Serum Osteoprotegerin Levels and the Vascular Reactivity Index in Patients with Hypertension.

Background and Objectives: Osteoprotegerin (OPG), a soluble glycoprotein found in serum, has been associated with both the presence and severity of atherosclerosis. OPG is regarded as the mediator in the process of vascular endothelial dysfunction. Impaired endothelial function has an intimate link with hypertension (HTN) and is associated with significant morbidity and mortality. This study was to investigate the connection between OPG and endothelial dysfunction in patients having HTN. Materials and Methods: There are 102 patients with HTN included. For the purpose of determining the levels of OPG, a commercial enzyme-linked immunosorbent test kit was applied. The vascular reactivity index (VRI), which is assessed via the digital thermal monitoring, provides information on endothelial function. Results: Ten patients with HTN (9.8%) were classified as having poor vascular reactivity (VRI < 1.0), 46 HTN patients (45.1%) as having intermediate vascular reactivity (1.0 ≤ VRI < 2.0), and 46 HTN patients (45.1%) were classified as having high vascular reactivity (VRI ≥ 2.0). A greater serum OPG level (p < 0.001) and older age (p = 0.022) were linked to impaired vascular reactivity. The estimated glomerular filtration rate (r = 0.196, p = 0.048) was positively correlated with VRI values in hypertensive participants, while advanced age (r = -0.222, p = 0.025) and the log-transformed OPG level (log-OPG, r = -0.357, p < 0.001) were negatively correlated with VRI. Serum log-OPG level was shown to be strongly and independently correlated with VRI values in HTN individuals after multivariable forward stepwise linear regression analysis (ß = -0.357, adjusted R2 change = 0.119, p < 0.001). Conclusions: In patients with HTN, serum OPG levels were adversely correlated with VRI and probably had a role in endothelial dysfunction.

Two RNA Tunnel Inhibitors Bind in Highly Conserved Sites in Dengue Virus NS5 Polymerase: Structural and Functional Studies.

Dengue virus (DENV) NS5 RNA-dependent RNA polymerase (RdRp), an important drug target, synthesizes viral RNA and is essential for viral replication. While a number of allosteric inhibitors have been reported for hepatitis C virus RdRp, few have been described for DENV RdRp. Following a diverse compound screening campaign and a rigorous hit-to-lead flowchart combining biochemical and biophysical approaches, two DENV RdRp nonnucleoside inhibitors were identified and characterized. These inhibitors show low- to high-micromolar inhibition in DENV RNA polymerization and cell-based assays. X-ray crystallography reveals that they bind in the enzyme RNA template tunnel. One compound (NITD-434) induced an allosteric pocket at the junction of the fingers and palm subdomains by displacing residue V603 in motif B. Binding of another compound (NITD-640) ordered the fingers loop preceding the F motif, close to the RNA template entrance. Most of the amino acid residues that interacted with these compounds are highly conserved in flaviviruses. Both sites are important for polymerase de novo initiation and elongation activities and essential for viral replication. This work provides evidence that the RNA tunnel in DENV RdRp offers interesting target sites for inhibition.IMPORTANCE Dengue virus (DENV), an important arthropod-transmitted human pathogen that causes a spectrum of diseases, has spread dramatically worldwide in recent years. Despite extensive efforts, the only commercial vaccine does not provide adequate protection to naive individuals. DENV NS5 polymerase is a promising drug target, as exemplified by the development of successful commercial drugs against hepatitis C virus (HCV) polymerase and HIV-1 reverse transcriptase. High-throughput screening of compound libraries against this enzyme enabled the discovery of inhibitors that induced binding sites in the RNA template channel. Characterizations by biochemical, biophysical, and reverse genetics approaches provide a better understanding of the biological relevance of these allosteric sites and the way forward to design more-potent inhibitors.

A Cyclic Phosphoramidate Prodrug of 2'-Deoxy-2'-Fluoro-2'-C-Methylguanosine for the Treatment of Dengue Virus Infection.

Monophosphate prodrug analogs of 2'-deoxy-2'-fluoro-2'-C-methylguanosine have been reported as potent inhibitors of hepatitis C virus (HCV) RNA-dependent RNA polymerase. These prodrugs also display potent anti-dengue virus activities in cellular assays although their prodrug moieties were designed to produce high levels of triphosphate in the liver. Since peripheral blood mononuclear cells (PBMCs) are among the major targets of dengue virus, different prodrug moieties were designed to effectively deliver 2'-deoxy-2'-fluoro-2'-C-methylguanosine monophosphate prodrugs and their corresponding triphosphates into PBMCs after oral administration. We identified a cyclic phosphoramidate, prodrug 17, demonstrating well-balanced anti-dengue virus cellular activity and in vitro stability profiles. We further determined the PBMC concentration of active triphosphate needed to inhibit virus replication by 50% (TP50). Compound 17 was assessed in an AG129 mouse model and demonstrated 1.6- and 2.2-log viremia reductions at 100 and 300 mg/kg twice a day (BID), respectively. At 100 mg/kg BID, the terminal triphosphate concentration in PBMCs exceeded the TP50 value, demonstrating TP50 as the target exposure for efficacy. In dogs, oral administration of compound 17 resulted in high PBMC triphosphate levels, exceeding the TP50 at 10 mg/kg. Unfortunately, 2-week dog toxicity studies at 30, 100, and 300 mg/kg/day showed that "no observed adverse effect level" (NOAEL) could not be achieved due to pulmonary inflammation and hemorrhage. The preclinical safety results suspended further development of compound 17. Nevertheless, present work has proven the concept that an efficacious monophosphate nucleoside prodrug could be developed for the potential treatment of dengue virus infection.

Structure-activity relationship of uridine-based nucleoside phosphoramidate prodrugs for inhibition of dengue virus RNA-dependent RNA polymerase.

To identify a potent and selective nucleoside inhibitor of dengue virus RNA-dependent RNA polymerase, a series of 2'- and/or 4'-ribose sugar modified uridine nucleoside phosphoramidate prodrugs and their corresponding triphosphates were synthesized and evaluated. Replacement of 2'-OH with 2'-F led to be a poor substrate for both dengue virus and human mitochondrial RNA polymerases. Instead of 2'-fluorination, the introduction of fluorine at the ribose 4'-position was found not to affect the inhibition of the dengue virus polymerase with a reduction in uptake by mitochondrial RNA polymerase. 2'-C-ethynyl-4'-F-uridine phosphoramidate prodrug displayed potent anti-dengue virus activity in the primary human peripheral blood mononuclear cell-based assay with no significant cytotoxicity in human hepatocellular liver carcinoma cell lines and no mitochondrial toxicity in the cell-based assay using human prostate cancer cell lines.

A Deterrence Approach to Regulate Nurses' Compliance with Electronic Medical Records Privacy Policy.

Hospitals have become increasingly aware that electronic medical records (EMR) may bring about tangible/intangible benefits to managing institutions, including reduced medical errors, improved quality-of-care, curtailed costs, and allowed access to patient information by healthcare professionals regardless of limitations. However, increased dependence on EMR has led to a corresponding increase in the influence of EMR breaches. Such incursions, which have been significantly facilitated by the introduction of mobile devices for accessing EMR, may induce tangible/intangible damage to both hospitals and concerned individuals. The purpose of this study was to explore factors which may tend to inhibit nurses' intentions to violate privacy policy concerning EMR based upon the deterrence theory perspective. Utilizing survey methodology, 262 responses were analyzed via structural equation modeling. Results revealed that punishment certainty, detection certainty, and subjective norm would most certainly and significantly reduce nurses' intentions to violate established EMR privacy policy. With these findings, recommendations for health administrators in planning and designing effective strategies which may potentially inhibit nurses from violating EMR privacy policy are discussed.

Synergistic suppression of dengue virus replication using a combination of nucleoside analogs and nucleoside synthesis inhibitors.

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Currently, there is no clinically approved vaccine or antiviral for DENV. Combination therapy is a common practice in antiviral treatment and a potential approach to search for new treatments for infectious pathogens. In this study, we performed a combination treatment in cell culture by using three distinct classes of inhibitors, including ribavirin (a guanosine analog with several antiviral mechanisms), brequinar (a pyrimidine biosynthesis inhibitor), and INX-08189 (a guanosine analog). The compound pairs were evaluated for antiviral activity by use of a DENV-2 luciferase replicon assay. Our result indicated that the combination of ribavirin and INX-08189 exhibited strong antiviral synergy. This result suggests that synergy can be achieved with compound pairs in which one compound suppresses the synthesis of the nucleoside for which the other compound is a corresponding nucleoside analog. In addition, we found that treatment of cells with brequinar alone could activate interferon-stimulated response elements (ISREs); furthermore, brequinar and NITD-982 (another pyrimidine biosynthesis inhibitor) potentiated interferon-induced ISRE activation. Compared to treatment with brequinar, treatment of cells with ribavirin alone could also induce ISRE activation, but to a lesser extent; however, when cells were cotreated with ribavirin and beta interferon, ribavirin did not augment the interferon-induced ISRE activation.

Activation of peripheral blood mononuclear cells by dengue virus infection depotentiates balapiravir.

In a recent clinical trial, balapiravir, a prodrug of a cytidine analog (R1479), failed to achieve efficacy (reducing viremia after treatment) in dengue patients, although the plasma trough concentration of R1479 remained above the 50% effective concentration (EC(50)). Here, we report experimental evidence to explain the discrepancy between the in vitro and in vivo results and its implication for drug development. R1479 lost its potency by 125-fold when balapiravir was used to treat primary human peripheral blood mononuclear cells (PBMCs; one of the major cells targeted for viral replication) that were preinfected with dengue virus. The elevated EC(50) was greater than the plasma trough concentration of R1479 observed in dengue patients treated with balapiravir and could possibly explain the efficacy failure. Mechanistically, dengue virus infection triggered PBMCs to generate cytokines, which decreased their efficiency of conversion of R1479 to its triphosphate form (the active antiviral ingredient), resulting in decreased antiviral potency. In contrast to the cytidine-based compound R1479, the potency of an adenosine-based inhibitor of dengue virus (NITD008) was much less affected. Taken together, our results demonstrate that viral infection in patients before treatment could significantly affect the conversion of the prodrug to its active form; such an effect should be calculated when estimating the dose efficacious for humans.

Effect of the precrack preparation with an ultrasonic instrument on the ceramic bracket removal.

BACKGROUND/PURPOSE: In terms of fracture mechanics, a precrack preparation may facilitate the propagation of a break through the expected fracture plane during the bracket debonding process. The purpose of this study was to evaluate the effect of an ultrasonic precrack preparation on the debonding force and failure modes of ceramic bracket removal. METHODS: Eighty extracted premolars were assigned to four groups: Inspire, precrack Inspire, Clarity, and precrack Clarity groups, with each group containing 20 teeth. The precrack preparations were made at the mesial gingival line angle of Inspire brackets and on the mesial side of Clarity brackets with an ultrasonic tip. Debonding force, failure modes, and bracket breakage score were measured and recorded. Fracture surfaces after bracket debonding were observed with scanning electron microscopy (SEM). RESULTS: We found that the ultrasonic precrack preparation could significantly decrease the average debonding force and the mean bracket breakage scores of both kinds of ceramic brackets. After bracket debonding, 80% of brackets in the precrack Inspire group and 100% of brackets in the precrack Clarity group showed no bracket failure. However, only 25% of brackets in the Inspire group and 75% of brackets in the Clarity group showed no bracket failure. SEM micrographs showed a precrack notch at the adhesive resin after precrack preparation, and no enamel damage was noted after the bracket debonding. CONCLUSION: The ultrasonic precrack preparation can significantly decrease the debonding force and may guide the bracket debonding through a favorable fracture plane without damage to either the bracket or the enamel.

Conformational flexibility of the Dengue virus RNA-dependent RNA polymerase revealed by a complex with an inhibitor.

We report a highly reproducible method to crystallize the RNA-dependent RNA polymerase (RdRp) domain of dengue virus serotype 3 (DENV-3), allowing structure refinement to a 1.79-Å resolution and revealing amino acids not seen previously. We also present a DENV-3 polymerase/inhibitor cocrystal structure at a 2.1-Å resolution. The inhibitor binds to the RdRp as a dimer and causes conformational changes in the protein. The improved crystallization conditions and new structural information should accelerate structure-based drug discovery.

Application and development of ultrasonics in dentistry.

Since the 1950s, dentistry's ultrasonic instruments have developed rapidly. Because of better visualization, operative convenience, and precise cutting ability, ultrasonic instruments are widely and efficiently applied in the dental field. This article describes the development and improvement of ultrasonic instruments in several dental fields. Although some issues still need clarification, the results of previous studies indicate that ultrasonic instruments have a high potential to become convenient and efficient dental tools and deserve further development.

Cyanotriazoles are selective topoisomerase II poisons that rapidly cure trypanosome infections.

Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo-electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.

Functional analysis of two cavities in flavivirus NS5 polymerase.

Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.

Synthesis and antiviral activity of 4,6-disubstituted pyrimido[4,5-b]indole ribonucleosides.

A series of new pyrimido[4,5-b]indole ribonucleosides bearing phenyl or hetaryl group at position 4 has been prepared by selective Pd-catalyzed cross-coupling reactions of the corresponding protected 4,6-dichloropyrimido[4,5-b]indole ribonucleoside with (het)arylboronic acids or stannanes followed by deprotection. Further cross-couplings under harsher conditions and employing X-Phos ligand proceeded at the position 6 leading to 4,6-disubstituted pyrimido[4,5-b]indole ribonucleosides. Some of these compounds displayed antiviral activity against Dengue virus.

An adenosine nucleoside inhibitor of dengue virus.

Dengue virus (DENV), a mosquito-borne flavivirus, is a major public health threat. The virus poses risk to 2.5 billion people worldwide and causes 50 to 100 million human infections each year. Neither a vaccine nor an antiviral therapy is currently available for prevention and treatment of DENV infection. Here, we report a previously undescribed adenosine analog, NITD008, that potently inhibits DENV both in vitro and in vivo. In addition to the 4 serotypes of DENV, NITD008 inhibits other flaviviruses, including West Nile virus, yellow fever virus, and Powassan virus. The compound also suppresses hepatitis C virus, but it does not inhibit nonflaviviruses, such as Western equine encephalitis virus and vesicular stomatitis virus. A triphosphate form of NITD008 directly inhibits the RNA-dependent RNA polymerase activity of DENV, indicating that the compound functions as a chain terminator during viral RNA synthesis. NITD008 has good in vivo pharmacokinetic properties and is biologically available through oral administration. Treatment of DENV-infected mice with NITD008 suppressed peak viremia, reduced cytokine elevation, and completely prevented the infected mice from death. No observed adverse effect level (NOAEL) was achieved when rats were orally dosed with NITD008 at 50 mg/kg daily for 1 week. However, NOAEL could not be accomplished when rats and dogs were dosed daily for 2 weeks. Nevertheless, our results have proved the concept that a nucleoside inhibitor could be developed for potential treatment of flavivirus infections.

Discovery of Novel Quinoline-Based Proteasome Inhibitors for Human African Trypanosomiasis (HAT).

Human African Trypanosomiasis (HAT) is a vector-borne disease caused by kinetoplastid parasites of the Trypanosoma genus. The disease proceeds in two stages, with a hemolymphatic blood stage and a meningo-encephalic brain stage. In the latter stage, the parasite causes irreversible damage to the brain leading to sleep cycle disruption and is fatal if untreated. An orally bioavailable treatment is highly desirable. In this study, we present a brain-penetrant, parasite-selective 20S proteasome inhibitor that was rapidly optimized from an HTS singleton hit to drug candidate compound 7 that showed cure in a stage II mouse efficacy model. Here, we describe hit expansion and lead optimization campaign guided by cryo-electron microscopy and an in silico model to predict the brain-to-plasma partition coefficient Kp as an important parameter to prioritize compounds for synthesis. The model combined with in vitro and in vivo experiments allowed us to advance compounds with favorable unbound brain-to-plasma ratios (Kp,uu) to cure a CNS disease such as HAT.

Discovery and Preclinical Pharmacology of INE963, a Potent and Fast-Acting Blood-Stage Antimalarial with a High Barrier to Resistance and Potential for Single-Dose Cures in Uncomplicated Malaria.

A series of 5-aryl-2-amino-imidazothiadiazole (ITD) derivatives were identified by a phenotype-based high-throughput screening using a blood stage Plasmodium falciparum (Pf) growth inhibition assay. A lead optimization program focused on improving antiplasmodium potency, selectivity against human kinases, and absorption, distribution, metabolism, excretion, and toxicity properties and extended pharmacological profiles culminated in the identification of INE963 (1), which demonstrates potent cellular activity against Pf 3D7 (EC50 = 0.006 µM) and achieves "artemisinin-like" kill kinetics in vitro with a parasite clearance time of <24 h. A single dose of 30 mg/kg is fully curative in the Pf-humanized severe combined immunodeficient mouse model. INE963 (1) also exhibits a high barrier to resistance in drug selection studies and a long half-life (T1/2) across species. These properties suggest the significant potential for INE963 (1) to provide a curative therapy for uncomplicated malaria with short dosing regimens. For these reasons, INE963 (1) was progressed through GLP toxicology studies and is now undergoing Ph1 clinical trials.

Inhibition of dengue virus polymerase by blocking of the RNA tunnel.

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)(20)]. Chemical modification of the initial "hit" improved the compound potency to an IC(50) (that is, a concentration that inhibits 50% RdRp activity) of 0.7 microM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC(50) of 5 microM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC(50)s of >100 microM against hepatitis C virus RdRp and human DNA polymerase alpha and beta. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.

High-accuracy thickness measurement of a transparent plate with the heterodyne central fringe identification technique.

In a modified Twyman-Green interferometer, the optical path variation is measured with the heterodyne central fringe identification technique, as the light beam is focused by a displaced microscopic objective on the front/rear surface of the test transparent plate. The optical path length variation is then measured similarly after the test plate is removed. The geometrical thickness of the test plate can be calculated under the consideration of dispersion effect. This method has a wide measurable range and a high accuracy in the measurable range.

NITD-688, a pan-serotype inhibitor of the dengue virus NS4B protein, shows favorable pharmacokinetics and efficacy in preclinical animal models.

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.

Inhibition of dengue virus by an ester prodrug of an adenosine analog.

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen that infects humans. Neither a vaccine nor an antiviral therapy is currently available for DENV. Here, we report an adenosine nucleoside prodrug that potently inhibits DENV replication both in cell culture and in a DENV mouse model. NITD449 (2'-C-acetylene-7-deaza-7-carbamoyladenosine) was initially identified as a parental compound that inhibits all four serotypes of DENV with low cytotoxicity. However, in vivo pharmacokinetic studies indicated that NITD449 had a low level of exposure in plasma when dosed orally. To increase the oral bioavailability, we covalently linked isobutyric acids to the 3'- and 5'-hydroxyl groups of ribose via ester linkage to NITD449, leading to the prodrug NITD203 (3',5'-O-diisobutyryl-2'-C-acetylene-7-deaza-7-carbamoyl-adenosin). Pharmacokinetic analysis showed that upon oral dosing of the prodrug, NITD203 was readily converted to NITD449, resulting in improved exposure of the parental compound in plasma in both mouse and rat. In DENV-infected AG129 mice, oral dosing of the prodrug at 25 mg/kg of body weight reduced peak viremia by 30-fold. Antiviral spectrum analysis showed that NITD203 inhibited various flaviviruses (DENV, yellow fever virus, and West Nile virus) and hepatitis C virus but not Chikungunya virus (an alphavirus). Mode-of-action analysis, using a luciferase-reporting replicon, indicated that NITD203 inhibited DENV RNA synthesis. Although NITD203 exhibited potent in vitro and in vivo efficacies, the compound could not reach a satisfactory no-observable-adverse-effect level (NOAEL) in a 2-week in vivo toxicity study. Nevertheless, our results demonstrate that a prodrug approach using a nucleoside analog could potentially be developed for flavivirus antiviral therapy.