Association of dietary habits with general and abdominal obesity in Korean children and adolescents: cluster analysis of nationwide population survey data.

Introduction: Childhood obesity is a growing global health concern, but few studies have investigated dietary factors specifically related to obesity and abdominal obesity in children and adolescents. Herein, we aimed to identify the dietary factors affecting childhood obesity in Korean children and adolescents. Methods: Data from the Korea National Health and Nutrition Survey (KNHANES) VIII were analyzed using K-means clustering analysis to identify distinct clusters based on nine variables related to dietary habit, nutritional status, and nutritional education. Multiple logistic regression analysis was used to examine the association between incident obesity risk and the different clusters. We enrolled 2,290 participants aged 6-18 years, and separated them into two distinct clusters; Healthy and Unhealthy Dietary Habit Groups, clusters 1 and 2, respectively. Results: Cluster 1 was characterized by a lower obesity prevalence, healthier dietary habits (regular breakfast consumption; fruit and vegetable, reduced total energy, and lower protein and fat intakes), and greater nutritional education than Cluster 2. After adjusting for confounders, compared with Cluster 1, Cluster 2 demonstrated a significantly higher prevalence (OR [95% CI]) of both general and abdominal obesity (1.49 [1.05-2.13], p=0.027 and 1.43 [1.09-1.88], p=0.009). Discussion: Maintaining optimal dietary quality and patterns are crucial to prevent childhood obesity. Further research is warranted to explore specific dietary interventions tailored to different clusters to effectively address childhood obesity.

Comparative study on structural and functional brain differences in mild cognitive impairment patients with tinnitus.

Objective: Tinnitus may be associated with various brain changes. However, the degenerative changes in patients with tinnitus have not been extensively investigated. We aimed to evaluate degenerative, structural, and functional brain changes in patients with mild cognitive impairment (MCI) who also suffer from tinnitus. Materials and methods: This study included participants aged 60 to 80 years with MCI and a hearing level better than 40 dB. The participants were classified into two groups: MCI with tinnitus (MCI-T) and MCI without tinnitus (MCI-NT). All patients underwent Tinnitus Handicap Inventory (THI), 3 T brain MRI, F18-florapronol PET, and F18-FDG PET. Results: The MCI-T group exhibited higher ß-amyloid deposition in the superior temporal gyrus, temporal pole, and middle temporal gyrus compared to the MCI-NT group (p < 0.05 for all). Additionally, the MCI-T group showed increased metabolism in the inferior frontal gyrus, insula, and anterior cingulate cortex (ACC) (p < 0.005 for all). The THI score was strongly correlated with increased volume in the insula, ACC, superior frontal gyrus, supplementary motor area, white matter near the hippocampus, and precentral gyrus (p < 0.05 for all). Moreover, the MCI-T group demonstrated higher metabolic activity in the default mode network (DMN) and lower activity in the executive control network (ECN) (p < 0.05 for all). In the MCI-T group, the posterior DMN was positively correlated with the visual network and negatively with the ECN, whereas in the MCI-NT group, it correlated positively with the ECN. Conclusion: The MCI-T group exhibited greater ß-amyloid accumulation in the auditory cortex and more extensive changes across various brain networks compared with the MCI-NT group, potentially leading to diverse clinical symptoms such as dementia with semantic deficits or depression. Tinnitus in MCI patients may serve as a biomarker for degenerative changes in the temporal lobe and alterations in brain network dynamics.

Discovery of novel disease-causing mutation in SSBP1 and its correction using adenine base editor to improve mitochondrial function.

Mutations in nuclear genes regulating mitochondrial DNA (mtDNA) replication are associated with mtDNA depletion syndromes. Using whole-genome sequencing, we identified a heterozygous mutation (c.272G>A:p.Arg91Gln) in single-stranded DNA-binding protein 1 (SSBP1), a crucial protein involved in mtDNA replisome. The proband manifested symptoms including sensorineural deafness, congenital cataract, optic atrophy, macular dystrophy, and myopathy. This mutation impeded multimer formation and DNA-binding affinity, leading to reduced efficiency of mtDNA replication, altered mitochondria dynamics, and compromised mitochondrial function. To correct this mutation, we tested two adenine base editor (ABE) variants on patient-derived fibroblasts. One variant, NG-Cas9-based ABE8e (NG-ABE8e), showed higher editing efficacy (≤30%) and enhanced mitochondrial replication and function, despite off-target editing frequencies; however, risks from bystander editing were limited due to silent mutations and off-target sites in non-translated regions. The other variant, NG-Cas9-based ABE8eWQ (NG-ABE8eWQ), had a safer therapeutic profile with very few off-target effects, but this came at the cost of lower editing efficacy (≤10% editing). Despite this, NG-ABE8eWQ-edited cells still restored replication and improved mtDNA copy number, which in turn recovery of compromised mitochondrial function. Taken together, base editing-based gene therapies may be a promising treatment for mitochondrial diseases, including those associated with SSBP1 mutations.

Full-cycle study on developing a novel structured micromixer and evaluating the nanoparticle products as mRNA delivery carriers.

Achieving precise control of nanoparticle size while maintaining consistency and high uniformity is of paramount importance for improving the efficacy of nanoparticle-based therapies and minimizing potential side effects. Although microfluidic technologies are widely used for reliable nanoparticle synthesis, they face challenges in meeting critical homogeneity requirements, mainly due to imperfect mixing efficiency. Furthermore, channel clogging during continuous operation presents a significant obstacle in terms of quality control, as it progressively impedes the mixing behavior necessary for consistent nanoparticle production for therapeutic delivery and complicates the scaling-up process. This study entailed the development of a 3D-printed novel micromixer embedded with hemispherical baffle microstructures, a dual vortex mixer (DVM), which integrates Dean vortices to generate two symmetrical counter-rotating intensified secondary flows. The DVM with a relatively large mixer volume showed rapid mixing characteristics even at a flow rate of several mL min-1 and produced highly uniform lipids, liposomes, and polymer nanoparticles in a size range (50-130 nm) and polydispersity index (PDI) values below 0.15. For the evaluation of products, SARS-CoV-2 Spike mRNA-loaded lipid nanoparticles were examined to verify protein expression in vitro and in vivo using firefly luciferase (FLuc) mRNA. This showed that the performance of the system is comparable to that of a commercial toroidal mixer. Moreover, the vigorous in-situ dispersion of nanoparticles by harnessing the power of vortex physically minimizes the occurrence of aggregation, ensuring consistent production performance without internal clogging of a half-day operation and facilitating quality control of the nanoparticles at desired scales.

Updates on Genetic Hearing Loss: From Diagnosis to Targeted Therapies.

Sensorineural hearing loss (SNHL) is the most common sensory disorder, with a high Mendelian genetic contribution. Considering the genotypic and phenotypic heterogeneity of SNHL, the advent of next-generation sequencing technologies has revolutionized knowledge on its genomic architecture. Nonetheless, the conventional application of panel and exome sequencing in real-world practice is being challenged by the emerging need to explore the diagnostic capability of whole-genome sequencing, which enables the detection of both noncoding and structural variations. Small molecules and gene therapies represent good examples of how breakthroughs in genetic understanding can be translated into targeted therapies for SNHL. For example, targeted small molecules have been used to ameliorate autoinflammatory hearing loss caused by gain-of-function variants of NLRP3 and inner ear proteinopathy with OSBPL2 variants underlying dysfunctional autophagy. Strikingly, the successful outcomes of the first-in-human trial of OTOF gene therapy highlighted its potential in the treatment of various forms of genetic hearing loss. clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies are currently being developed for site-specific genome editing to treat human genetic disorders. These advancements have led to an era of genotype- and mechanism-based precision medicine in SNHL practice.

CRISPR-based editing strategies to rectify EYA1 complex genomic rearrangement linked to haploinsufficiency.

Pathogenic structure variations (SVs) are associated with various types of cancer and rare genetic diseases. Recent studies have used Cas9 nuclease with paired guide RNAs (gRNAs) to generate targeted chromosomal rearrangements, focusing on producing fusion proteins that cause cancer, whereas research on precision genome editing for rectifying SVs is limited. In this study, we identified a novel complex genomic rearrangement (CGR), specifically an EYA1 inversion with a deletion, implicated in branchio-oto-renal/branchio-oto syndrome. To address this, two CRISPR-based approaches were tested. First, we used Cas9 nuclease and paired gRNAs tailored to the patient's genome. The dual CRISPR-Cas9 system induced efficient correction of paracentric inversion in patient-derived fibroblast, and effectively restored the expression of EYA1 mRNA and protein, along with its transcriptional activity required to regulate the target gene expression. Additionally, we used CRISPR activation (CRISPRa), which leads to the upregulation of EYA1 mRNA expression in patient-derived fibroblasts. Moreover, CRISPRa significantly improved EYA1 protein expression and transcriptional activity essential for target gene expression. This suggests that CRISPRa-based gene therapies could offer substantial translational potential for approximately 70% of disease-causing EYA1 variants responsible for haploinsufficiency. Our findings demonstrate the potential of CRISPR-guided genome editing for correcting SVs, including those with EYA1 CGR linked to haploinsufficiency.

Expanding Genotype-Phenotype Correlation of CLCNKA and CLCNKB Variants Linked to Hearing Loss.

The ClC-K channels CLCNKA and CLCNKB are crucial for the transepithelial transport processes required for sufficient urinary concentrations and sensory mechanoelectrical transduction in the cochlea. Loss-of-function alleles in these channels are associated with various clinical phenotypes, ranging from hypokalemic alkalosis to sensorineural hearing loss (SNHL) accompanied by severe renal conditions, i.e., Bartter's syndrome. Using a stepwise genetic approach encompassing whole-genome sequencing (WGS), we identified one family with compound heterozygous variants in the ClC-K channels, specifically a truncating variant in CLCNKA in trans with a contiguous deletion of CLCNKA and CLCNKB. Breakpoint PCR and Sanger sequencing elucidated the breakpoint junctions derived from WGS, and allele-specific droplet digital PCR confirmed one copy loss of the CLCNKA_CLCNKB contiguous deletion. The proband that harbors the CLCNKA_CLCNKB variants is characterized by SNHL without hypokalemic alkalosis and renal anomalies, suggesting a distinct phenotype in the ClC-K channels in whom SNHL predominantly occurs. These results expanded genotypes and phenotypes associated with ClC-K channels, including the disease entities associated with non-syndromic hearing loss. Repeated identification of deletions across various extents of CLCNKA_CLCNKB suggests a mutational hotspot allele, highlighting the need for an in-depth analysis of the CLCNKA_CLCNKB intergenic region, especially in undiagnosed SNHL patients with a single hit in CLCNKA.

Novel autosomal dominant TMC1 variants linked to hearing loss: insight into protein-lipid interactions.

BACKGROUND: TMC1, which encodes transmembrane channel-like protein 1, forms the mechanoelectrical transduction (MET) channel in auditory hair cells, necessary for auditory function. TMC1 variants are known to cause autosomal dominant (DFNA36) and autosomal recessive (DFNB7/11) non-syndromic hearing loss, but only a handful of TMC1 variants underlying DFNA36 have been reported, hampering analysis of genotype-phenotype correlations. METHODS: In this study, we retrospectively reviewed 338 probands in an in-house database of genetic hearing loss, evaluating the clinical phenotypes and genotypes of novel TMC1 variants associated with DFNA36. To analyze the structural impact of these variants, we generated two structural models of human TMC1, utilizing the Cryo-EM structure of C. elegans TMC1 as a template and AlphaFold protein structure database. Specifically, the lipid bilayer-embedded protein database was used to construct membrane-embedded models of TMC1. We then examined the effect of TMC1 variants on intramolecular interactions and predicted their potential pathogenicity. RESULTS: We identified two novel TMC1 variants related to DFNA36 (c.1256T > C:p.Phe419Ser and c.1444T > C:p.Trp482Arg). The affected subjects had bilateral, moderate, late-onset, progressive sensorineural hearing loss with a down-sloping configuration. The Phe419 residue located in the transmembrane domain 4 of TMC1 faces outward towards the channel pore and is in close proximity to the hydrophobic tail of the lipid bilayer. The non-polar-to-polar variant (p.Phe419Ser) alters the hydrophobicity in the membrane, compromising protein-lipid interactions. On the other hand, the Trp482 residue located in the extracellular linker region between transmembrane domains 5 and 6 is anchored to the membrane interfaces via its aromatic rings, mediating several molecular interactions that stabilize the structure of TMC1. This type of aromatic ring-based anchoring is also observed in homologous transmembrane proteins such as OSCA1.2. Conversely, the substitution of Trp with Arg (Trp482Arg) disrupts the cation-π interaction with phospholipids located in the outer leaflet of the phospholipid bilayer, destabilizing protein-lipid interactions. Additionally, Trp482Arg collapses the CH-π interaction between Trp482 and Pro511, possibly reducing the overall stability of the protein. In parallel with the molecular modeling, the two mutants degraded significantly faster compared to the wild-type protein, compromising protein stability. CONCLUSIONS: This results expand the genetic spectrum of disease-causing TMC1 variants related to DFNA36 and provide insight into TMC1 transmembrane protein-lipid interactions.

Ramifications of POU4F3 variants associated with autosomal dominant hearing loss in various molecular aspects.

POU4F3, a member of the POU family of transcription factors, commonly causes autosomal dominant deafness. Exome sequencing was used to identify four novel variants in POU4F3 (NM_002700.2), including c.564dupA: p.Ala189SerfsTer26, c.743T > C:p.Leu248Pro, c.879C > A:p.Phe293Leu, and c.952G > A:p.Val318Met, and diverse aspects of the molecular consequences of their protein expression, stability, subcellular localization, and transcriptional activity were investigated. The expression of three mutant proteins, encoded by missense variants, was reduced compared to the wild-type protein, demonstrating that the mutants were unstable and vulnerable to degradation. Additionally, all the mutant proteins had distinct subcellular localization patterns. A mutant protein carrying p.Ala189SerfsTer26, in which both mono- and bi-partite nuclear localization signals were disrupted, showed abnormal subcellular localization. Resultantly, all the mutant proteins significantly reduced the transcriptional activity required to regulate the downstream target gene expression. Furthermore, we identified the altered expression of 14 downstream target genes associated with inner ear development using patient-derived lymphoblastoid cell lines. There was a significant correlation of the expression profile between patient-derived cells and the cochlear hair cells, which provided a breakthrough for cases where the collection of human cochlear samples for transcriptome studies was unfeasible. This study expanded the genotypic spectrum of POU4F3 in DFNA15, and further refined the molecular mechanisms underlying POU4F3-associated DFNA15.

Phenotypic and molecular basis of SIX1 variants linked to non-syndromic deafness and atypical branchio-otic syndrome in South Korea.

Branchio-oto-renal (BOR)/branchio-otic (BO) syndrome is a rare disorder and exhibits clinically heterogenous phenotypes, marked by abnormalities in the ear, branchial arch, and renal system. Sporadic cases of atypical BOR/BO syndrome have been recently reported; however, evidence on genotype-phenotype correlations and molecular mechanisms of those cases is lacking. We herein identified five SIX1 heterozygous variants (c.307dupC:p.Leu103Profs*51, c.373G>A:p.Glu125Lys, c.386_391del:p.Tyr129_Cys130del, c.397_399del:p.Glu133del, and c.501G>C:p.Gln167His), including three novel variants, through whole-exome sequencing in five unrelated Korean families. All eight affected individuals with SIX1 variants displayed non-syndromic hearing loss (DFNA23) or atypical BO syndrome. The prevalence of major and minor criteria for BOR/BO syndrome was significantly reduced among individuals with SIX1 variants, compared to 15 BOR/BO syndrome families with EYA1 variants. All SIX1 variants interacted with the EYA1 wild-type; their complexes were localized in the nucleus except for the p.Leu103Profs*51 variant. All mutants also showed obvious but varying degrees of reduction in DNA binding affinity, leading to a significant decrease in transcriptional activity. This study presents the first report of SIX1 variants in South Korea, expanding the genotypic and phenotypic spectrum of SIX1 variants, characterized by DFNA23 or atypical BO syndrome, and refines the diverse molecular aspects of SIX1 variants according to the EYA1-SIX1-DNA complex theory.

ECPAS/Ecm29-mediated 26S proteasome disassembly is an adaptive response to glucose starvation.

The 26S proteasome comprises 20S catalytic and 19S regulatory complexes. Approximately half of the proteasomes in cells exist as free 20S complexes; however, our mechanistic understanding of what determines the ratio of 26S to 20S species remains incomplete. Here, we show that glucose starvation uncouples 26S holoenzymes into 20S and 19S subcomplexes. Subcomplex affinity purification and quantitative mass spectrometry reveal that Ecm29 proteasome adaptor and scaffold (ECPAS) mediates this structural remodeling. The loss of ECPAS abrogates 26S dissociation, reducing degradation of 20S proteasome substrates, including puromycylated polypeptides. In silico modeling suggests that ECPAS conformational changes commence the disassembly process. ECPAS is also essential for endoplasmic reticulum stress response and cell survival during glucose starvation. In vivo xenograft model analysis reveals elevated 20S proteasome levels in glucose-deprived tumors. Our results demonstrate that the 20S-19S disassembly is a mechanism adapting global proteolysis to physiological needs and countering proteotoxic stress.

Daily injection of melatonin inhibits insulin resistance induced by chronic mealtime shift.

Shift work disorders have become an emerging concern worldwide. Shift disorders encompass a wide range of illnesses that have yet to be identified. The study focused on the relationship between shift work disorders and insulin resistance. Previously, it was reported that advancing the usual mealtime of mice triggered insulin resistance. Here, the hypothesis that chronic mealtime shifts induce oxidative damage leading to chronic diseases such as type 2 diabetes was tested. It was found that mealtime shift causes imbalances between anti-oxidative capacity and reactive oxygen species (ROS) levels, indicating increased oxidative damage during the light/rest phase. This study further demonstrated that daily supplementation of antioxidants at the appropriate time of day inhibited insulin resistance caused by chronic mealtime shifts, suggesting significant and chronic health implications for shift workers. In conclusion, it was confirmed that increased ROS levels caused by mealtime shift induce insulin resistance, which is inhibited by the antioxidant melatonin.

Development and Evaluation of an In Silico Dermal Absorption Model Relevant for Children.

The higher skin surface area to body weight ratio in children and the prematurity of skin in neonates may lead to higher chemical exposure as compared to adults. The objectives of this study were: (i) to provide a comprehensive review of the age-dependent anatomical and physiological changes in pediatric skin, and (ii) to construct and evaluate an age-dependent pediatric dermal absorption model. A comprehensive review was conducted to gather data quantifying the differences in the anatomy and physiology of child and adult skin. Maturation functions were developed for model parameters that were found to be age-dependent. A pediatric dermal absorption model was constructed by updating a MoBi implementation of the Dancik et al. 2013 skin permeation model with these maturation functions. Using a workflow for adult-to-child model extrapolation, the predictive performance of the model was evaluated by comparing its predicted rates of flux of diamorphine, phenobarbital and buprenorphine against experimental observations using neonatal skin. For diamorphine and phenobarbital, the model provided reasonable predictions. The ratios of predicted:observed flux in neonates for diamorphine ranged from 0.55 to 1.40. For phenobarbital, the ratios ranged from 0.93 to 1.26. For buprenorphine, the model showed acceptable predictive performance. Overall, the physiologically based pediatric dermal absorption model demonstrated satisfactory prediction accuracy. The prediction of dermal absorption in neonates using a model-based approach will be useful for both drug development and human health risk assessment.

Evaluation of Quantitative Structure Property Relationship Algorithms for Predicting Plasma Protein Binding in Humans.

The extent of plasma protein binding is an important compound-specific property that influences a compound's pharmacokinetic behavior and is a critical input parameter for predicting exposure in physiologically based pharmacokinetic (PBPK) modeling. When experimentally determined fraction unbound in plasma (fup) data are not available, quantitative structure-property relationship (QSPR) models can be used for prediction. Because available QSPR models were developed based on training sets containing pharmaceutical-like compounds, we compared their prediction accuracy for environmentally relevant and pharmaceutical compounds. Fup values were calculated using Ingle et al., Watanabe et al. and ADMET Predictor (Simulation Plus). The test set included 818 pharmaceutical and environmentally relevant compounds with fup values ranging from 0.01 to 1. Overall, the three QSPR models resulted in over-prediction of fup for highly binding compounds and under-prediction for low or moderately binding compounds. For highly binding compounds (0.01≤ fup ≤ 0.25), Watanabe et al. performed better with a lower mean absolute error (MAE) of 6.7% and a lower mean absolute relative prediction error (RPE) of 171.7 % than other methods. For low to moderately binding compounds, both Ingle et al. and ADMET Predictor performed better than Watanabe et al. with superior MAE and RPE values. The positive polar surface area, the number of basic functional groups and lipophilicity were the most important chemical descriptors for predicting fup. This study demonstrated that the prediction of fup was the most uncertain for highly binding compounds. This suggested that QSPR-predicted fup values should be used with caution in PBPK modeling.

Evaluation of models for predicting pediatric fraction unbound in plasma for human health risk assessment.

Pediatric physiologically based pharmacokinetic (PBPK) models facilitate the prediction of PK parameters in children under specific exposure conditions. Pharmacokinetic outcomes are highly sensitive to fraction unbound in plasma (fup) as incorporated into PBPK models. Rarely is fup in children (fupchild) experimentally derived and prediction is based upon fup in adults (fupadult) as well as a ratio of plasma protein concentrations between children and adults. The objectives were to (i) evaluate protein concentration vs. age profile derived from ontogeny models, (ii) assess predictive performances of fup ontogeny models, and (iii) determine overall uncertainty in fupchild prediction resulting from a combination of quantitative structure-property relationship (QSPR) model and ontogeny models. The plasma albumin and alpha-acid glycoprotein (AAG) concentration data for pediatrics and fupchild and fupadult data were obtained from literature. The protein concentration vs. age profile derived from ontogeny models were compared to observed levels. Fupchild values were calculated according to ontogeny models using both observed and QSPR-predicted fupadult as inputs and predictive performances of ontogeny models assessed by comparing predicted fupchild to observed values. Protein concentrations vs. age profiles derived from non-linear equations were more congruent with observed albumin levels than linear or step-wise models. When observed fupadult values were used as input, the fupchild data were under-predicted with average fold error (AFE) amounts ranging 0.79-0.81 and 0.77-0.97 for albumin and AAG ontogeny models, respectively. When QSPR-predicted fupadult values were used as input, AFE of fupchild ranged 1.2-1.35 and 0.98-1.2 for albumin and AAG models, respectively. The choice of ontogeny model with respect to prediction accuracy is more important for AAG, highly bound compounds and infants. For these compounds and scenarios, experimental determination of fupchild for inclusion into a pediatric PBPK model is necessary to have confidence in PBPK model outputs.

Proteasome Activity in the Plasma as a Novel Biomarker in Mild Cognitive Impairment with Chronic Tinnitus.

BACKGROUND: Although the existence of proteasomes in human blood, termed circulating proteasomes (c-proteasomes), has been reported previously, their origin and pathophysiological functions remain largely unknown. OBJECTIVE: Given that c-proteasome activity was significantly reduced in Alzheimer's disease model mice and relatively high frequency of mild cognitive impairment (MCI) is accompanied by chronic tinnitus in aged patients, we examined whether c-proteasome activity in human plasma was associated with cognitive function in patients with chronic tinnitus. METHODS: c-Proteasome activity in the plasma of tinnitus patients (N = 55) was measured with fluorogenic reporter substrate, suc-LLVY-AMC. To assess MCI, the Montreal Cognitive Assessment was conducted with a cut-off score of 22/23. All patients underwent audiological and psychoacoustic analyses. Levels of c-proteasomes, Aß42, and Aß40 were measured using ELISA, and their association with c-proteasome activity was evaluated. RESULTS: The activity of circulating proteasomes was significantly lower in patients with chronic tinnitus and MCI (p = 0.042), whereas activities of other plasma enzymes showed little correlation. In addition, c-proteasome activity was negatively associated with the level of plasma Aß and was directly dependent on its own concentration in the plasma of patients with chronic tinnitus. CONCLUSION: Our current work provides a new perspective for understanding the potential relationship between circulating proteasomes in the plasma and cognitive dysfunction, suggesting a novel, non-invasive biomarker in the context of MCI diagnosis.

Aggresomal sequestration and STUB1-mediated ubiquitylation during mammalian proteaphagy of inhibited proteasomes.

The 26S proteasome, a self-compartmentalized protease complex, plays a crucial role in protein quality control. Multiple levels of regulatory systems modulate proteasomal activity for substrate hydrolysis. However, the destruction mechanism of mammalian proteasomes is poorly understood. We found that inhibited proteasomes are sequestered into the insoluble aggresome via HDAC6- and dynein-mediated transport. These proteasomes colocalized with the autophagic receptor SQSTM1 and cleared through selective macroautophagy, linking aggresomal segregation to autophagic degradation. This proteaphagic pathway was counterbalanced with the recovery of proteasomal activity and was critical for reducing cellular proteasomal stress. Changes in associated proteins and polyubiquitylation on inhibited 26S proteasomes participated in the targeting mechanism to the aggresome and autophagosome. The STUB1 E3 Ub ligase specifically ubiquitylated purified human proteasomes in vitro, mainly via Lys63-linked chains. Genetic and chemical inhibition of STUB1 activity significantly impaired proteasome processing and reduced resistance to proteasomal stress. These data demonstrate that aggresomal sequestration is the crucial upstream event for proteasome quality control and overall protein homeostasis in mammals.

Reduced chronic restraint stress in mice overexpressing hyperactive proteasomes in the forebrain.

While chronic restraint stress (CRS) results in depression-like behaviors possibly through oxidative stress in the brain, its molecular etiology and the development of therapeutic strategies remain elusive. Since oxidized proteins can be targeted by the ubiquitin-proteasome system, we investigated whether increased proteasome activity might affect the stress response in mice. Transgenic mice, expressing the N-terminally deleted version of α3 subunit (α3ΔN) of the proteasome, which has been shown to generate open-gated mutant proteasomes, in the forebrain were viable and fertile, but showed higher proteasome activity. After being challenged with CRS for 14 d, the mutant mice with hyperactive proteasomes showed significantly less immobility time in the forced swimming test compared with their wild-type littermates, suggesting that the α3ΔN transgenic mice are resistant to CRS. The accumulation of ER stress markers, such as polyubiquitin conjugates and phospho-IRE1α, was also significantly delayed in the hippocampus of the mutants. Notably, α3ΔN mice exhibited little deficits in other behavioral tasks, suggesting that stress resilience is likely due to the degradation of misfolded proteins by the open-gated proteasomes. These data strongly indicate that not only is the proteasome a critical modulator of stress response in vivo but also a possible therapeutic target for reducing chronic stress.

Model qualification of the PK-Sim® pediatric module for pediatric exposure assessment of CYP450 metabolized compounds.

Pediatric physiologically based pharmacokinetic (PBPK) models facilitate the estimation of pharmacokinetic (PK) parameters in children under specific exposure conditions. In human health risk assessment, PBPK modeling has been used to determine a chemical-specific human kinetic adjustment factor (HKAF). Due to increased demands in regulatory assessment, model evaluation and qualification have gained growing attention. The aim of this study was to undertake model qualification of pediatric PBPK models for compounds that are primarily metabolized by cytochrome P450 (CYP) enzymes. The objectives were to determine the appropriateness of the virtual individual creating algorithm in PK-Sim® in predicting PK parameters and their variability in children and identify critical system-specific inputs. PBPK models in adults were constructed for several pharmaceuticals (grouped by major clearance process such as CYP3A4). Several age groups of virtual individuals were created to represent children in pediatric clinical studies. The mean and variance of clearance (CL) from virtual populations were compared to observed values. Sensitivity analysis on area under the curve (AUC) was performed. System-specific parameters of virtual children that contribute to inter-individual PK properties were assessed. Eighty-one percent of the comparisons between simulated and observed clearance values were within twofold error. The mean fold errors were 1.1, 1, 0.7 and 1.8 in adolescents, children, infants and neonates, respectively. CL variability was reasonably predicted for 70% of the comparisons with comparable coefficients of variation between observed and predicted. The sensitivity analysis revealed that fraction unbound in plasma, parameters related to CYP enzyme-mediated metabolism and liver volumewere most important in the estimation of pediatric exposure. A comparison of variabilities in weight, height and liver volume in virtual children showed reliable agreement with observed data. The presented results of predictive performance and properties of virtual populations provide confidence in the use of PK-Sim for pediatric PBPK modeling in toxicological applications including PBPK-based-HKAF derivation.

Acidiphilamides A-E, Modified Peptides as Autophagy Inhibitors from an Acidophilic Actinobacterium, Streptacidiphilus rugosus.

Five new tripeptides, acidiphilamides A-E (1-5), were discovered along with two previously reported compounds, l-isoleucinamide (6) and l-valinamide (7), from Streptacidiphilus rugosus AM-16, an acidophilic actinobacterial strain isolated from acidic forest soil. The structures of 1-5 were elucidated as modified tripeptides bearing phenylalaninol or methioninol fragments with C3-C5 acyl chains based mainly on NMR and mass spectroscopic data. The absolute configurations of the amine units were established by advanced Marfey's method and GITC (2,3,4,6-tetra- O-acetyl-ß-d-glucopyranosyl isothiocyanate) derivatization followed by LC/MS analysis. Acidiphilamides A and B (1 and 2), the first secondary metabolites isolated from the rare actinobacterial genus Streptacidiphilus, significantly inhibited autophagic flux but not proteasome activity in HeLa cells. These compounds appeared to block mainly the autophagosome-lysosome fusion step in the late stage of cellular autophagy.