The role of high cholesterol in SARS-CoV-2 infectivity.

Coronavirus disease 2019 (COVID-19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2. The virus binds to angiotensinogen converting enzyme 2 (ACE2), which mediates viral entry into mammalian cells. COVID-19 is notably severe in the elderly and in those with underlying chronic conditions. The cause of selective severity is not well understood. Here we show cholesterol and the signaling lipid phosphatidyl-inositol 4,5 bisphosphate (PIP2) regulate viral infectivity through the localization of ACE2's into nanoscopic (<200 nm) lipid clusters. Uptake of cholesterol into cell membranes (a condition common to chronic disease) causes ACE2 to move from PIP2 lipids to endocytic ganglioside (GM1) lipids, where the virus is optimally located for viral entry. In mice, age and high-fat diet increase lung tissue cholesterol by up to 40%. And in smokers with chronic disease, cholesterol is elevated 2-fold, a magnitude of change that dramatically increases infectivity of virus in cell culture. We conclude increasing the ACE2 location near endocytic lipids increases viral infectivity and may help explain the selective severity of COVID-19 in aged and diseased populations.

Bictegravir nanomicelles and anionic pullulan loaded vaginal film: Dual mechanistic pre-exposure prophylaxis (PrEP) for HIV.

Locally delivered pre-exposure prophylaxis (PrEP) has proven to be a promising strategy to combat Human immunodeficiency virus (HIV) transmission but several findings encountered toxicities or proved to be marginally effective in clinical settings. Therefore, innovative, multifunctional, and safer alternatives are being progressively investigated. Herein, we explored negatively charged carbohydrate, anionic pullulan (AP) as a rapidly soluble film-former and novel anti-HIV agent. Additionally, Bictegravir (BCT), an HIV integrase inhibitor was co-delivered in the form of nanomicelles for sustained antiviral activity. BCT-loaded PLGA-PEG polymeric nanomicelles (BN) were incorporated into PVA/pullulan-based film matrix comprising of 2 % w/v AP (BN-AP film). In cell-based assays, biocompatibility and TEER values for BN-AP films were similar to control while the commercial vaginal contraceptive film (VCF®) showed severe cytotoxicity and drastically reduced the tight junction integrity. Rapid disintegration of BN-AP film with >85 % drug release was observed in simulated vaginal and seminal fluid. Most importantly, AP and BN-AP film significantly inhibited HIV-1 replication with IC50 at as low as 91 µg/mL and 0.708 nM, respectively. Therefore, this study entails successful development of BN-AP film that functioned as an effective, biocompatible dual-acting PrEP formulation.

Hydroxychloroquine blocks SARS-CoV-2 entry into the endocytic pathway in mammalian cell culture.

Hydroxychloroquine (HCQ), a drug used to treat lupus and malaria, was proposed as a treatment for SARS-coronavirus-2 (SARS-CoV-2) infection, albeit with controversy. In vitro, HCQ effectively inhibits viral entry, but its use in the clinic has been hampered by conflicting results. A better understanding of HCQ's mechanism of actions in vitro is needed. Recently, anesthetics were shown to disrupt ordered clusters of monosialotetrahexosylganglioside1 (GM1) lipid. These same lipid clusters recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to endocytic lipids, away from phosphatidylinositol 4,5 bisphosphate (PIP2) clusters. Here we employed super-resolution imaging of cultured mammalian cells (VeroE6, A549, H1793, and HEK293T) to show HCQ directly perturbs clustering of ACE2 receptor with both endocytic lipids and PIP2 clusters. In elevated (high) cholesterol, HCQ moves ACE2 nanoscopic distances away from endocytic lipids. In cells with resting (low) cholesterol, ACE2 primarily associates with PIP2 clusters, and HCQ moves ACE2 away from PIP2 clusters-erythromycin has a similar effect. We conclude HCQ inhibits viral entry through two distinct mechanisms in high and low tissue cholesterol and does so prior to inhibiting cathepsin-L. HCQ clinical trials and animal studies will need to account for tissue cholesterol levels when evaluating dosing and efficacy.

Identification of potent small molecule inhibitors of SARS-CoV-2 entry.

The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.

The role of high cholesterol in age-related COVID19 lethality.

Coronavirus disease 2019 (COVID19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originating in Wuhan, China in 2019. The disease is notably severe in elderly and those with underlying chronic conditions. A molecular mechanism that explains why the elderly are vulnerable and why children are resistant is largely unknown. Here we show loading cells with cholesterol from blood serum using the cholesterol transport protein apolipoprotein E (apoE) enhances the entry of pseudotyped SARS-CoV-2 and the infectivity of the virion. Super resolution imaging of the SARS-CoV-2 entry point with high cholesterol shows almost twice the total number of endocytic entry points. Cholesterol concomitantly traffics angiotensinogen converting enzyme (ACE2) to the endocytic entry site where SARS-CoV-2 presumably docks to efficiently exploit entry into the cell. Furthermore, in cells producing virus, cholesterol optimally positions furin for priming SARS-CoV-2, producing a more infectious virion with improved binding to the ACE2 receptor. In vivo, age and high fat diet induces cholesterol loading by up to 40% and trafficking of ACE2 to endocytic entry sites in lung tissue from mice. We propose a component of COVID19 severity based on tissue cholesterol level and the sensitivity of ACE2 and furin to cholesterol. Molecules that reduce cholesterol or disrupt ACE2 localization with viral entry points or furin localization in the producer cells, may reduce the severity of COVID19 in obese patients.

ß-cyclodextrin polymer/Soluplus® encapsulated Ebselen ternary complex (EßpolySol) as a potential therapy for vaginal candidiasis and pre-exposure prophylactic for HIV.

Epidemiological findings have discussed recurrent and persistent vulvovaginal candidiasis to be a major manifestation of HIV infected women. Conversely, women with vulvovaginal candidiasis have higher risk of acquiring HIV transmitted during intercourse. Common treatments for such conditions include combined antiretroviral and antifungal therapy. Drug-Drug interaction is a major problem encountered due to common CYP450 metabolic pathway of azoles and antiretroviral drugs. Ebselen (EB), lipophilic, organo-selenium compound has demonstrated promising anti-HIV and anti-fungal activity. The aim of current research was to develop and characterize a rapidly soluble and non-cytotoxic vaginal film of ebselen which could serve dual purpose of treating vulvovaginal candidiasis and pre-exposure prophylactic (PrEP) against HIV. Ebselen/cyclodextrin polymer/Soluplus® (1:10:10) ternary complex (EßpolySol) showed 200 fold enhancement in aqueous solubility and no degradation of EB in thermogravimetry analysis. EßpolySol film with tensile strength of 33.12 ± 1.98 N/cm2 disintegrated within 30 sec, presented instant drug release with no apparent precipitation in simulated vaginal fluid. EßpolySol film showed compatibility with HEC-1A monolayer and HeLa cells compared to VCF®. EßpolySol film showed MIC of 20 µM against Candida species and IC50 of 0.71 µM against HIV.

Efavirenz nanomicelles loaded vaginal film (EZ film) for preexposure prophylaxis (PrEP) of HIV.

Preexposure prophylaxis (PrEP) using oral or vaginal microbicide is an emerging and effective strategy to prevent HIV transmission. Vaginal film is becoming more acceptable and a convenient dosage form compared to cream, gel and suppository. Extremely poor aqueous solubility of efavirenz (EFV) limits its use as vaginal microbicide. The aim of this study was to develop and evaluate a monomeric surfactant free, rapidly soluble vaginal film of EFV (EZ film). EZ film was prepared using a tetrafunctional block polymer (Tetronic 1107), carrageenan and polyvinyl alcohol (PVA) by solvent evaporation method. First, different solubilizers were screened for EFV solubility, in vitro cytotoxicity and cell membrane integrity assay on HeLa cells. Optimized film was characterized for solid state, mechanical strength, epithelial integrity, in vitro drug release in simulated vaginal fluid (SVF), simulated seminal fluid (SSF) and in vitro anti-HIV activity. Optimized EZ film showed a particle size of 48 ±â€¯3.8 nm with PDI of 0.299. Differential scanning colorimetry (DSC) thermogram suggested the complete amorphization of EFV within the film. EZ film rapidly disintegrated (30 s) with complete release of EFV in SVF and SSF. The film was found to be non-toxic to HeLa cells and showed similar anti-HIV-1 activity as that of EFV in DMSO. EZ film did not show any significant change in the TEER value in HEC 1A cell line. Hence, the findings from the current study strongly suggest that the EZ film could be a cost-effective and convenient dosage form for PrEP of HIV.

Comparative levels of carbonyl delivery between mass-market cigars and cigarettes.

The recent 2016 deeming of cigars by the US Food and Drug Administration (FDA) has led to increased interest in cigar science, including ways to accurately measure the harmful and potentially harmful constituents (HPHCs) found within mainstream cigar smoke. At present, there are standardized methods for evaluating HPHCs in mainstream cigarette smoke but none specific to cigar analysis except for nicotine and carbon monoxide. This study sought to analyze carbonyl delivery in marketed cigars and cigarillos and compare them against levels found in cigarettes. To accomplish this the standard cigarette method, CORESTA recommended method 74 (CRM-74), was optimized for cigar smoking including an evaluation of the trapping efficiency and the stability of the carbonyl-hydrazone adducts due to the increased smoke time required for cigar collection. On a per product basis, carbonyl delivery from cigars smoked under CRM-64 conditions was found to yield similar levels of formaldehyde and greater levels of acetaldehyde, acrolein and crotonaldehyde than measured in mainstream cigarette smoke collected under conditions prescribed under ISO standard 3308. Furthermore, on a per product basis, cigarettes smoked under the ISO 20778 intense smoking regime delivered higher levels of formaldehyde, acrolein and crotonaldehyde as compared to cigars smoked under the CORESTA regime, while acetaldehyde was found to be higher in mainstream cigar smoke. Given the recent deeming, this work expands upon previously reported work, limited in scope by either number of products or analytes reported, through the analysis of carbonyl delivery found in the mainstream smoke for 12 brands of cigars and cigarillos.

Migration of Quaternary Ammonium Cations from Exfoliated Clay/Low-Density Polyethylene Nanocomposites into Food Simulants.

Clay/polymer nanocomposites (CPNs) are polymers incorporating refined clay particles that are frequently functionalized with quaternary ammonium cations (QACs) as dispersion aids. There is interest in commercializing CPNs for food contact applications because they have improved strength and barrier properties, but there are few studies on the potential for QACs in CPNs to transfer to foods under conditions of intended use. In this study, we manufactured low-density poly(ethylene) (LDPE)-based CPNs and assessed whether QACs can migrate into several food simulants under accelerated storage conditions. QACs were found to migrate to a fatty food simulant (ethanol) at levels of ∼1.1 µg mg-1 CPN mass after 10 days at 40 °C, constituting about 4% total migration (proportion of the initial QAC content in the CPN that migrated to the simulant). QAC migration into ethanol was ∼16× higher from LDPE containing approximately the same concentration of QACs but no clay, suggesting that most QACs in the CPN are tightly bound to clay particles and are immobile. Negligible QACs were found to migrate into aqueous, alcoholic, or acidic simulants from CPNs, and the amount of migrated QACs was also found to scale with the temperature and the initial clay concentration. The migration data were compared to a theoretical diffusion model, and it was found that the diffusion constant for QACs in the CPN was several orders of magnitude slower than predicted, which we attributed to the potential for QACs to migrate as dimers or other aggregates rather than as individual ions. Nevertheless, the use of the migration model resulted in a conservative estimate of the mass transfer of QAC from the CPN test specimens.

Resistance to the Tat Inhibitor Didehydro-Cortistatin A Is Mediated by Heightened Basal HIV-1 Transcription.

Human immunodeficiency virus type 1 (HIV-1) Tat binds the viral RNA structure transactivation-responsive element (TAR) and recruits transcriptional cofactors, amplifying viral mRNA expression. The Tat inhibitor didehydro-cortistatin A (dCA) promotes a state of persistent latency, refractory to viral reactivation. Here we investigated mechanisms of HIV-1 resistance to dCA in vitro Mutations in Tat and TAR were not identified, consistent with the high level of conservation of these elements. Instead, viruses resistant to dCA developed higher Tat-independent basal transcription. We identified a combination of mutations in the HIV-1 promoter that increased basal transcriptional activity and modifications in viral Nef and Vpr proteins that increased NF-κB activity. Importantly, these variants are unlikely to enter latency due to accrued transcriptional fitness and loss of sensitivity to Tat feedback loop regulation. Furthermore, cells infected with these variants become more susceptible to cytopathic effects and immune-mediated clearance. This is the first report of viral escape to a Tat inhibitor resulting in heightened Tat-independent activity, all while maintaining wild-type Tat and TAR.IMPORTANCE HIV-1 Tat enhances viral RNA transcription by binding to TAR and recruiting activating factors. Tat enhances its own transcription via a positive-feedback loop. Didehydro-cortistatin A (dCA) is a potent Tat inhibitor, reducing HIV-1 transcription and preventing viral rebound. dCA activity demonstrates the potential of the "block-and-lock" functional cure approaches. We investigated the viral genetic barrier to dCA resistance in vitro While mutations in Tat and TAR were not identified, mutations in the promoter and in the Nef and Vpr proteins promoted high Tat-independent activity. Promoter mutations increased the basal transcription, while Nef and Vpr mutations increased NF-κB nuclear translocation. This heightened transcriptional activity renders CD4+ T cells infected with these viruses more susceptible to cytotoxic T cell-mediated killing and to cell death by cytopathic effects. Results provide insights on drug resistance to a novel class of antiretrovirals and reveal novel aspects of viral transcriptional regulation.

The Tat inhibitor didehydro-cortistatin A suppresses SIV replication and reactivation.

The HIV-1 transactivation protein (Tat) binds the HIV mRNA transactivation responsive element (TAR), regulating transcription and reactivation from latency. Drugs against Tat are unfortunately not clinically available. We reported that didehydro-cortistatin A (dCA) inhibits HIV-1 Tat activity. In human CD4+ T cells isolated from aviremic individuals and in the humanized mouse model of latency, combining dCA with antiretroviral therapy accelerates HIV-1 suppression and delays viral rebound upon treatment interruption. This drug class is amenable to block-and-lock functional cure approaches, aimed at a durable state of latency. Simian immunodeficiency virus (SIV) infection of rhesus macaques (RhMs) is the best-characterized model for AIDS research. Here, we demonstrate, using in vitro and cell-based assays, that dCA directly binds to SIV Tat's basic domain. dCA specifically inhibits SIV Tat binding to TAR, but not a Tat-Rev fusion protein, which activates transcription when Rev binds to its cognate RNA binding site replacing the apical region of TAR. Tat-TAR inhibition results in loss of RNA polymerase II recruitment to the SIV promoter. Importantly, dCA potently inhibits SIV reactivation from latently infected Hut78 cells and from primary CD4+ T cells explanted from SIVmac239-infected RhMs. In sum, dCA's remarkable breadth of activity encourages SIV-infected RhM use for dCA preclinical evaluation.-Mediouni, S., Kessing, C. F., Jablonski, J. A., Thenin-Houssier, S., Clementz, M., Kovach, M. D., Mousseau, G., de Vera, I.M.S., Li, C., Kojetin, D. J., Evans, D. T., Valente, S. T. The Tat inhibitor didehydro-cortistatin A suppresses SIV replication and reactivation.

Factors Affecting Transfer of the Heavy Metals Arsenic, Lead, and Cadmium from Diatomaceous-Earth Filter Aids to Alcoholic Beverages during Laboratory-Scale Filtration.

Filtration methods for alcoholic fermented beverages often use filter aids such as diatomaceous earth (DE), which may contain elevated amounts of the heavy metals arsenic (As), lead (Pb), and cadmium (Cd). Here, we evaluated factors affecting transfer of these heavy metals from DE to beer and wine. A laboratory-scale filtration system was used to process unfiltered ale, lager, red wine, and white wine with three types of food-grade DE. Filtrate and DE were analyzed for heavy metals using ICP-MS, in addition to LC-ICP-MS for As-speciation analysis. Use of 2 g/L DE containing 5.4 mg/kg soluble inorganic As (iAs) for filtering beer and wine resulted in significant ( p < 0.05) increases of 11.2-13.7 µg/L iAs in the filtered beverage. There was a significant ( p < 0.05) effect from the DE quantity used in filtration on the transfer of iAs in all beverage types, whereas no alterations were observed for Pb and Cd levels. Methods to wash DE using water, citric acid, or EDTA all significantly ( p < 0.05) reduced iAs concentrations, whereas only EDTA significantly reduced Pb levels. Cd concentrations were not affected by any wash method. These data indicate that specific steps can be taken to limit heavy-metal transfer from DE filter aids to beer and wine.

Didehydro-Cortistatin A Inhibits HIV-1 by Specifically Binding to the Unstructured Basic Region of Tat.

The intrinsically disordered HIV-1 Tat protein binds the viral RNA transactivation response structure (TAR), which recruits transcriptional cofactors, amplifying viral mRNA expression. Limited Tat transactivation correlates with HIV-1 latency. Unfortunately, Tat inhibitors are not clinically available. The small molecule didehydro-cortistatin A (dCA) inhibits Tat, locking HIV-1 in persistent latency, blocking viral rebound. We generated chemical derivatives of dCA that rationalized molecular docking of dCA to an active and specific Tat conformer. These revealed the importance of the cycloheptene ring and the isoquinoline nitrogen's positioning in the interaction with specific residues of Tat's basic domain. These features are distinct from the ones required for inhibition of cyclin-dependent kinase 8 (CDK8), the only other known ligand of dCA. Besides, we demonstrated that dCA activity on HIV-1 transcription is independent of CDK8. The binding of dCA to Tat with nanomolar affinity alters the local protein environment, rendering Tat more resistant to proteolytic digestion. dCA thus locks a transient conformer of Tat, specifically blocking functions dependent of its basic domain, namely the Tat-TAR interaction; while proteins with similar basic patches are unaffected by dCA. Our results improve our knowledge of the mode of action of dCA and support structure-based design strategies targeting Tat, to help advance development of dCA, as well as novel Tat inhibitors.IMPORTANCE Tat activates virus production, and limited Tat transactivation correlates with HIV-1 latency. The Tat inhibitor dCA locks HIV in persistent latency. This drug class enables block-and-lock functional cure approaches, aimed at reducing residual viremia during therapy and limiting viral rebound. dCA may also have additional therapeutic benefits since Tat is also neurotoxic. Unfortunately, Tat inhibitors are not clinically available. We generated chemical derivatives and rationalized binding to an active and specific Tat conformer. dCA features required for Tat inhibition are distinct from features needed for inhibition of cyclin-dependent kinase 8 (CDK8), the only other known target of dCA. Furthermore, knockdown of CDK8 did not impact dCA's activity on HIV-1 transcription. Binding of dCA to Tat's basic domain altered the local protein environment and rendered Tat more resistant to proteolytic digestion. dCA locks a transient conformer of Tat, blocking functions dependent on its basic domain, namely its ability to amplify viral transcription. Our results define dCA's mode of action, support structure-based-design strategies targeting Tat, and provide valuable information for drug development around the dCA pharmacophore.

High Throughput Quantification of Quaternary Ammonium Cations in Food Simulants by Flow-Injection Mass Spectrometry.

Background: A flow-injection MS (FI/MS) method was evaluated for the quantitation of quaternary ammonium cations (QACs) in simple food simulants. Methods: The calibration standard was dimethyldioctadecyl ammonium ion (C18-C18), and the internal standard was benzyldimethylhexadecyl (BDMHD) ammonium ion. Calibration standards based on the C18-C18 ion were prepared in ethanol with a range of 5 to 500 ppb and contained 100 ppb BDMHD. The mobile phase was 90 + 10 (v/v) acetonitrile-5 mM aqueous ammonium acetate and flowed directly into an electrospray source of the mass spectrometer. Detection was accomplished by single ion recording (SIR) in positive mode. Results: Calibration curves were linear with coefficients of determination above 0.995, and the LOQ was 5 ppb. Recoveries of four QACs derived from Arquad 2HT-75, a commercially available surfactant, were measured in common food simulants: ethanol, water, 10% (v/v) ethanol in water, and 3% (v/v) aqueous acetic acid. A solvent exchange procedure was employed for the three aqueous solvents, which included complete evaporation of the sample followed by reconstitution in ethanol prior to injection. The solvent exchange method minimized losses because of QAC adsorption on glass surfaces. Recoveries ranged from 74.4 ± 4.0 to 106.7 ± 6.6% for the two most abundant Arquad 2HT-75 component cations, dimethyldioctadecyl ammonium and dimethyloctadecyl-hexadecyl ammonium. Conclusions: This method is suitable to quantify trace levels of QACs in food simulants as part of exposure evaluations related to their use in emerging food contact materials.

Effects of the Adulteration Technique on the Near-Infrared Detection of Melamine in Milk Powder.

The United States Pharmacopeial Convention has led an international collaborative project to develop a toolbox of screening methods and reference standards for the detection of milk powder adulteration. During the development of adulterated milk powder reference standards, blending methods used to combine melamine and milk had unanticipated strong effects on the near-infrared (NIR) spectrum of melamine. The prominent absorbance band at 1468 nm of melamine was retained when it was dry-blended with skim milk powder but disappeared in wet-blended mixtures, where spray-dried milk powder samples were prepared from solution. Analyses using polarized light microscopy, Raman spectroscopy, dielectric relaxation spectroscopy, X-ray diffraction, and mass spectrometry indicated that wet blending promoted reversible and early Maillard reactions with lactose that are responsible for differences in melamine NIR spectra between wet- and dry-blended samples. Targeted detection estimates based solely on dry-blended reference standards are likely to overestimate NIR detection capabilities in wet-blended samples as a result of previously overlooked matrix effects arising from changes in melamine hydrogen-bonding status, covalent complexation with lactose, and the lower but more homogeneous melamine local concentration distribution produced in wet-blended samples. Techniques used to incorporate potential adulterants can determine the suitability of milk reference standards for use with rapid detection methods.

Factors Affecting the Levels of Heavy Metals in Juices Processed with Filter Aids.

This study investigated factors that may contribute to the presence of arsenic and other heavy metals in apple and grape juices processed with filter aids. Different types and grades of filter aids were analyzed for arsenic, lead, and cadmium with inductively coupled plasma-tandem mass spectrometry. Potential factors affecting the transfer of heavy metals to juices during filtration treatments were evaluated. Effects of washing treatments on removal of heavy metals from filter aids were also determined. Results showed that diatomaceous earth (DE) generally contained a higher level of arsenic than perlite, whereas perlite had a higher lead content than DE. Cellulose contained the lowest level of arsenic among the surveyed filter aids. All samples of food-grade filter aids contained arsenic and lead levels that were below the U.S. Pharmacopeia and National Formulary limits of 10 ppm of total leachable arsenic and lead for food-grade DE filter aids. Two samples of arsenic-rich (>3 ppm) food-grade filter aids raised the level of arsenic in apple and grape juices during laboratory-scale filtration treatments, whereas three samples of low-arsenic (<1 ppm) food-grade filter aids did not affect arsenic levels in filtered juices. Filtration tests with simulated juices (pH 2.9 to 4.1, Brix [°Bx] 8.2 to 18.1, total suspended solids [TSS] 0.1 to 0.5%) showed that pH or sugar content had no effect on arsenic levels of filtered juices, whereas arsenic content of filtered juice was elevated when higher amounts of filter aid were used for filtration. Authentic unfiltered apple juice (pH 3.6, °Bx 12.9, TSS 0.4%) and grape juice (pH 3.3, °Bx 16.2, TSS 0.05%) were used to verify results obtained with simulated juices. However, body feed ratio did not affect the arsenic content of filtered authentic juices. Washing treatments were effective at reducing arsenic, but not cadmium or lead, concentrations in a DE filter aid. This study identified ways to reduce the amount of arsenic transferred to juices during filtration.

Targeted and non-targeted detection of lemon juice adulteration by LC-MS and chemometrics.

Economically motivated adulteration (EMA) of lemon juice was detected by LC-MS and principal component analysis (PCA). Twenty-two batches of freshly squeezed lemon juice were adulterated by adding an aqueous solution containing 5% citric acid and 6% sucrose to pure lemon juice to obtain 30%, 60% and 100% lemon juice samples. Their total titratable acidities, °Brix and pH values were measured, and then all the lemon juice samples were subject to LC-MS analysis. Concentrations of hesperidin and eriocitrin, major phenolic components of lemon juice, were quantified. The PCA score plots for LC-MS datasets were used to preview the classification of pure and adulterated lemon juice samples. Results showed a large inherent variability in the chemical properties among 22 batches of 100% lemon juice samples. Measurement or quantitation of one or several chemical properties (targeted detection) was not effective in detecting lemon juice adulteration. However, by using the LC-MS datasets, including both chromatographic and mass spectrometric information, 100% lemon juice samples were successfully differentiated from adulterated samples containing 30% lemon juice in the PCA score plot. LC-MS coupled with chemometric analysis can be a complement to existing methods for detecting juice adulteration.

Use of Nondisclosure Agreements in Medical Malpractice Settlements by a Large Academic Health Care System.

IMPORTANCE: Honesty and transparency are essential aspects of health care, including in physicians' and hospitals' responses to medical error. Biases and habits associated with medical malpractice litigation, however, may work at cross-purposes with compassion in clinical care and with efforts to improve patient safety. OBJECTIVE: To determine the frequency of nondisclosure agreements in medical malpractice settlements and the extent to which the restrictions in these agreements seem incompatible with good patient care. DESIGN, SETTING, AND PARTICIPANTS: We performed a retrospective review of medical malpractice claim files, including settlement agreements, for claims closed before (fiscal year 2001-2002), during (fiscal year 2006-2007), and after (fiscal years 2009-2012) the implementation of tort reform in Texas. We studied The University of Texas System, which self-insures malpractice claims that involve 6000 physicians at 6 medical campuses in 5 cities. MAIN OUTCOMES AND MEASURES: Nondisclosure provisions in medical malpractice settlements. RESULTS: During the 5 study years, The University of Texas System closed 715 malpractice claims and made 150 settlement payments. For the 124 cases that met our selection criteria, the median compensation paid by the university was $100,000 (range, $500-$1.25 million), and the mean compensation was $185,372. A total of 110 settlement agreements (88.7%) included nondisclosure provisions. All the nondisclosure clauses prohibited disclosure of the settlement terms and amount, 61 (55.5%) prohibited disclosure that the settlement had been reached, 51 (46.4%) prohibited disclosure of the facts of the claim, 29 (26.4%) prohibited reporting to regulatory agencies, and 10 (9.1%) prohibited disclosure by the settling physicians and hospitals, not only by the claimant. Three agreements (2.7%) included specific language that prohibited the claimant from disparaging the physicians or hospitals. The 50 settlement agreements signed after tort reform took full effect in Texas (2009-2012) had stricter nondisclosure provisions than the 60 signed in earlier years: settlements after tort reform were more likely to prohibit disclosure of the event of settlement (36 [72.0%] vs 25 [41.7%]; P < .001), to prohibit disclosure of the facts of the claims (31 [62.0%] vs 20 [33.3%]; P = .003), and to prohibit reporting to regulatory bodies (25 [50.0%] vs 4 [6.7%]; P < .001). CONCLUSIONS AND RELEVANCE: An academic health system with a declared commitment to patient safety and transparency used nondisclosure clauses in most malpractice settlement agreements but with little standardization or consistency. The scope of nondisclosure was often broader than seemed needed to protect physicians and hospitals from disparagement by the plaintiff or to avoid publicizing settlement amounts that might attract other claimants. Some agreements prohibited reporting to regulatory agencies, a practice that the health system changed in response to our findings.