method was optimized by varying dithizone dose, 2-mercaptoethanol concentration, and sample volume. In addition, the effect of pH, coexisting interfering ions, and salt effect on the enrichment was also discussed. Under the optimized conditions, the detection limits of Hg species for 5 mL water sample were 0.15 ng/L for Hg(II), 0.07 ng/L for CH3Hg(II), and 0.04 ng/L for C2H5Hg(II) with recoveries in the range of 85%-100%. The developed dithizone-functionalized C18 SPE column can be reused after a single functionalization, which significantly simplifies the enrichment step. Moreover, the stability of Hg species enriched on the SPE column demonstrated its suitability for field sampling of Hg species for later laboratory analysis. This environment-friendly method offers a robust tool to detect ultra-trace Hg species in cereals and environmental samples.

In Situ Tracking Photodegradation of Trace Graphene Oxide by the Online Coupling of Photoinduced Chemical Vapor Generation with a Point Discharge Optical Emission Spectrometer.

The photostability of graphene oxide (GO) strongly affects the performance of its products in optics and photonics. However, the photostability of GO, especially at trace levels, remains largely unexplored mainly because of the lack of available techniques. Herein, we developed a novel online system consisting of a highly efficient photoinduced chemical vapor generation reactor and an in situ measurement technique using a miniaturized and sensitive point discharge optical emission spectrometer. On the basis of the results of inorganic carbon species, abundant oxygen-containing functional groups on GO nanosheets made the degradation much easier than graphene. Under the optimized conditions (e.g., initial pH of 2.8 and binary photocatalysts dose of 200 mM H2O2, 1.0 mM Fe3+ ions, and 50 mg/L TiO2 NPs), the limit of detection for GO was 87.5 µg/L C with a linear range of 0.5-10 mg/L C. Specifically, the accuracy and reliability of the developed system was verified by quantifying self-prepared GO as well as aggregated GO in natural organic matter-rich water samples. Finally, the sunlight-induced photodegradation of GO under simulated environmental conditions was successfully tracked. The developed system is a promising platform for in-time quality control of GO-based products as well as predicting the occurrence, transformation, and fate of GO at environmentally relevant concentrations in the natural aquatic environment.

Synthesis and preliminary evaluation of a novel positron emission tomography (PET) ligand for imaging fatty acid amide hydrolase (FAAH).

Fatty acid amide hydrolase (FAAH) exerts its main function in the catabolism of the endogenous chemical messenger anandamide (AEA), thus modulating the endocannabinoid (eCB) pathway. Inhibition of FAAH may serve as an effective strategy to relieve anxiety and possibly other central nervous system (CNS)-related disorders. Positron emission tomography (PET) would facilitate us to better understand the relationship between FAAH in certain disease conditions, and accelerate clinical translation of FAAH inhibitors by providing in vivo quantitative information. So far, most PET tracers show irreversible binding patterns with FAAH, which would result in complicated quantitative processes. Herein, we have identified a new FAAH inhibitor (1-((1-methyl-1H-indol-2-yl)methyl)piperidin-4-yl)(oxazol-2-yl)methanone (8) which inhibits the hydrolysis of AEA in the brain with high potency (IC50 value 11 nM at a substrate concentration of 0.5 µM), and without showing time-dependency. The PET tracer [11C]8 (also called [11C]FAAH-1906) was successfully radiolabeled with [11C]MeI in 17 ± 6% decay-corrected radiochemical yield (n = 7) with >74.0 GBq/µmol (2 Ci/µmol) molar activity and >99% radiochemical purity. Ex vivo biodistribution and blocking studies of [11C]8 in normal mice were also conducted, indicating good brain penetration, high brain target selectivity, and modest to excellent target selectivity in peripheral tissues. Thus, [11C]8 is a potentially useful PET ligand with enzyme inhibitory and target binding properties consistent with a reversible mode of action.

A population stereotaxic positron emission tomography brain template for the macaque and its application to ischemic model.

PURPOSE: Positron emission tomography (PET) is a non-invasive imaging tool for the evaluation of brain function and neuronal activity in normal and diseased conditions with high sensitivity. The macaque monkey serves as a valuable model system in the field of translational medicine, for its phylogenetic proximity to man. To translation of non-human primate neuro-PET studies, an effective and objective data analysis platform for neuro-PET studies is needed. MATERIALS AND METHODS: A set of stereotaxic templates of macaque brain, namely the Institute of High Energy Physics & Jinan University Macaque Template (HJT), was constructed by iteratively registration and averaging, based on 30 healthy rhesus monkeys. A brain atlas image was created in HJT space by combining sub-anatomical regions and defining new 88 bilateral functional regions, in which a unique integer was assigned for each sub-anatomical region. RESULTS: The HJT comprised a structural MRI T1 weighted image (T1WI) template image, a functional FDG-PET template image, intracranial tissue segmentations accompanied with a digital macaque brain atlas image. It is compatible with various commercially available software tools, such as SPM and PMOD. Data analysis was performed on a stroke model compared with a group of healthy controls to demonstrate the usage of HJT. CONCLUSION: We have constructed a stereotaxic template set of macaque brain named HJT, which standardizes macaque neuroimaging data analysis, supports novel radiotracer development and facilitates translational neuro-disorders research.

Isotope Tracers To Study the Environmental Fate and Bioaccumulation of Metal-Containing Engineered Nanoparticles: Techniques and Applications.

The rapidly growing applicability of metal-containing engineered nanoparticles (MENPs) has made their environmental fate, biouptake, and transformation important research topics. However, considering the relatively low concentration of MENPs and the high concentration of background metals in the environment and in organisms, tracking the fate of MENPs in environment-related scenarios remains a challenge. Intrinsic labeling of MENPs with radioactive or stable isotopes is a useful tool for the highly sensitive and selective detection of MENPs in the environment and organisms, thus enabling tracing of their transformation, uptake, distribution, and clearance. In this review, we focus on radioactive/stable isotope labeling of MENPs for their environmental and biological tracing. We summarize the advantages of intrinsic radioactive/stable isotopes for MENP labeling and discuss the considerations in labeling isotope selection and preparation of labeled MENPs, as well as exposure routes and detection of labeled MENPs. In addition, current practice in the use of radioactive/stable isotope labeling of MENPs to study their environmental fate and bioaccumulation is reviewed. Future perspectives and potential applications are also discussed, including imaging techniques for radioactive- and stable-isotope-labeled MENPs, hyphenated multistable isotope tracers with speciation analysis, and isotope fractionation as a MENP tracer. It is expected that this critical review could provide the necessary background information to further advance the applications of isotope tracers to study the environmental fate and bioaccumulation of MENPs.

Freezing Facilitates Formation of Silver Nanoparticles under Natural and Simulated Sunlight Conditions.

Freezing is essential in the light-mediated transformation of organic pollutants. However, the effects of the freezing process on the reduction of Ag+ by natural organic matter (NOM) remains unclear, causing significant uncertainties in the natural formation of silver nanoparticles (AgNPs). This study investigated the sunlight-induced reduction of Ag+ by NOM under natural or controlled freezing processes. Natural (outdoor) freezing experiments demonstrated intense aggregation and precipitation of AgNPs in three aqueous media, including a NOM solution and two river water samples, under natural sunlight irradiation. Indoor experiments under simulated sunlight irradiation and controlled freezing processes showed that freezing at -20 °C and repeated freeze-thaw cycles (-20 to 4 °C) drastically accelerated the formation and growth of AgNPs compared to maintenance at 4 °C. Finally, under the natural freezing process, commercial AgNPs were found to influence the redox reduction of Ag+ probably through a reduction in dissolution rates and homoaggregation with AgNPs newly formed in the river water samples. Additionally, the enhancement effect of freezing on AgNP formation was confirmed in the presence of Ag+ and AgNPs both at environmentally relevant concentration levels, especially upon light irradiation. This work emphasizes the importance of freezing processes on the natural formation of AgNPs.

Enhanced removal of Cr(VI) by biochar with Fe as electron shuttles.

Biochar is extensively used as an effective soil amendment for environmental remediation. In addition to its strong contaminant sorption capability, biochar also plays an important role in chemical transformation of contaminant due to its inherent redox-active moieties. However, the transformation efficiency of inorganic contaminants is generally very limited when the direct adsorption of contaminants on biochar is inefficient. The present study demonstrates the role of Fe ion as an electron shuttle to enhance Cr(VI) reduction by biochars. Batch experiments were conducted to examine the effects of Fe(III) levels, pyrolysis temperature of biochar, initial solution pH, and biochar dosage on the efficiency of Cr(VI) removal. Results showed a significant enhancement in Cr(VI) reduction with an increase in Fe(III) concentration and a decrease of initial pH. Biochar produced at higher pyrolysis temperatures (e.g., 700°C) favored Cr(VI) removal, especially in the presence of Fe(III), while a higher biochar dosage proved unfavorable likely due to the agglomeration or precipitation of biochar. Speciation analysis of Fe and Cr elements on the surface of biochar and in the solution further confirmed the role of Fe ion as an electron shuttle between biochar and Cr(VI). The present findings provide a potential strategy for the advanced treatment of Cr(VI) at low concentrations as well as an insight into the environmental fate of Cr(VI) and other micro-pollutants in soil or aqueous compartments containing Fe and natural or engineered carbonaceous materials.

Environmentally Relevant Freeze-Thaw Cycles Enhance the Redox-Mediated Morphological Changes of Silver Nanoparticles.

Silver nanoparticles (AgNPs) are inevitably released into natural systems, particularly into aquatic environments, where they are oxidized and release Ag+, which is reduced back to AgNPs. Environmental freeze-thaw cycles or freezing may accelerate the dynamic transformation between AgNPs and Ag+. Herein, the significant morphological changes caused by freezing treatments were assessed by UV-vis spectroscopy and high-resolution transmission electron microscopy, which revealed that reductive regeneration, particle fusion, and coalescence of the AgNPs occurred. In addition, a stable Ag isotope was used to track the AgNP redox reaction, which was found to be accelerated under freezing and freeze-thaw cycles relative to the reaction of particles stored at a normal temperature (4 °C, 25 °C). Furthermore, natural organic matter was found to stabilize the particle morphology. Ca2+ and Cl- intensified the morphological changes and redox reaction through Ca2+-induced particle coalescence and Cl--enhanced reduction of Ag+ during the freeze-thaw treatment. These physicochemical changes also occurred for an environmentally relevant concentration of AgNPs (50 ng L-1) in simulated environmental conditions and natural water samples after freeze-thaw cycles. Since the morphological changes and redox acceleration induced by environmental freezing conditions could dramatically influence the mobility, bioavailability, toxicity, and environmental fate of AgNPs, the freeze-thaw-induced effects should be considered in the environmental risk assessment of AgNPs.

Correlation between the expression of miR150 and FOXO4 and the local recurrence and metastasis of nasopharyngeal carcinoma after intensive radiotherapy.

PURPOSE: To investigate the relationship between the expression of miR150 and FOXO4 in nasopharyngeal carcinoma (NPC) and the local recurrence and metastasis after intensive radiotherapy. METHODS: 94 patients with NPC were selected in Hunan Provincial People's Hospital from May 2011 to May 2013. All patients received intensive radiotherapy. Thirty healthy controls were also included. The expression levels of miR150 and FOXO4 mRNA in blood lymphocytes were detected by RT-PCR. All patients with NPC were followed up for 36 months. Blood was drawn from patients to analyze the expression of miR150 and FOXO4. MiR150 inhibitor was used to treat NPC cells, and FOXO4 overexpression cell lines were established. Transwell invasion assay was performed to investigate the effects of miR150 expression inhibition and FOXO4 overexpression on cell invasion. Protein levels were detected by western blot. RESULTS: Compared with healthy controls, the levels of miR150 mRNA in NPC patients were significantly increased, while FOXO4 mRNA levels were significantly decreased (p<0.05). The levels of miR150 and FOXO4 were significantly correlated with distant metastasis and tumor recurrence (p<0.05). High expression level of miR150 or low expression level of FOXO4 significantly shortened the overall survival (OS) of patients (p<0.05). Cox's proportional hazards model showed that miR150 and FOXO4 were potential independent risk factors for NPC (p<0.05). The level of miR150 in patients with tumor recurrence was significantly higher than that in patients without tumor recurrence, while the level of FOXO4 in patients with tumor recurrence was lower than that patients without tumor recurrence (p<0.05). MiR150 expression inhibition or FOXO4 overexpression significantly reduced the invasion abilities of CNE1 and CNE2 cells and protein levels of matrix metalloproteinase2 (MMP2) and MMP9 (p<0.05). CONCLUSION: MiR150 and FOXO4 are closely related to the metastasis and recurrence of NPC, and are independent prognostic factors for NPC. MiR150 and FOXO4 are of clinical significance in predicting NPC prognosis.

Tracking the Transformation of Nanoparticulate and Ionic Silver at Environmentally Relevant Concentration Levels by Hollow Fiber Flow Field-Flow Fractionation Coupled to ICPMS.

It is a great challenge to monitor the physical and chemical transformation of nanoparticles at environmentally relevant concentration levels, mainly because the commonly used techniques like dynamic light scattering and transmission electron microscopy are unable to characterize and quantify trace level nanoparticles in complex matrices. Herein, we demonstrate the on-line coupled system of hollow fiber flow field-flow fractionation (HF5), minicolumn concentration, and inductively coupled plasma mass spectrometry (ICPMS) detection as an efficient approach to study the aggregation and chemical transformation of silver nanoparticles (AgNPs) and ionic Ag species in the aqueous environment at ng/mL levels. Taking advantage of the in-line dialysis of HF5, the selective capture of Ag(I) species by the resin in minicolumn, and the high selectivity and sensitivity of ICPMS detection, we recorded the aggregation of 10 ng/mL AgNPs in complex matrices (e.g., NOM, Na+/Ca2+), revealing an interesting tiny AgNPs formation process of photoreduction of trace level Ag(I) that is different from larger AgNPs generated at high concentration of Ag(I) by accurate characterization and respectively identifying and quantifying new thiol-complexed Ag(I) and residual Ag(I) in the intertransformation of Ag(I) and AgNPs in domestic wastewater by simultaneously detecting the S and Ag signals via ICPMS.

Deep sequencing of protease inhibitor resistant HIV patient isolates reveals patterns of correlated mutations in Gag and protease.

While the role of drug resistance mutations in HIV protease has been studied comprehensively, mutations in its substrate, Gag, have not been extensively cataloged. Using deep sequencing, we analyzed a unique collection of longitudinal viral samples from 93 patients who have been treated with therapies containing protease inhibitors (PIs). Due to the high sequence coverage within each sample, the frequencies of mutations at individual positions were calculated with high precision. We used this information to characterize the variability in the Gag polyprotein and its effects on PI-therapy outcomes. To examine covariation of mutations between two different sites using deep sequencing data, we developed an approach to estimate the tight bounds on the two-site bivariate probabilities in each viral sample, and the mutual information between pairs of positions based on all the bounds. Utilizing the new methodology we found that mutations in the matrix and p6 proteins contribute to continued therapy failure and have a major role in the network of strongly correlated mutations in the Gag polyprotein, as well as between Gag and protease. Although covariation is not direct evidence of structural propensities, we found the strongest correlations between residues on capsid and matrix of the same Gag protein were often due to structural proximity. This suggests that some of the strongest inter-protein Gag correlations are the result of structural proximity. Moreover, the strong covariation between residues in matrix and capsid at the N-terminus with p1 and p6 at the C-terminus is consistent with residue-residue contacts between these proteins at some point in the viral life cycle.

Locally weighted histogram analysis and stochastic solution for large-scale multi-state free energy estimation.

The weighted histogram analysis method (WHAM) including its binless extension has been developed independently in several different contexts, and widely used in chemistry, physics, and statistics, for computing free energies and expectations from multiple ensembles. However, this method, while statistically efficient, is computationally costly or even infeasible when a large number, hundreds or more, of distributions are studied. We develop a locally WHAM (local WHAM) from the perspective of simulations of simulations (SOS), using generalized serial tempering (GST) to resample simulated data from multiple ensembles. The local WHAM equations based on one jump attempt per GST cycle can be solved by optimization algorithms orders of magnitude faster than standard implementations of global WHAM, but yield similarly accurate estimates of free energies to global WHAM estimates. Moreover, we propose an adaptive SOS procedure for solving local WHAM equations stochastically when multiple jump attempts are performed per GST cycle. Such a stochastic procedure can lead to more accurate estimates of equilibrium distributions than local WHAM with one jump attempt per cycle. The proposed methods are broadly applicable when the original data to be "WHAMMED" are obtained properly by any sampling algorithm including serial tempering and parallel tempering (replica exchange). To illustrate the methods, we estimated absolute binding free energies and binding energy distributions using the binding energy distribution analysis method from one and two dimensional replica exchange molecular dynamics simulations for the beta-cyclodextrin-heptanoate host-guest system. In addition to the computational advantage of handling large datasets, our two dimensional WHAM analysis also demonstrates that accurate results similar to those from well-converged data can be obtained from simulations for which sampling is limited and not fully equilibrated.