Ontogeny of Human Liver Aldehyde Oxidase: Developmental Changes and Implications for Drug Metabolism.

Despite the increasing importance of aldehyde oxidase (AO) in the drug metabolism of clinical candidates, ontogeny data for AO are limited. The objective of our study was to characterize the age-dependent AO content and activity in the human liver cytosolic fraction (HLC) and human hepatocytes (HH). HLC (n = 121 donors) and HH (n = 50 donors) were analyzed for (1) AO protein content by quantitative proteomics and (2) enzyme activity using carbazeran as a probe substrate. AO activity showed high technical variability and poor correlation with the content in HLC samples, whereas hepatocyte samples showed a strong correlation between the content and activity. Similarly, AO content and activity showed no significant age-dependent differences in HLC samples, whereas the average AO content and activity in hepatocytes increased significantly (∼20-40-fold) from the neonatal levels (0-28 days). Based on the hepatocyte data, the age at which 50% of the adult AO content is reached (age50) was 3.15 years (0.32-13.97 years, 95% CI). Metabolite profiling of carbazeran revealed age-dependent metabolic switching and the role of non-AO mechanisms (glucuronidation and desmethylation) in carbazeran elimination. The content-activity correlation in hepatocytes improved significantly (R2 = 0.95; p < 0.0001) in samples showing <10% contribution of glucuronidation toward the overall metabolism, confirming that AO-mediated oxidation and glucuronidation are the key routes of carbazeran metabolism. Considering the confounding effect of glucuronidation on AO activity, AO content-based ontogeny data are a more direct reflection of developmental changes in protein expression. The comprehensive ontogeny data of AO in HH samples are more reliable than HLC data, which are important for developing robust physiologically based pharmacokinetic models for predicting AO-mediated metabolism in children.

Retrospective Evaluation of Moderate Sedation Visits That Used Oral Meperidine and Hydroxyzine With Oral or Intranasal Midazolam.

Purpose: To examine the influence of substituting intranasal (IN) midazolam (MID) for oral (PO) MID, within the three-drug combination of meperidine (MEP), hydroxyzine (H) and MID, on sedation treatment outcomes. Methods: A retrospective, cross-sectional analysis examined patient variables and sedation outcomes in 508 pediatric dental patients sedated by single- and multi-drug sedation regimens (MEP-H; MEP-H-(PO)-MID; MEP-H-(IN)-MID; single-agent MID). The outcome assessment examined sedation visit effectiveness, sedation treatment completion, treatment time and medication administration to discharge time. Multivariable logistic regression analyses assessed predictive variables associated with sedation visit effectiveness. Results: Both three-drug combinations (MEP-H-(PO)-MID; MEP-H-(IN)-MID) were used for behavior guidance in children of a similar age (median age=7.1 and 6.5 years, respectively, for the two drug combinations) and weight (median weight = 23.7 and 23.5 kg, respectively, for the two drug combinations). These three-drug combinations had a higher likelihood of sedation effectiveness over the reference sedation regimen of single-agent midazolam (MEP-H-(PO)-MID adjusted odds ratio [OR] = 2.65; 95 percent confidence interval [95% CI]=1.09 to 6.45; P=0.032; and MEP-H-(IN)-MID OR=2.08; 95% CI=1.03 to 4.18; P=0.039). MEP-H-(IN)MID was associated with a shorter medication administration to discharge time for patients by 23 minutes (interquartile range [IQR]=9.5 to 34 minutes) compared to MEP-H-(PO) MID (P<0.05) while providing a comparable number of teeth treated (median=five). All sedation drug regimens, including MEP-H-(IN)MID, had high levels of oxygen saturation during all sedation appointments. Conclusion: Substituting IN for PO MID in MEP-H-MID was associated with a shorter total time to discharge while demonstrating comparable efficacy during sedation.

Challenges and Opportunities for In Vitro-In Vivo Extrapolation of Aldehyde Oxidase-Mediated Clearance: Toward a Roadmap for Quantitative Translation.

Underestimation of aldehyde oxidase (AO)-mediated clearance by current in vitro assays leads to uncertainty in human dose projections, thereby reducing the likelihood of success in drug development. In the present study we first evaluated the current drug development practices for AO substrates. Next, the overall predictive performance of in vitro-in vivo extrapolation of unbound hepatic intrinsic clearance (CLint,u) and unbound hepatic intrinsic clearance by AO (CLint,u,AO) was assessed using a comprehensive literature database of in vitro (human cytosol/S9/hepatocytes) and in vivo (intravenous/oral) data collated for 22 AO substrates (total of 100 datapoints from multiple studies). Correction for unbound fraction in the incubation was done by experimental data or in silico predictions. The fraction metabolized by AO (fmAO) determined via in vitro/in vivo approaches was found to be highly variable. The geometric mean fold errors (gmfe) for scaled CLint,u (mL/min/kg) were 10.4 for human hepatocytes, 5.6 for human liver cytosols, and 5.0 for human liver S9, respectively. Application of these gmfe's as empirical scaling factors improved predictions (45%-57% within twofold of observed) compared with no correction (11%-27% within twofold), with the scaling factors qualified by leave-one-out cross-validation. A road map for quantitative translation was then proposed following a critical evaluation on the in vitro and clinical methodology to estimate in vivo fmAO In conclusion, the study provides the most robust system-specific empirical scaling factors to date as a pragmatic approach for the prediction of in vivo CLint,u,AO in the early stages of drug development. SIGNIFICANCE STATEMENT: Confidence remains low when predicting in vivo clearance of AO substrates using in vitro systems, leading to de-prioritization of AO substrates from the drug development pipeline to mitigate risk of unexpected and costly in vivo impact. The current study establishes a set of empirical scaling factors as a pragmatic tool to improve predictability of in vivo AO clearance. Developing clinical pharmacology strategies for AO substrates by utilizing mass balance/clinical drug-drug interaction data will help build confidence in fmAO.

Analysis of the Bile Acid Composition in a Fibroblast Growth Factor 19-Expressing Liver-Humanized Mouse Model and Its Use for CYP3A4-Mediated Drug-Drug Interaction Studies.

Numerous biomedical applications have been described for liver-humanized mouse models, such as in drug metabolism or drug-drug interaction (DDI) studies. However, the strong enlargement of the bile acid (BA) pool due to lack of recognition of murine intestine-derived fibroblast growth factor-15 by human hepatocytes and a resulting upregulation in the rate-controlling enzyme for BA synthesis, cytochrome P450 (CYP) 7A1, may pose a challenge in interpreting the results obtained from such mice. To address this challenge, the human fibroblast growth factor-19 (FGF19) gene was inserted into the Fah-/- , Rag2-/- , Il2rg-/- NOD (FRGN) mouse model, allowing repopulation with human hepatocytes capable of responding to FGF19. While a decrease in CYP7A1 expression in human hepatocytes from humanized FRGN19 mice (huFRGN19) and a concomitant reduction in BA production was previously shown, a detailed analysis of the BA pool in these animals has not been elucidated. Furthermore, there are sparse data on the use of this model to assess potential clinical DDI. In the present work, the change in BA composition in huFRGN19 compared with huFRGN control animals was systematically evaluated, and the ability of the model to recapitulate a clinically described CYP3A4-mediated DDI was assessed. In addition to a massive reduction in the total amount of BA, FGF19 expression in huFRGN19 mice resulted in significant changes in the profile of various primary, secondary, and sulfated BAs in serum and feces. Moreover, as observed clinically, administration of the pregnane X receptor agonist rifampicin reduced the oral exposure of the CYP3A4 substrate triazolam. SIGNIFICANCE STATEMENT: Transgenic expression of FGF19 normalizes the unphysiologically high level of bile acids in a chimeric liver-humanized mouse model and leads to massive changes in bile acid composition. These adaptations could overcome one of the potential impediments in the use of these mouse models for drug-drug interaction studies.

Dissecting Parameters Contributing to the Underprediction of Aldehyde Oxidase-Mediated Metabolic Clearance of Drugs.

We investigated the effect of variability and instability in aldehyde oxidase (AO) content and activity on the scaling of in vitro metabolism data. AO content and activity in human liver cytosol (HLC) and five recombinant human AO preparations (rAO) were determined using targeted proteomics and carbazeran oxidation assay, respectively. AO content was highly variable as indicated by the relative expression factor (REF; i.e., HLC to rAO content) ranging from 0.001 to 1.7 across different in vitro systems. The activity of AO in HLC degrades at a 10-fold higher rate in the presence of the substrate as compared with the activity performed after preincubation without substrate. To scale the metabolic activity from rAO to HLC, a protein-normalized activity factor (pnAF) was proposed wherein the activity was corrected by AO content, which revealed up to sixfold higher AO activity in HLC versus rAO systems. A similar value of pnAF was observed for another substrate, ripasudil. Physiologically based pharmacokinetic (PBPK) modeling revealed a significant additional clearance (CL; 66%), which allowed for the successful prediction of in vivo CL of four other substrates, i.e., O-benzyl guanine, BIBX1382, zaleplon, and zoniporide. For carbazeran, the metabolite identification study showed that the direct glucuronidation may be contributing to around 12% elimination. Taken together, this study identified differential protein content, instability of in vitro activity, role of additional AO clearance, and unaccounted metabolic pathways as plausible reasons for the underprediction of AO-mediated drug metabolism. Consideration of these factors and integration of REF and pnAF in PBPK models will allow better prediction of AO metabolism. SIGNIFICANCE STATEMENT: This study elucidated the plausible reasons for the underprediction of aldehyde oxidase (AO)-mediated drug metabolism and provided recommendations to address them. It demonstrated that integrating protein content and activity differences and accounting for the loss of AO activity, as well as consideration of extrahepatic clearance and additional pathways, would improve the in vitro to in vivo extrapolation of AO-mediated drug metabolism using physiologically based pharmacokinetic modeling.

Comparisons between human and rodent hepatic glutathione S-Transferase activities reveal sex and species differences.

Glutathione S-transferases (GSTs) are conjugating enzymes involved in drug metabolism, antioxidant defence, and cell signalling. Herein, we investigated hepatic GST conjugation in several mouse and rat strains, including both sexes, with a direct comparison to humans.Using general and isoform-selective substrates, all mouse strains had significantly greater activities than humans for total cytosolic GST, GST-M, GST-T, and microsomal GST activities. Some strains had significantly greater GST-P activities compared to humans. Sex differences between males and females were evident in all strains for total cytosolic GST, GST-M, and GST-P, and sex differences in GST-T and microsomal GST activities within strains were noted.All rats had significantly greater activities than humans for GST-M and GST-T; only some strains were significantly greater than humans for GST-P, total cytosolic GST, and microsomal GST. Sex differences within strains showed significantly greater GST-M and GST-T activities in males compared to females. Select strains showed sex differences for total cytosolic and microsomal GST activities; there were no sex differences in GST-P activities.Significant differences in glutathione conjugation between humans and rodents exist, including sex differences. This highlights the need for careful animal selection in pre-clinical studies where GSTs are the primary metabolic pathway.

Fluoride and gallein inhibit polyphosphate accumulation by oral pathogen Rothia dentocariosa.

The uptake and storage of extracellular orthophosphate (Pi) by polyphosphate (polyP) accumulating bacteria may contribute to mineral dissolution in the oral cavity. To test the effect of potential inhibitors of polyP kinases on Rothia dentocariosa, gallein (0, 25, 50, and 100 µM) and fluoride (0, 50, and 100 ppm) were added to R. dentocariosa cultures grown in brain-heart infusion broth. At a late log growth phase (8 h), extracellular Pi was measured using an ascorbic acid assay, and polyP was isolated from bacterial cells treated with RNA/DNAases using a neutral phenol/chloroform extraction. Extracts were hydrolyzed and quantified as above. Gallein and fluoride had minor effects on bacterial growth with NaF having a direct effect on media pH. Gallein (≥25 µM) and fluoride (≥50 ppm) attenuated the bacterial drawdown of extracellular Pi by 56.7% (P < 0.05) and 37.3% (P < 0.01). There was a corresponding polyP synthesis decrease of 73.2% (P < 0.0001) from gallein and 83.1% (P < 0.0001) from fluoride. Attenuated total reflectance-Fourier-transform infrared spectroscopy validated the presence of polyP and its reduced concentration in R. dentocariosa bacterial cells following gallein and fluoride treatment. Rothia dentocariosa can directly change extracellular Pi and accumulate intracellular polyP, but the mechanism is attenuated by gallein and NaF.

Weighted Blanket Use as an Alternative to Protective Stabilization During Moderate Sedation.

Purpose: The purpose of this retrospective cross-sectional study was to examine protective stabilization (PS) patterns before and after the availability of weighted blankets (WBs) as a behavioral guidance approach during in-office dental moderate sedation. Methods: A retrospective chart review evaluated pediatric patient sedation records after six-pound lead-free WBs were introduced into the dental clinic and compared clinical outcomes to a time before WBs were available. Multivariable logistic regression analyses assessed variables associated with the occurrence of PS use during a sedation visit. Results: PS (PS) usage decreased from 78.7 percent before to 32.8 percent after the availability of WBs during sedation visits (chi-square, P<0.001). Increase in age (adjusted odds ratio [OR] equals 0.69, 95 percent confidence interval [95% CI] equals 0.53 to 0.90, P=0.006) and WB use reduced PS management (adjusted OR equals 0.067, 95% CI equals 0.020 to 0.22, P<0.001). Body mass index, gender, treatment amount, and sedation regimen did not predict the occurrence of PS. The number of completed teeth treated was not found to be statistically different between cases managed with PS versus those managed without restraint. Children managed with PS but without WBs had statistically higher heart rate changes (20.26±23.17) during treatment than children managed without restraint (8.12±15.15). Conclusions: An increase in age and weighted blanket use was associated with a reduction in the occurrence of protective stabilization during moderate sedation dental visits at the university pediatric dental clinic. Clinical practice sedation protocols should consider weighted blanket use as an alternative to PS.

Short Signature rpoB Gene Sequence to Differentiate Species in Mycobacterium abscessus Group.

Mycobacterium abscessus group (MAG) are rapidly growing acid-fast bacteria that consist of three closely related species: M. abscessus (Ma), M. bolletii (Mb), and M. massiliense (Mm). Differentiation of these species can be difficult but is increasingly requested owing to recent infectious outbreaks and their differential drug resistance. We developed a novel and rapid pyrosequencing method using short signature sequences (35 to 45 bp) at a hypervariable site in the rpoB gene to differentiate the three MAG species, along with M. chelonae (Mc), and M. immunogenum (Mi). This method was evaluated using 111 M. chelonae-abscessus complex (MCAC) isolates, including six reference strains. All isolates were successfully differentiated to the species level (69 Ma, four Mb, six Mm, 23 Mc, and nine Mi). The species identifications by this method had 100% agreement with Sanger sequencing as well as an in-silico rpoB typing method. This short signature sequencing (SSS) method is rapid (6 to 7 h), accurately differentiates MAG species, and is useful for informing antimicrobial therapy decision. IMPORTANCE Mycobacterium abscessus group (MAG) are rapidly growing acid-fast bacteria that include three species: M. abscessus, M. massiliense, and M. bolletii. These species are among the leading causes of nontuberculosis mycobacteria infections in humans but difficult to differentiate using commonly used methods. The differences of drug resistance among the species shape the treatment regimens and make it significant for them to be differentiated accurately and quickly. We developed and evaluated a novel short signature sequencing (SSS) method utilizing a gene called rpoB to differentiate the three MAG species, as well as other two species (M. chelonae and M. immunogenum). The identification results had 100% agreement with both the reference method of Sanger sequencing and rpoB typing method via a computer-simulated analysis. This SSS method was accurate and quick (6 to 7 h) for species differentiation, which will benefit patient care. The technology used for this method is affordable and easy to operate.

Application of empirical scalars to enable early prediction of human hepatic clearance using IVIVE in drug discovery: an evaluation of 173 drugs.

The utilization of in vitro data to predict drug pharmacokinetics (PK) in vivo has been a consistent practice in early drug discovery for decades. However, its success is hampered by mispredictions attributed to uncharacterized biological phenomena/experimental artifacts. Predicted drug clearance (CL) from experimental data (i.e. hepatocyte intrinsic clearance: CLint, fraction unbound in plasma: fu,p) is often systematically underpredicted using the well-stirred model (WSM). The objective of this study was to evaluate using empirical scalars in the WSM to correct for CL mispredictions. Drugs (N=28) were used to generate numerical scalars on CLint (α), and fu,p (ß) to minimize the error (AAFE) for CL predictions. These scalars were validated using an additional dataset (N=28 drugs) and applied to a non-redundant AstraZeneca (AZ) dataset available in the literature (N=117 drugs) for a total of 173 compounds. CL predictions using the WSM were improved for most compounds using an α value of 3.66 (~64%<2-fold) compared to no scaling (~46%<2-fold). Similarly, using a ß value of 0.55 or combination of α and ß scalars (values of 1.74 and 0.66, respectively) resulted in a similar improvement in predictions (~64%<2-fold and ~65%<2-fold, respectively). For highly bound compounds (fu,p{less than or equal to}0.01), AAFE was substantially reduced across all scaling methods. Using the ß scalar alone or a combination of α and ß appeared optimal; and produce larger magnitude corrections for highly-bound compounds. Some drugs are still disproportionally mispredicted, however the improvements in prediction error and simplicity of applying these scalars suggests its utility for early-stage CL predictions. Significance Statement In early drug discovery, prediction of human clearance using in vitro experimental data plays an essential role in triaging compounds prior to in vivo studies. These predictions have been systematically underestimated. Here we introduce empirical scalars calibrated on the extent of plasma protein binding that appear to improve clearance prediction across multiple datasets. This approach can be used in early phases of drug discovery prior to the availability of pre-clinical data for early quantitative predictions of human clearance.

Untargeted metabolomics identifies the potential role of monocarboxylate transporter 6 (MCT6/SLC16A5) in lipid and amino acid metabolism pathways.

Monocarboxylate transporter 6 (MCT6; SLC16A5) is an orphan transporter protein with expression in multiple tissues. The endogenous function of MCT6 related to human health and disease remains unknown. Our previous transcriptomic and proteomic analyses in Mct6 knockout (KO) mice suggested that MCT6 may play a role in lipid and glucose homeostasis, but additional evidence is required. Thus, the objective of this study was to further explore the impact of MCT6 on metabolic function using untargeted metabolomic analysis in Mct6 KO mice. The plasma from male and female mice and livers from male mice were submitted for global metabolomics analysis to assess the relative changes in endogenous small molecules across the liver and systemic circulation associated with absence of Mct6. More than 782 compounds were detected with 101 and 51 metabolites significantly changed in plasma of male and female mice, respectively, and 100 metabolites significantly changed in the livers of male mice (p < .05). Significant perturbations in lipid metabolism were annotated in the plasma and liver metabolome, with additional alterations in the amino acid metabolism pathway in plasma samples from male and female mice. Elevated lipid diacylglycerol and altered fatty acid metabolite concentrations were found in liver and plasma samples of male Mct6 KO mice. Significant reduction of N-terminal acetylated amino acids was found in plasma samples of male and female Mct6 KO mice. In summary, the present study confirmed the significant role of MCT6 in lipid and amino acid homeostasis, suggesting its contribution in metabolic diseases.

Methacrylate Polymers With "Flipped External" Ester Groups: A Review.

Current resin composites have favorable handling and upon polymerization initial physical properties that allow for efficient material replacement of removed carious tooth structure. Dental resin composites have long term durability limitations due to the hydrolysis of ester bonds within the methacrylate based polymer matrix. This article outlines the importance of ester bonds positioned internal to the carbon-carbon double bond in current methacrylate monomers. Water and promiscuous salivary/bacterial esterase activity can initiate ester bond hydrolysis that can sever the polymer backbone throughout the material. Recent studies have custom synthesized, with the latest advances in modern organic chemical synthesis, a novel molecule named ethylene glycol bis (ethyl methacrylate) (EGEMA). EGEMA was designed to retain the reactive acrylate units. Upon intermolecular polymerization of vinyl groups, EGEMA ester groups are positioned outside the backbone of the polymer chain. This review highlights investigation into the degradation resistance of EGEMA using buffer, esterase, and microbial storage assays. Material samples of EGEMA had superior final physical and mechanical properties than traditional ethylene glycol dimethacrylate (EGDMA) in all degradation assays. Integrating bioinformatics-based biodegradation predictions to the experimental results of storage media analyzed by LC/GC-MS revealed that hydrolysis of EGEMA generated small amounts of ethanol while preserving the strength bearing polymer backbone. Prior studies support investigation into additional custom synthesized methacrylate polymers with "flipped external" ester groups. The long term goal is to improve clinical durability compared to current methacrylates while retaining inherent advantages of acrylic based chemistry, which may ease implementation of these novel methacrylates into clinical practice.

Utilizing a degradation prediction pathway system to understand how a novel methacrylate derivative polymer with flipped external ester groups retains physico-mechanical properties following esterase exposure.

OBJECTIVE: The region of failure for current methacrylates (i.e. derivatives of acrylates) are ester bond linkages that hydrolyze in the presence of salivary and bacterial esterases that break the polymer network backbone. This effect decreases the mechanical properties of methacrylate-based materials. METHODS: The ethylene glycol dimethacrylate (EGDMA) or novel ethylene glycol ethyl methacrylate (EGEMA) discs were prepared using 40 µL of the curing mixture containing photo/co-initiators for 40 s in a PTFE mold at 1000 mW/cm2. The degree of conversion was used as a quality control measure for the prepared discs, followed by physical, mechanical, and chemical characterization of discs properties before and after cholesterol esterase treatment. RESULTS: After 9 weeks of standardized cholesterol esterase (CEase) exposure, EGDMA discs showed exponential loss of material (p = 0.0296), strength (p = 0.0014) and increased water sorption (p = 0.0002) compared to EGEMA discs. We integrated a degradation prediction pathway system to LC/MS and GC/MS analyses to elucidate the degradation by-products of both EGEMA and EGDMA polymers. GC/MS analysis demonstrated that the esterase catalysis was directed to central polymer backbone breakage, producing ethylene glycol, for EGDMA, and to side chain breakage, producing ethanol, for EGEMA. The flipped external ester group linkage design is attributed to EGEMA showing higher resistance to esterase biodegradation and changes in mechanical and physical properties than EGDMA. SIGNIFICANCE: EGEMA is a potential substitute for common macromer diluents, such as EGDMA, based on its resistance to biodegradation effects. This work inspires the flipped external group design to be applied to analogs of current larger, hydrophobic strength bearing macromers used in future dental material formulations.

A novel methacrylate derivative polymer that resists bacterial cell-mediated biodegradation.

This study tests biodegradation resistance of a custom synthesized novel ethylene glycol ethyl methacrylate (EGEMA) with ester bond linkages that are external to the central polymer backbone when polymerized. Ethylene glycol dimethacrylate (EGDMA) with internal ester bond linkages and EGEMA discs were prepared in a polytetrafluoroethylene (PTFE) mold using 40 µl macromer and photo/co-initiator mixture cured for 40 s at 1000 mW/cm2 . The discs were stored in the constant presence of Streptococcus mutans (S. mutans) in Todd Hewitt Yeast + Glucose (THYE+G) media up to 9 weeks (n = 8 for each macromer type) and physical/mechanical properties were assessed. Initial measurements EGEMA versus EGDMA polymer discs showed equivalent degree of conversion (45.69% ± 2.38 vs. 46.79% ± 4.64), diametral tensile stress (DTS; 8.12± 2.92 MPa vs. 6.02 ± 1.48 MPa), and low subsurface optical defects (0.41% ± 0.254% vs. 0.11% ± 0.074%). The initial surface wettability (contact angle) was slightly higher (p ≤ .012) for EGEMA (62.02° ± 3.56) than EGDMA (53.86° ± 5.61°). EGDMA showed higher initial Vicker's hardness than EGEMA (8.03 ± 0.88 HV vs. 5.93 ± 0.69 HV; p ≤ .001). After 9 weeks of S. mutans exposure, EGEMA (ΔDTS-1.30 MPa) showed higher resistance to biodegradation effects with a superior DTS than EGDMA (ΔDTS-6.39 MPa) (p = .0039). Visible and scanning electron microscopy images of EGEMA show less surface cracking and defects than EGDMA. EGDMA had higher loss of material (18.9% vs. 8.5%, p = .0009), relative changes to fracture toughness (92.5% vs. 49.2%, p = .0022) and increased water sorption (6.1% vs. 1.9%, p = .0022) compared to EGEMA discs. The flipped external ester group linkage design is attributed to EGEMA showing higher resistance to bacterial degradation effects than an internal ester group linkage design methacrylate.

Recovery and Its Dynamics of Filamentous Fungi from Clinical Specimen Cultures: An Extensive Study.

The culture method remains vital in diagnosing fungal infections, but extensive data-based evaluation of the method, especially for filamentous fungi (molds), is minimal. The purpose of this study was to characterize mold recoveries from fungal cultures and the impact of media and incubation duration. Clinical specimens for fungal cultures were submitted primarily from the eastern and central United States, and mold isolation data were prospectively collected and analyzed. A total of 1,821 molds in 59 genera were isolated from 1,687 positive specimens, accounting for approximately 5.6% of our cohort of 30,000 fungal cultures. Within 2 weeks, nearly 90% of molds and 97.3% of Aspergillus fumigatus complex were recovered (>95% confidence interval [CI]). All Mucorales fungi were recovered within 11 days of incubation. The recovery peak time was day 3 for Mucorales fungi, day 4 for hyaline molds, day 5 for dematiaceous molds, and day 7 for Onygenales fungi. The recovery of Histoplasma capsulatum and Trichophyton species in the fourth week of incubation reveals that a 3-week incubation time is insufficient. Inhibitory mold agar was the best medium for recovering all mold types among all tested specimen types, yielding nearly 78% of mold growth overall, indicating the necessity of selective medium for fungal cultures. IMPORTANCE Fungal culture is the gold standard method of diagnosing fungal infections, but important information, such as the impact of media and incubation times on fungal recovery, is not well documented. This study addressed these gaps using extensive data-based evaluation focused on molds. We identified the best medium types and incubation times for better fungal culture practice. We analyzed 1,821 molds from 1,687 positive specimens in our cohort of approximately 30,000 fungal cultures. Mold recovery peaked between 3 and 7 days of incubation, dependent upon the type of mold. Some well-defined fungal pathogens, such as Histoplasma capsulatum and Trichophyton species, were isolated in the fourth week of incubation. Inhibitory mold agar was identified as the best medium for recovering all mold types among all tested specimen sources. As we are aware, this is the largest study of fungal culture methods and supports 4 weeks of incubation for optimal mold recovery.

The experiences of therapists providing psychological treatment for adults with depression and intellectual disabilities as part of a randomised controlled trial.

BACKGROUND: Health professionals were trained to deliver adapted psychological interventions for depression to people with learning disabilities and depression alongside a supporter. Exploring the delivery of psychological interventions can help increase access to therapy. METHOD: Twenty-seven participants took part in six focus groups, and the data were subject to a Framework Analysis. RESULTS: The structure and focus of the manualised therapies, and the use of specific techniques were perceived as key to service-user engagement. Supporters' involvement was valued by therapists if they had a good relationship and regular contact with the individual they supported. Regular clinical supervision was regarded as vital in understanding their role, assessing progress and delivering the interventions. CONCLUSIONS: The findings highlight that health professionals can embrace a focussed therapeutic role and increase access to psychological therapies for people with intellectual disabilities.

Conceptual Model for Using Imidazoline Derivative Solutions in Pulpal Management.

Alpha-adrenergic agonists, such as the Imidazoline derivatives (ImDs) of oxymetazoline and xylometazoline, are highly effective hemostatic agents. ImDs have not been widely used in dentistry but their use in medicine, specifically in ophthalmology and otolaryngology, warrants consideration for pulpal hemostasis. This review presents dental healthcare professionals with an overview of ImDs in medicine. ImD solutions have the potential to be more effective and biocompatible than existing topical hemostatic compounds in pulpal management. Through a comprehensive analysis of the pharmacology of ImDs and the microphysiology of hemostasis regulation in oral tissues, a conceptual model of pulpal management by ImD solutions is presented.

Evaluation of a Competitive Equilibrium Dialysis Approach for Assessing the Impact of Protein Binding on Clearance Predictions.

Fraction unbound (fu) is an important consideration when characterizing the ADME properties of drug candidates. For highly bound compounds, there can be low confidence in quantifying fu introducing uncertainty in certain parameter estimations. Specifically, predictions of clearance (CL) rely on accurate fu values measured in plasma (fu,p) and microsomes (fu,mic) to scale in vitro intrinsic CL to in vivo CL. However, determining the ratio of fu,p/fu,mic may circumvent the need to measure discrete binding values. The purpose of this study was to evaluate a plasma-to-microsome competitive equilibrium dialysis (cED) method to determine fu,p/fu,mic ratio (fuR) for nine physiochemically-distinct compounds, and to investigate the impact of altering microsomal concentrations on fuR. The values of fuR were comparable to ratios calculated from discretely measured fu,p and fu,mic values. Furthermore, increasing microsomal concentrations increased fuR for basic and neutral compounds. When using fuR values, there was a good in vitro-in vivo correlation (IVIVC) (≤3-fold observed in vivo CL). These results suggest that the cED method used to determine fuR may be an appropriate, alternative IVIVC approach. Application of cED may extend beyond IVIVC of CL to evaluate other parameters such as species differences in protein binding and free tissue to plasma ratios.

Parameterization of Microsomal and Cytosolic Scaling Factors: Methodological and Biological Considerations for Scalar Derivation and Validation.

Mathematical models that can predict the kinetics of compounds have been increasingly adopted for drug development and risk assessment. Data for these models may be generated from in vitro experimental systems containing enzymes contributing to metabolic clearance, such as subcellular tissue fractions including microsomes and cytosol. Extrapolation from these systems is facilitated by common scaling factors, known as microsomal protein per gram (MPPG) and cytosolic protein per gram (CPPG). Historically, parameterization of MPPG and CPPG has employed the use of recovery factors, commonly benchmarked to cytochromes P450 which work well in some contexts, but could be problematic for other enzymes. Here, we propose absolute quantification of protein content and supplementary assays to evaluate microsomal/cytosolic purity that should be employed. Examples include calculation of microsomal latency by mannose-6-phosphatase activity and immunoblotting of subcellular fractions with fraction-specific markers. Further considerations include tissue source, as disease states can affect enzyme expression and activity, and the methodology used for scalar parameterization. Regional- and organ-specific expression of enzymes, in addition to differences in organ physiology, is another important consideration. Because most efforts have focused on the liver that is, for the most part, homogeneous, derived scalars may not capture the heterogeneity of other major tissues contributing to xenobiotic metabolism including the kidneys and small intestine. Better understanding of these scalars, and how to appropriately derive them from extrahepatic tissues can provide support to the inferences made with physiologically based pharmacokinetic modeling, increase its accuracy in characterizing in vivo drug pharmacokinetics, and improve confidence in go-no-go decisions for clinical trials.

A Novel Dental Polymer with a Flipped External Ester Group Design that Resists Degradation via Polymer Backbone Preservation.

Current dental sealants with methacrylate based chemistry are prone to hydrolytic degradation. A conventional ethylene glycol dimethacrylate (EGDMA) was compared to a novel methacrylate monomer with a flipped external ester group (ethylene glycol ethyl methacrylate - EGEMA) that was designed to resist polymer degradation effects. Fourier transform infrared spectroscopy and water contact angle confirmed a comparable degree of initial conversion and surface wettability for EGDMA and EGEMA. EGDMA disks initially performed better compared to EGEMA as suggested by higher surface hardness and 1.5 times higher diametral tensile strength (DTS). After 15 weeks of hydrolytic and accelerated aging, EGDMA and EGEMA DTS was reduced by 88% and 44% respectively. This accelerated aging model resulted in 3.3 times higher water sorption for EDGMA than EGEMA disks. EGDMA had an increase in grain boundary defects and visible erosion sites with accelerated aging, while for EGEMA the changes were not significant.