Physicochemical characterization and quantification of nanoplastics: applicability, limitations and complementarity of batch and fractionation methods.

A comprehensive physicochemical characterization of heterogeneous nanoplastic (NPL) samples remains an analytical challenge requiring a combination of orthogonal measurement techniques to improve the accuracy and robustness of the results. Here, batch methods, including dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM), as well as separation/fractionation methods such as centrifugal liquid sedimentation (CLS) and field-flow fractionation (FFF)-multi-angle light scattering (MALS) combined with pyrolysis gas chromatography mass spectrometry (pyGC-MS) or Raman microspectroscopy (RM) were evaluated for NPL size, shape, and chemical composition measurements and for quantification. A set of representative/test particles of different chemical natures, including (i) polydisperse polyethylene (PE), (ii) (doped) polystyrene (PS) NPLs, (iii) titanium dioxide, and (iv) iron oxide nanoparticles (spherical and elongated), was used to assess the applicability and limitations of the selected methodologies. Particle sizes and number-based concentrations obtained by orthogonal batch methods (DLS, NTA, TRPS) were comparable for monodisperse spherical samples, while higher deviations were observed for polydisperse, agglomerated samples and for non-spherical particles, especially for light scattering methods. CLS and TRPS offer further insight with increased size resolution, while detailed morphological information can be derived by electron microscopy (EM)-based approaches. Combined techniques such as FFF coupled to MALS and RM can provide complementary information on physical and chemical properties by online measurements, while pyGC-MS analysis of FFF fractions can be used for the identification of polymer particles (vs. inorganic particles) and for their offline (semi)quantification. However, NPL analysis in complex samples will continue to present a serious challenge for the evaluated techniques without significant improvements in sample preparation.

Preclinical Characterization of NVR 3-778, a First-in-Class Capsid Assembly Modulator against Hepatitis B Virus.

NVR 3-778 is the first capsid assembly modulator (CAM) that has demonstrated antiviral activity in hepatitis B virus (HBV)-infected patients. NVR 3-778 inhibited the generation of infectious HBV DNA-containing virus particles with a mean antiviral 50% effective concentration (EC50) of 0.40 µM in HepG2.2.15 cells. The antiviral profile of NVR 3-778 indicates pan-genotypic antiviral activity and a lack of cross-resistance with nucleos(t)ide inhibitors of HBV replication. The combination of NVR 3-778 with nucleos(t)ide analogs in vitro resulted in additive or synergistic antiviral activity. Mutations within the hydrophobic pocket at the dimer-dimer interface of the core protein could confer resistance to NVR 3-778, which is consistent with the ability of the compound to bind to core and to induce capsid assembly. By targeting core, NVR 3-778 inhibits pregenomic RNA encapsidation, viral replication, and the production of HBV DNA- and HBV RNA-containing particles. NVR 3-778 also inhibited de novo infection and viral replication in primary human hepatocytes with EC50 values of 0.81 µM against HBV DNA and between 3.7 and 4.8 µM against the production of HBV antigens and intracellular HBV RNA. NVR 3-778 showed favorable pharmacokinetics and safety in animal species, allowing serum levels in excess of 100 µM to be achieved in mice and, thus, enabling efficacy studies in vivo The overall preclinical profile of NVR 3-778 predicts antiviral activity in vivo and supports its further evaluation for safety, pharmacokinetics, and antiviral activity in HBV-infected patients.

Particle-by-Particle Charge Analysis of DNA-Modified Nanoparticles Using Tunable Resistive Pulse Sensing.

Resistive pulse sensors, RPS, are allowing the transport mechanism of molecules, proteins and even nanoparticles to be characterized as they traverse pores. Previous work using RPS has shown that the size, concentration and zeta potential of the analyte can be measured. Here we use tunable resistive pulse sensing (TRPS) which utilizes a tunable pore to monitor the translocation times of nanoparticles with DNA modified surfaces. We start by demonstrating that the translocation times of particles can be used to infer the zeta potential of known standards and then apply the method to measure the change in zeta potential of DNA modified particles. By measuring the translocation times of DNA modified nanoparticles as a function of packing density, length, structure, and hybridization time, we observe a clear difference in zeta potential using both mean values and population distributions as a function of the DNA structure. We demonstrate the ability to resolve the signals for ssDNA, dsDNA, small changes in base length for nucleotides between 15 and 40 bases long, and even the discrimination between partial and fully complementary target sequences. Such a method has potential and applications in sensors for the monitoring of nanoparticles in both medical and environmental samples.

Determination of in Vivo Enzyme Occupancy Utilizing Inhibitor Dissociation Kinetics.

During drug discovery, assessment of in vivo target occupancy by therapeutic candidates is often required for predicting clinical efficacy. Current strategies for determining target occupancy include using radiolabeled or irreversible surrogates, which can be technically challenging, and the results are often not sufficiently quantitative. We developed a straightforward method by applying slow-dissociation kinetics to quantitatively determine enzyme occupancy without using specialized reagents. We applied this method to determine occupancy of Cathepsin K inhibitors in bone tissues harvested from rabbit femurs. Tissues from dosed animals were harvested, flash frozen, lysed, then analyzed by a jump-dilution assay with substrate. The rate of substrate turnover was monitored continuously until reaching steady state and progress curves were fit with the equation [product] = vst + ((vi - vs)/kobs)(1 - exp(-kobst)). The initial rate vi represents the residual activity of the enzyme before inhibitor dissociation; vs is the reaction rate after dissociation of the inhibitor. Occupancy is derived from the ratio of vi/vs. A significant benefit of the method is that data from both the occupied and unoccupied states are obtained in the same assay under identical conditions, which provides greater consistency between studies. The Cat K inhibitor MK-0674 (in vitro IC50 1 nM) was tested in young rabbits (<6 month old) and showed a dose-dependent increase in occupancy, reaching essentially complete occupancy at 1.0 mg/kg. In addition the method enables measurement of the total Cat K in the target tissue. Results confirmed complete occupancy even as the osteoclasts responded to higher doses with increased enzyme production.

The effect of music-based listening interventions on the volume, fat content, and caloric content of breast milk-produced by mothers of premature and critically ill infants.

PURPOSE: Maternal breast milk is considered the nutritional "gold standard" for all infants, especially premature infants. However, preterm mothers are at risk of not producing adequate milk. Multiple factors affect the production of milk, including stress, fatigue, and the separation of the breastfeeding dyad-for example, when mother or infant is hospitalized. The purpose of this study was to examine the effects of listening and visual interventions on the quantity and quality of breast milk produced by mothers using a double electric breast pump. SUBJECTS: Mothers of 162 preterm infants were randomly assigned to 1 of 4 groups. METHODS: The control group received standard nursing care, whereas mothers in the 3 experimental groups additionally listened to a recording of 1 of 3 music-based listening interventions while using the pump. RESULTS: Mothers in the experimental groups produced significantly more milk (P < .0012). Mothers in these groups also produced milk with significantly higher fat content during the first 6 days of the study.

Divergent effects of joyful and anxiety-provoking music on endothelial vasoreactivity.

OBJECTIVE: To evaluate the extent to which music may affect endothelial function. In previous research, a link between music and physiologic parameters such as heart rate and blood pressure has been observed. METHODS: Randomized four-phase crossover and counterbalanced trial in ten healthy, nonsmoking volunteers (70% male; mean age, 35.6 years) that included self-selections of music evoking joy or provoking anxiety. Two additional phases included watching video clips to induce laughter and listening to audio tapes to promote relaxation. To minimize emotional desensitization, subjects were asked to refrain from using self-selected tapes and images for at least 2 weeks before the assigned study phase. Endothelial function was assessed by brachial artery flow-mediated dilation (FMD) and measured as percent diameter change after an overnight fast. After baseline FMD measurements, subjects were randomized to a 30-minute phase of the testing stimulus followed by poststudy FMD; they returned a minimum of 1 week later for the subsequent task. A total of 160 FMD measurements were obtained. RESULTS: Compared with baseline, music that evoked joy was associated with increases in mean upper arm FMD (2.7% absolute increase; p < .001), whereas reductions in FMD were observed after listening to music that elicited anxiety (0.6% absolute decrease; p = .005 difference between joyful and anxiety-provoking music). Self-selected joyful music was associated with increased FMD to a magnitude previously observed with aerobic activity or statin therapy. CONCLUSION: Listening to joyful music may be an adjunctive life-style intervention for the promotion of vascular health.

Optimal vascular protection: a case for combination antihypertensive therapy.

Endothelial dysfunction is an important factor in the pathogenesis of atherosclerosis, hypertension, and heart failure. The endothelium mediates vascular tone, structure, and function by the release and regulation of multiple vasoactive substances that promote or inhibit vasodilation, vasoconstriction, cell growth, and other mechanisms. The effect of antihypertensive drugs on endothelial function may be an important indicator of their ability to reduce risks for cardiovascular morbidity and mortality. Endothelium-dependent vasodilation induced by various antihypertensive drugs is accurately measured with high-resolution ultrasound of flow-mediated dilation of the brachial artery. Calcium channel blockers and angiotensin-converting enzyme inhibitors have been shown to reverse endothelial dysfunction. The benefits of angiotensin-converting enzyme inhibitors and calcium channel blockers on the endothelium are believed to derive from their effects on nitric oxide production and antioxidant effects, possibly independent of blood pressure reduction. Due to their complementary mechanisms of action, it has been hypothesized that the combination of an angiotensin-converting enzyme inhibitor and a calcium channel blocker will provide superior cardiovascular protection, in part by producing an additive effect of increased nitric oxide availability, when compared with either agent alone.

Rat tail skin temperature regulation by estrogen, phytoestrogens and tamoxifen.

OBJECTIVE: Develop a rat model for the evaluation of estrogenic agents on estrogen deficiency-induced changes in thermoregulation. METHODS: OVX rats are impaired in thermoregulation which manifests itself as an elevation in basal tail skin temperature (TST) and are less able to respond to temperature changes than intact rats. RESULTS: Administration of estrogen subcutaneously to estrogen-depleted rats either as depot formulation, biodegradable pellets, or daily injections, suppressed the increased TST. OVX rats maintained on a diet devoid of phytoestrogens had a higher TST by several degrees than OVX rats fed normal chow, offering greater ability to test estrogenic agents on thermoregulation. Depletion of estrogen in intact rats via chronic administration of leuprolide acetate, a GnRH agonist, also increased TST, which was in turn suppressed by estrogen. In intact rats, tamoxifen exhibited estrogen antagonistic activity elevating TST, while in OVX rats, tamoxifen acted as an agonist by suppressing TST. CONCLUSION: OVX rats kept on a diet devoid of phytoestrogens are a sensitive model for estrogen-dependent thermoregulation.

Effect of supplemental phytonutrients on impairment of the flow-mediated brachial artery vasoactivity after a single high-fat meal.

OBJECTIVES: Our objective was to determine if long-term daily administration of phytonutrient supplements can prevent the immediate adverse impact of a high-fat meal and increase the production of nitric oxide. BACKGROUND: Ingestion of a high-fat meal impairs flow-mediated vasodilation of the brachial artery for at least 4 h; however, co-ingestion of vitamin antioxidants or a green salad has been shown to prevent this effect. METHODS: Flow-mediated brachial artery reactivity test (BART) both before and 3 h after a 900 calorie 50 g fat meal was evaluated in 38 healthy volunteers (age 36.4 +/- 10.1 years). Subjects were randomized to four weeks of daily supplementation with a powdered fruit vegetable juice concentrate (Juice Plus [JP]) along with a complex supplement providing nutritional antioxidants and various herbal extracts (Vineyard [V]), JP alone, or a matching placebo. At three and four weeks, BART was repeated both before and after the high-fat meal. Serum nitrate/nitrite concentrations were measured at baseline and at four weeks. RESULTS: Four weeks of the JP-V combination blunted the detrimental effect of the high-fat meal (-47.5 +/- 23.4% at baseline vs. -1.7 +/- 9.7% at four weeks [p < 0.05]). Four weeks of JP alone had a similar beneficial effect (-45.1 +/- 19.7% at baseline vs. -16.6 +/- 10.3% at four weeks [p < 0.05]), whereas there was no substantial effect of the placebo. In the subjects treated with supplements, concentrations of serum nitrate/nitrite increased from 78 +/- 39 to 114 +/- 62 microm/l (p < 0.02). CONCLUSIONS: Daily ingestion of modest amounts of a fruit/vegetable juice concentrate with or without adjunctive phytonutrient supplementation can reduce the immediate adverse impact of high-fat meals on flow-mediated vasoactivity and increase nitrate/nitrite blood concentration.