Potential Health Risk of Aluminum in Four Camellia sinensis Cultivars and Its Content as a Function of Leaf Position.

Tea plants can accumulate aluminum (Al) in their leaves to a greater extent than most other edible plants. Few studies, however, address the Al concentration in leaves at different positions, which is important information for tea quality control. Leaves from four different cultivars of Camellia sinensis L. grown in Hawaii were analyzed for Al concentrations at 10 different leaf positions. Each cultivar was harvested in the winter and summer to determine seasonal variations of Al concentrations in the leaves. The results showed that Al concentrations in the winter leaves were an average of 1.2-fold higher than those in the summer leaves, although the seasonal variations were not statistically significant. The total Al concentration of successively lower leaves showed an exponential increase (R2 ≥ 0.900) for all four cultivars in the summer season, whereas those of the winter leaves fit a bi-phase linear regression (R2 ≥ 0.968). The regression of the Al concentrations against the top-5 leaf positions in the winter season fit one linear regression, while that against leaf positions 6-11 fit another linear regression. The average Al concentrations between the third leaf and the shoot plus first two leaves increased approximately 2.7-fold and 1.9-fold for all cultivars in the winter and summer months, respectively. The Al concentrations in the rest of the leaves increased approximately 1.5-fold in a sequential order. The target hazard quotient being between 1.69 × 10-2 and 5.06 × 10-1 in the tea leaf samples of the four cultivars in Hawaii were all less than 1, suggesting negligible health risks for consumers. The results of this study may be useful for directing harvest practices and estimating tea quality.

Griffithsin Inhibits Nipah Virus Entry and Fusion and Can Protect Syrian Golden Hamsters From Lethal Nipah Virus Challenge.

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis and respiratory disease in humans. There is currently no approved therapeutic for human use against NiV infection. Griffithsin (GRFT) is high-mannose oligosaccharide binding lectin that has shown in vivo broad-spectrum activity against viruses, including severe acute respiratory syndrome coronavirus, human immunodeficiency virus 1, hepatitis C virus, and Japanese encephalitis virus. In this study, we evaluated the in vitro antiviral activities of GRFT and its synthetic trimeric tandemer (3mG) against NiV and other viruses from 4 virus families. The 3mG had comparatively greater potency than GRFT against NiV due to its enhanced ability to block NiV glycoprotein-induced syncytia formation. Our initial in vivo prophylactic evaluation of an oxidation-resistant GRFT (Q-GRFT) showed significant protection against lethal NiV challenge in Syrian golden hamsters. Our results warrant further development of Q-GRFT and 3mG as potential NiV therapeutics.

Selective Metal-Phenolic Assembly from Complex Multicomponent Mixtures.

Selective self-assembly in multicomponent mixtures offers a method for isolating desired components from complex systems for the rapid production of functional materials. Developing approaches capable of selective assembly of "target" components into intended three-dimensional structures is challenging because of the intrinsically high complexity of multicomponent systems. Herein, we report the selective coordination-driven self-assembly of metal-phenolic networks (MPNs) from a series of complex multicomponent systems (including crude plant extracts) into thin films via metal chelation with phenolic ligands. The metal (FeIII) selectively assembles low abundant phenolic components (e.g., myricetrin and quercetrin) from plant extracts into thin films. This selective metal-phenolic assembly is independent of the substrate properties (e.g., size, surface charge, and shape). Moreover, the high selectivity is consistent across different target phenolic ligands in model mixtures, even though each individual component can form thin films from single-component systems. A computational simulation of film formation suggests that the driving force for the selective behavior stems from differences in the number of chelating sites in the phenolic structures. The MPN films are shown to demonstrate improved antioxidant properties compared with the corresponding phenolic compounds in their free form, therefore exhibiting potential as free-standing antioxidant films.

Attenuation and efficacy of live-attenuated Rift Valley fever virus vaccine candidates in non-human primates.

Rift Valley fever virus (RVFV) is an important mosquito-borne veterinary and human pathogen that has caused large outbreaks of severe disease throughout Africa and the Arabian Peninsula. Currently, no licensed vaccine or therapeutics exists to treat this potentially deadly disease. The explosive nature of RVFV outbreaks and the severe consequences of its accidental or intentional introduction into RVFV-free areas provide the impetus for the development of novel vaccine candidates for use in both livestock and humans. Rationally designed vaccine candidates using reverse genetics have been used to develop deletion mutants of two known RVFV virulence factors, the NSs and NSm genes. These recombinant viruses were demonstrated to be protective and immunogenic in rats, mice, and sheep, without producing clinical illness in these animals. Here, we expand upon those findings and evaluate the single deletion mutant (ΔNSs rRVFV) and double deletion mutant (ΔNSs-ΔNSm rRVFV) vaccine candidates in the common marmoset (Callithrix jacchus), a non-human primate (NHP) model resembling severe human RVF disease. We demonstrate that both the ΔNSs and ΔNSs-ΔNSm rRVFV vaccine candidates were found to be safe and immunogenic in the current study. The vaccinated animals received a single dose of vaccine that led to the development of a robust antibody response. No vaccine-induced adverse reactions, signs of clinical illness or infectious virus were detected in the vaccinated marmosets. All vaccinated animals that were subsequently challenged with RVFV were protected against viremia and liver disease. In summary, our results provide the basis for further development of the ΔNSs and ΔNSs-ΔNSm rRVFV as safe and effective human RVFV vaccines for this significant public health threat.

Discovery of novel benzothienoazepine derivatives as potent inhibitors of respiratory syncytial virus.

The development of novel non-nucleoside inhibitors of the RSV polymerase complex is of significant clinical interest. Compounds derived from the benzothienoazepine core, such as AZ-27, are potent inhibitors of RSV viruses of the A-subgroup, but are only moderately active against the B serotype and as yet have not demonstrated activity in vivo. Herein we report the discovery of several novel families of C-2 arylated benzothienoazepine derivatives that are highly potent RSV polymerase inhibitors and reveal an exemplary structure, compound 4a, which shows low nanomolar activity against both RSV A and B viral subtypes. Furthermore, this compound is effective at suppressing viral replication, when administered intranasally, in a rodent model of RSV infection. These results suggest that compounds belonging to this chemotypes have the potential to provide superior anti-RSV agents than those currently available for clinical use.

The lipid moiety of brincidofovir is required for in vitro antiviral activity against Ebola virus.

Brincidofovir (BCV) is the 3-hexadecyloxy-1-propanol (HDP) lipid conjugate of the acyclic nucleoside phosphonate cidofovir (CDV). BCV has established broad-spectrum activity against double-stranded DNA (dsDNA) viruses; however, its activity against RNA viruses has been less thoroughly evaluated. Here, we report that BCV inhibited infection of Ebola virus in multiple human cell lines. Unlike the mechanism of action for BCV against cytomegalovirus and other dsDNA viruses, phosphorylation of CDV to the diphosphate form appeared unnecessary. Instead, antiviral activity required the lipid moiety and in vitro activity against EBOV was observed for several HDP-nucleotide conjugates.

Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein.

Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014-2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies.

Treatment of congenital pulmonary lymphangiectasia using ethiodized oil lymphangiography.

Historically, congenital pulmonary lymphangiectasia (CPL) has had poor treatment outcomes despite aggressive therapy. There are recent reports of ethiodized oil (Lipiodol) lymphangiography successfully treating lymphatic leakage in adults. In this report, we describe two infants with CPL complicated by chylothoraces successfully treated by instillation of ethiodized oil into the lymphatic system. Congenital atresia of the thoracic duct was demonstrated on the lymphangiogram in both patients before treatment. Both patients have shown good short-term outcomes without supplemental oxygen or fat restricted diets at 9 months of age. Ethiodized oil lymphangiography represents a new treatment modality for some patients with CPL.

High-throughput, luciferase-based reverse genetics systems for identifying inhibitors of Marburg and Ebola viruses.

Marburg virus (MARV) and Ebola virus (EBOV), members of the family Filoviridae, represent a significant challenge to global public health. Currently, no licensed therapies exist to treat filovirus infections, which cause up to 90% mortality in human cases. To facilitate development of antivirals against these viruses, we established two distinct screening platforms based on MARV and EBOV reverse genetics systems that express secreted Gaussia luciferase (gLuc). The first platform is a mini-genome replicon to screen viral replication inhibitors using gLuc quantification in a BSL-2 setting. The second platform is complementary to the first and expresses gLuc as a reporter gene product encoded in recombinant infectious MARV and EBOV, thereby allowing for rapid quantification of viral growth during treatment with antiviral compounds. We characterized these viruses by comparing luciferase activity to virus production, and validated luciferase activity as an authentic real-time measure of viral growth. As proof of concept, we adapt both mini-genome and infectious virus platforms to high-throughput formats, and demonstrate efficacy of several antiviral compounds. We anticipate that both approaches will prove highly useful in the development of anti-filovirus therapies, as well as in basic research on the filovirus life cycle.

Evaluation of luciferase and GFP-expressing Nipah viruses for rapid quantitative antiviral screening.

Nipah virus (NiV) outbreaks have occurred in Malaysia, India, and Bangladesh, and the virus continues to cause annual outbreaks of fatal human encephalitis in Bangladesh due to spillover from its bat host reservoir. Due to its high pathogenicity, its potential use for bio/agro-terrorism, and to the current lack of approved therapeutics, NiV is designated as an overlap select agent requiring biosafety level-4 containment. Although the development of therapeutic monoclonal antibodies and soluble protein subunit vaccines have shown great promise, the paucity of effective antiviral drugs against NiV merits further exploration of compound libraries using rapid quantitative antiviral assays. As a proof-of-concept study, we evaluated the use of fluorescent and luminescent reporter NiVs for antiviral screening. We constructed and rescued NiVs expressing either Renilla luciferase or green fluorescent protein, and characterized their reporter signal kinetics in different cell types as well as in the presence of several inhibitors. The 50% effective concentrations (EC50s) derived for inhibitors against both reporter viruses are within range of EC50s derived from virus yield-based dose-response assays against wild-type NiV (within 1Log10), thus demonstrating that both reporter NiVs can serve as robust antiviral screening tools. Utilizing these live NiV-based reporter assays requires modest instrumentation, and circumvents the time and labor-intensive steps associated with cytopathic effect or viral antigen-based assays. These reporter NiVs will not only facilitate antiviral screening, but also the study of host cell components that influence the virus life cycle.

Vitamin D supplementation: what's known, what to do, and what's needed.

The use of vitamin D supplements to prevent and treat a wide range of illnesses has increased substantially over the last decade. Epidemiologic evidence links vitamin D deficiency to autoimmune disease, cancer, cardiovascular disease, depression, dementia, infectious diseases, musculoskeletal decline, and more. The Institute of Medicine published an exhaustive report in 2010 that concluded that vitamin D supplementation for indications other than musculoskeletal health was not adequately supported by evidence and that most North Americans receive sufficient vitamin D from their diet and sun exposure. These conclusions are at odds with some clinical practice guidelines; thus, we sought to summarize the best available evidence regarding the benefits of vitamin D supplementation, to examine the potential risks, and to provide practical dosing advice. The adequacy of vitamin D stores is determined by measuring the 25-hydroxyvitamin D serum concentrations. The demarcations between deficiency (< 20 ng/ml), insufficiency (20-30 ng/ml), and optimal (30-80 ng/ml) serum concentrations are controversial. Vitamin D in doses of 800-5000 IU/day improve musculoskeletal health (e.g., reduces the rate of fractures and falls in older adults (aged ≥ 65 yrs). In patients with documented vitamin D deficiency, a cumulative dose of at least 600,000 IU administered over several weeks appears to be necessary to replenish vitamin D stores. Single large doses of 300,000-500,000 IU should be avoided. Vitamin D supplementation should not be offered routinely to other patient populations. Although results from some prospective clinical trials are promising, most have not been robustly designed and executed. The decision by young, otherwise healthy adults to take vitamin D in doses of 2000 IU/day or lower is unlikely to cause harm. For patients who are not at risk for developing vitamin D deficiency, sensible sun exposure is an inexpensive and enjoyable way to maintain vitamin D stores.

Hypoglycemic effect of bitter melon compared with metformin in newly diagnosed type 2 diabetes patients.

ETHNOPHARMACOLOGICAL RELEVANCE: Bitter melon (Momordica charantia L.) has been widely used as an traditional medicine treatment for diabetic patients in Asia. In vitro and animal studies suggested its hypoglycemic activity, but limited human studies are available to support its use. AIM OF STUDY: This study was conducted to assess the efficacy and safety of three doses of bitter melon compared with metformin. MATERIALS AND METHODS: This is a 4-week, multicenter, randomized, double-blind, active-control trial. Patients were randomized into 4 groups to receive bitter melon 500 mg/day, 1,000 mg/day, and 2,000 mg/day or metformin 1,000 mg/day. All patients were followed for 4 weeks. RESULTS: There was a significant decline in fructosamine at week 4 of the metformin group (-16.8; 95% CI, -31.2, -2.4 µmol/L) and the bitter melon 2,000 mg/day group (-10.2; 95% CI, -19.1, -1.3 µmol/L). Bitter melon 500 and 1,000 mg/day did not significantly decrease fructosamine levels (-3.5; 95% CI -11.7, 4.6 and -10.3; 95% CI -22.7, 2.2 µmol/L, respectively). CONCLUSIONS: Bitter melon had a modest hypoglycemic effect and significantly reduced fructosamine levels from baseline among patients with type 2 diabetes who received 2,000 mg/day. However, the hypoglycemic effect of bitter melon was less than metformin 1,000 mg/day.

Evaluation of effect of ephedrine on the transport of drugs from the nasal cavity to the systemic circulation and the central nervous system.

It has been shown that vasoconstrictive drugs such as ephedrine derivatives are able to decrease systemic absorption of drugs administered by mucosal surfaces. The present paper set out to evaluate in the rat model the effect of co-administered nasal ephedrine on the absorption of GR138950 in a simple and in a pectin self-gelling formulation. It was hypothetised that a decrease in nasal systemic absorption would lead to an increase in direct nose-to-brain transport as demonstrated by the drug concentration in the olfactory lobes of the brain. It was found that ephedrine administered nasally with the drug in a simple aqueous solution resulted in a significant increase in nasal systemic absorption and also an increase in brain delivery; however, this trend was not observed with the pectin formulations. The pectin formulation with ephedrine resulted in lower systemic absorption of GR138950 and lower brain uptake compared to the simple solution formulation containing ephedrine.

Generation of eGFP expressing recombinant Zaire ebolavirus for analysis of early pathogenesis events and high-throughput antiviral drug screening.

Zaire ebolavirus causes large outbreaks of severe and usually fatal hemorrhagic disease in humans for which there is no effective treatment or cure. To facilitate examination of early critical events in viral pathogenesis and to identify antiviral compounds, a recombinant Zaire ebolavirus was engineered to express a foreign protein, eGFP, to provide a rapid and sensitive means to monitor virus replication in infected cells. This genetically engineered virus represents the first insertion of a foreign gene into ebolavirus. We show that Ebola-eGFP virus (EboZ-eGFP) infects known early targets of human infections and serves as an ideal model to screen antiviral compounds in less time than any previously published assay.