Strategies to deal with genetic analyzer-specific DNA methylation measurements.

Targeted bisulfite sequencing using single-base extension (SBE) can be used to measure DNA methylation via capillary electrophoresis on genetic analyzers in forensic labs. Several accurate age prediction models have been reported using this method. However, using different genetic analyzers with different software settings can generate different methylation values, leading to significant errors in age prediction. To address this issue, the study proposes and compares four methods as follows: (1) adjusting methylation values using numerous actual body fluid DNA samples, (2) adjusting methylation values using control DNAs with varying methylation ratios, (3) constructing new age prediction models for each genetic analyzer type, and (4) constructing new age prediction models that could be applied to all types of genetic analyzers. To test the methods for adjusting values using actual body fluid DNA samples, previously reported adjusting equations were used for blood/saliva DNA age prediction markers (ELOVL2, FHL2, KLF14, MIR29B2CHG/C1orf132, and TRIM59). New equations were generated for semen DNA age prediction markers (TTC7B, LOC401324/cg12837463, and LOC729960/NOX4) by drawing polynomial regression lines between the results of the three types of genetic analyzers (3130, 3500, and SeqStudio). The same method was applied to obtain adjustment equations using 11 control DNA samples. To develop new age prediction models for each genetic analyzer type, linear regression analysis was conducted using DNA methylation data from 150 blood, 150 saliva, and 62 semen samples. For the genetic analyzer-independent models, control DNAs were used to formulate equations for calibrating the bias of the data from each genetic analyzer, and linear regression analysis was performed using calibrated body fluid DNA data. In the comparison results, the genetic analyzer-specific models showed the highest accuracy. However, genetic analyzer-independent models through bias adjustment also provided accurate age prediction results, suggesting its use as an alternative in situations with multiple constraints.

The inhibitory effect of ovomucoid from egg white on biofilm formation by Streptococcus mutans.

BACKGROUND: Streptococcus mutans, the main pathogen associated with tooth decay, forms cariogenic biofilms on tooth surfaces. Therefore, controlling oral biofilm helps prevent dental caries. Hen's egg is a nutrient-dense food, and egg white is a good source of protein. Ovomucoid is one of the major proteins in egg white, with a 28 kDa molecular weight. The present study aimed to investigate the inhibitory effects of ovomucoid on the biofilm formation of S. mutans by suppressing virulence factors, including bacterial adherence, cellular aggregation and exopolysaccharide (EPS) production. RESULTS: Crystal violet staining showed that biofilm formation by S. mutans was inhibited by ovomucoid at 0.25-1 mg mL-1 levels. Field emission scanning electron microscopy also confirmed this inhibition. In addition, ovomucoid reduced mature biofilm, water-insoluble EPS synthesis and the metabolic activity of bacterial cells in the biofilm. The bacterial adhesion and aggregation abilities of S. mutans were also decreased in the presence of ovomucoid. Ovomucoid downregulated the expression of comDE and vicR genes involved in the two-component signal transduction system and gtfA and ftf genes involved in EPS production. CONCLUSION: Ovomucoid has the potential for use as an anti-biofilm agent for dental caries treatment because of its inhibitory effects on the virulence factors of S. mutans. © 2023 Society of Chemical Industry.

Sustained Exosome-Guided Macrophage Polarization Using Hydrolytically Degradable PEG Hydrogels for Cutaneous Wound Healing: Identification of Key Proteins and MiRNAs, and Sustained Release Formulation.

Macrophages (Mφs) are characterized by remarkable plasticity, an essential component of chronic inflammation. Thus, an appropriate and timely transition from proinflammatory (M1) to anti-inflammatory (M2) Mφs during wound healing is vital to promoting resolution of acute inflammation and enhancing tissue repair. Herein, exosomes derived from M2-Mφs (M2-Exos), which contain putative key regulators driving Mφ polarization, are used as local microenvironmental cues to induce reprogramming of M1-Mφs toward M2-Mφs for effective wound management. As an injectable controlled release depot for exosomes, hydrolytically degradable poly(ethylene glycol) (PEG) hydrogels (Exogels) are designed and employed for encapsulating M2-Exos to maximize their therapeutic effects in cutaneous wound healing. The degradation time of the hydrogels is adjustable from 6 days or up to 27 days by controlling the crosslinking density and tightness. The localization of M2-Exos leads to a successful local transition from M1-Mφs to M2-Mφs within the lesion for more than 6 days, followed by enhanced therapeutic effects including rapid wound closure and increased healing quality in an animal model for cutaneous wound healing. Collectively, the hydrolytically degradable PEG hydrogel-based exosome delivery system may serve as a potential tool in regulating local polarization state of Mφs, which is crucial for tissue homeostasis and wound repair.

Fibulin 5, a human Wharton's jelly-derived mesenchymal stem cells-secreted paracrine factor, attenuates peripheral nervous system myelination defects through the Integrin-RAC1 signaling axis.

In the peripheral nervous system (PNS), proper development of Schwann cells (SCs) contributing to axonal myelination is critical for neuronal function. Impairments of SCs or neuronal axons give rise to several myelin-related disorders, including dysmyelinating and demyelinating diseases. Pathological mechanisms, however, have been understood at the elementary level and targeted therapeutics has remained undeveloped. Here, we identify Fibulin 5 (FBLN5), an extracellular matrix (ECM) protein, as a key paracrine factor of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) to control the development of SCs. We show that co-culture with WJ-MSCs or treatment of recombinant FBLN5 promotes the proliferation of SCs through ERK activation, whereas FBLN5-depleted WJ-MSCs do not. We further reveal that during myelination of SCs, FBLN5 binds to Integrin and modulates actin remodeling, such as the formation of lamellipodia and filopodia, through RAC1 activity. Finally, we show that FBLN5 effectively restores the myelination defects of SCs in the zebrafish model of Charcot-Marie-Tooth (CMT) type 1, a representative demyelinating disease. Overall, our data propose human WJ-MSCs or FBLN5 protein as a potential treatment for myelin-related diseases, including CMT.

A serotonin voltammetric biosensor composed of carbon nanocomposites and DNA aptamer.

A sensitive and selective voltammetric biosensor composed of layer-by-layer (LbL) self-assembly of positively charged poly(diallyldimethylammonium)-wrapped oxidized single-walled carbon nanotubes (PDDA-oSWCNTs), negatively charged serotonin (5-hydroxytryptamine, 5-HT)-specific aptamer, and tyrosinase on Au nanoparticles deposited screen printed carbon electrode was developed for measurement of 5-HT. Surface characteristics of 5-HT biosensor were explored using scanning electron microscopy, X-ray photoelectron spectroscopy, and electrochemical impedance spectroscopy. The respective effects of 5-HT-specific aptamer and oSWCNTs on the detection of 5-HT were investigated by differential pulse voltammetry (DPV). The peak current at the potential of 0.29 V (vs. Ag/AgCl) increased with respect to 5-HT concentration resulting in two dynamic ranges from 0.05 to 0.5 and 1 to 20 µM with a limit of detection of 2 nM from the LbL biosensor in buffer solution, which were better than those without the LbL of aptamer and oSWCNTs. The developed biosensor was applied to the direct determination of 5-HT concentrations in undiluted healthy control and Internet gaming disorder serum samples. The results were verified by comparison with those from liquid chromatography-mass spectrometric analyses.

Compound heterozygous mutations of SH3TC2 in Charcot-Marie-Tooth disease type 4C patients.

Charcot-Marie-Tooth disease type 4C (CMT4C) is an autosomal recessive neuropathy caused by SH3TC2 mutations, characterized by spine deformities and cranial nerve involvement. This study identified four CMT4C families with compound heterozygous SH3TC2 mutations from 504 Korean demyelinating or intermediate CMT patients. The frequency of the CMT4C was calculated as 0.79% in demyelinating and intermediate patients (n = 504), but it was calculated as 2.02% in patients without PMP22 duplication (n = 198). The CMT4C frequency was similar to patients in Japan, but it was relatively low compared to those patients in other populations. The symptom was less severe and slowly progressed compared to the other AR-CMT. A patient harboring an intermediate neuropathy showed cranial nerve involvement but did not have scoliosis. This study will be helpful in making molecular diagnoses of demyelinating or intermediate CMT due to SH3TC2 mutations.

Analysis of Alveolar Bone Morphology of the Maxillary Central and Lateral Incisors with Normal Occlusion.

Background and objectives: This study investigated the morphology of the labial and palatal bony wall of the maxillary central and lateral incisors using cone-beam computed tomography (CBCT). The difference between males and females and the measurement between right and left sides were measured. Materials and Methods: Twenty participants, consisting of 11 females and 9 males having normal occlusion, were used for the analysis. The mean age was 21.9 ± 3.0 years. The thickness of the labial bony wall and palatal bony wall, perpendicular to the long axis of the root, were evaluated at 3 and 5 mm apical from the cemento-enamel junction (CEJ) and at the root apex. The available bony wall below the apex of the central and lateral incisors, and the angulation between the long axis of the tested tooth and outer surface of the labial bone were measured. Results: The mean labial bony wall thickness at the 3 and 5 mm apical from the CEJ were 1.1 ± 0.3 mm and 1.0 ± 0.4 mm for central incisors, respectively, as well as 1.2 ± 0.4 mm and 1.0 ± 0.4 mm for lateral incisors, respectively. The mean palatal bony wall thickness at 5 mm from the CEJ was above 2 mm in the central and lateral incisors. The percentage of labial bony wall thickness 2 mm or greater at the root apex in central incisors was higher than in lateral incisors (62.5% vs. 55.0%). The percentage of palatal bony wall thickness ≥2 mm at 3 mm apical from the CEJ in the central incisors was higher than in the lateral incisors (37.5% vs. 15.0%). The results on the left and right sides did not show statistically significant differences, except in the labial and palatal bony wall thickness at 3 mm from the CEJ in the lateral incisor. Generally, no significant differences were seen between males and females, but males had a significantly higher labial bony wall thickness at 3 and 5 mm from the CEJ in the central and lateral incisors when compared with females. Conclusions: This study showed that a majority of the cases of Korean participants had less than 2 mm of labial bony wall thickness at 3 and 5 mm apical from the CEJ at central and lateral incisors, and this should be kept in mind while performing dental practices, including tooth extraction or immediate implantation in anterior regions. Preoperative analysis using CBCT may be beneficial for establishing the treatment plan.

Functional efficacy of human recombinant FGF-2s tagged with (His)6 and (His-Asn)6 at the N- and C-termini in human gingival fibroblast and periodontal ligament-derived cells.

Fibroblast growth factor (FGF) is a multifunctional growth factor that induces cell proliferation, survival, migration, and differentiation in various cell types and tissues. With these biological functions, FGF-2 has been evaluated for clinical use in the regeneration of damaged tissues. The expression of hFGF-2 in Escherichia coli and a purification system using the immobilized metal affinity chromatography (IMAC) is well established to generate a continuous supply of FGF-2. Although hexa-histidine tag (H6) is commonly used for IMAC purification, hexa-histidine-asparagine tag (HN6) is also efficient for purification as it is easily exposed on the surface of the protein. In this study, four different tagging constructs of hFGF-2 based on tag positions and types (H6-FGF2, FGF2-H6, HN6-FGF2, and FGF2-HN6) were designed and expressed under the inducible T7 expression system in E. coli. The experimental conditions of expression and purification of each recombinant protein were optimized. The effective dosages of the recombinant proteins were determined based on the increase of cell proliferation in human gingival fibroblast. ED50s of H6-FGF2, FGF2-H6, HN6-FGF2, and FGF2-HN6 were determined (4.42 ng/ml, 3.55 ng/ml, 3.54 ng/ml, and 4.14 ng/ml, respectively) and found to be comparable to commercial FGF-2 (3.67 ng/ml). All the recombinant hFGF-2s inhibit the osteogenic induction and mineralization in human periodontal ligament-derived cells. Our data suggested that biological activities of the recombinant hFGF-2 are irrelevant to types and positions of tags, but may have an influence on the expression efficiency and solubility.

DGAT2 Mutation in a Family with Autosomal-Dominant Early-Onset Axonal Charcot-Marie-Tooth Disease.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy and is a genetically and clinically heterogeneous disorder. We examined a Korean family in which two individuals had an autosomal-dominant axonal CMT with early-onset, sensory ataxia, tremor, and slow disease progression. Pedigree analysis and exome sequencing identified a de novo missense mutation (p.Y223H) in the diacylglycerol O-acyltransferase 2 (DGAT2) gene. DGAT2 encodes an endoplasmic reticulum-mitochondrial-associated membrane protein, acyl-CoA:diacylglycerol acyltransferase, which catalyzes the final step of the triglyceride (TG) biosynthesis pathway. The patient showed consistently decreased serum TG levels, and overexpression of the mutant DGAT2 significantly inhibited the proliferation of mouse motor neuron cells. Moreover, the variant form of human DGAT2 inhibited the axonal branching in the peripheral nervous system of zebrafish. We suggest that mutation of DGAT2 is the novel underlying cause of an autosomal-dominant axonal CMT2 neuropathy. This study will help provide a better understanding of the pathophysiology of axonal CMT and contribute to the molecular diagnostics of peripheral neuropathies.

Effect of Asiasari radix on osteoblastic differentiation of stem cells derived from gingiva.

OBJECTIVE: To examine the dose-dependent impact of Asiasari Radix (A. radix) on the cell viability, differentiation and mineralization of stem cells derived from gingiva. METHODS: Stem cells that were derived from gingiva were grown in the presence of A. radix at final concentrations that ranged from 0.001 to 10 µg/mL. The morphology of the cells was viewed under an inverted microscope and the analysis of cell proliferation was performed by using Cell Counting Kit-8 (CCK-8) on day 1. The alkaline phosphatase activity test was used to assess differentiation and Alizarin red S staining was used to assess mineralization of treated cells. RESULTS: The control group showed spindleshaped, fibroblast-like morphology and the shapes of the cells in 0.001, 0.01, 0.1, 1 and 10 µg/mL of A. radix were similar to that of the control group at day 1. The cultures growing in the presence of 0.001 µg/mL of A. radix at day 1 showed an increase in the CCK-8 value (P < 0.05). Cultures growing in the presence of 0.001 µg/mL of A. radix presented the highest value for alkaline phosphatase activity (P > 0.05). Mineralized extracellular deposits were observed after Alizarin Red S staining and the cultures grown in the presence of 0.001 µg/mL of A. radix showed the highest value for quantitative results for bound dye (P < 0.05). CONCLUSION: Within the limits of this study, A. radix influenced the proliferation of stem cells derived from the gingiva and low concentrations of A. radix might enhance osteogenic differentiation of the stem cells.

Comparison of scleral buckling and vitrectomy using wide angle viewing system for rhegmatogenous retinal detachment in patients older than 35 years.

BACKGROUND: To compare scleral buckling (SB) and pars plana vitrectomy (PPV) using a wide angle viewing system (WAVS) for uncomplicated phakic rhegmatogenous retinal detachment (RRD). METHODS: The medical records of patients with uncomplicated phakic RRD were retrospectively reviewed. Eyes with pseudophakic or attached fovea were excluded. Patients treated with SB were classified as group B, and PPV using WAVS as group V. Primary success rate, visual acuity (VA), macular complications, and sustained subretinal fluid (SRF) were compared between groups. RESULTS: Seventy-two eyes were included in group B and 57 eyes in group V. Group B had better preoperative VA (1.38 ± 0.87 vs 1.84 ± 0.97 in LogMAR, P = 0.010), but worse final VA (0.51 ± 0.48 vs 0.30 ± 0.23, P = 0.012) than group V. The primary success rate of 94.7 % in group V was higher than 77.8 % in group B (P = 0.010). Final success rate was 100 % in both groups. There was no significant difference in macular complications between groups (P = 0.087). Sustained SRF was found in 22 eyes in group B (38.6 %), while only two eyes in group V exhibited sustained SRF (2.8 %, P < 0.001). CONCLUSIONS: Pars plana vitrectomy using WAVS was more efficacious than SB for treating uncomplicated phakic RRD.

The thickness of alveolar bone at the mandibular canine and premolar teeth in normal occlusion.

PURPOSE: The purpose of this study was to investigate the alveolar bone thickness on the buccal and lingual aspects of mandibular canines and premolars using cone-beam computed tomography (CBCT). The differences between the left side and the right side and that in male and female measurements were reviewed. PATIENTS AND METHODS: Three-dimensional CBCT of 20 subjects with normal occlusion (9 males and 11 females; mean [SD] age, 21.9 [3.0] y) were used. The thickness of the buccal and lingual bone walls, perpendicular to the long axis of the root, was evaluated at 3 and 5 mm apical to the cementoenamel junction (CEJ) and at the root apex. RESULTS: The mean buccal bone thickness measured at 3 and 5 mm apical to the CEJ was less than 2 mm on the canines and the premolars. The buccal bone thickness of the second premolar at 3 and 5 mm from the CEJ was significantly greater than that of the canine and the first premolar. There were no significant differences between the left and right sides, and the overall measurements of the alveolar bone thickness did not show significant male/female differences. CONCLUSIONS: This study presented the thickness of the buccal and lingual bone in different locations apical to the CEJ in subjects with normal occlusion and the frequency distribution of thick buccal bone wall (≥ 2 mm). The second premolar had the highest frequency distribution of thick buccal bone (≥ 2 mm) when compared with canine and the first premolar. The teeth with thin buccal bone (< 2 mm) should be treated with care for the implant because a thin buccal bone may be damaged more easily and buccal bone resorption may occur. This study may provide estimated value for patients with normal occlusion during tooth extraction and implant installation in the canine and premolar area of the mandible. Preoperative radiographic analysis, with care in using CBCT, may be applied for tooth extraction and implant therapy.

Anti-skin-aging benefits of exopolymers from Aureobasidium pullulans SM2001.

BACKGROUND: There have been many attempts to search for affordable and effective functional cosmetic ingredients, especially from natural sources. OBJECTIVES: As research into developing a functional cosmetic ingredient, we investigated whether exopolymers from Aureobasidium pullulans SM2001 (E-AP-SM2001) exert antioxidant, antiwrinkle, whitening, and skin moisturizing effects. METHODS: Antioxidant effects of E-AP-SM2001 were determined by measuring free radical scavenging capacity and superoxide dismutase (SOD)-like activity. Antiwrinkle effects were assessed through the inhibition of hyaluronidase, elastase, collagenase, and matrix metalloproteinase (MMP)-1. Whitening effects were measured by tyrosinase inhibition assay, and by melanin formation test in B16/F10 melanoma cells. Skin moisturizing effects were detected by mouse skin water content test. RESULTS: E-AP-SM2001 showed potent DPPH radical scavenging activity and SOD-like effects. Additionally, hyaluronidase, elastase, collagenase, and MMP-1 activities were significantly inhibited by E-AP-SM2001. We also observed that E-AP-SM2001 effectively reduced melanin production by B16/F10 melanoma cells and mushroom tyrosinase activities. Furthermore, significant increases in skin water content were detected in E-AP-SM2001- treated mouse skin, as compared with vehicle-treated control skin. Notably, a mask pack containing E-AP-SM2001 showed a >twofold more extensive moisturizing effect compared with one containing Saccharomycopsis ferment filtrate. CONCLUSIONS: Our results suggest that E-AP-SM2001 has adequate antiaging, antiwrinkle, and whitening benefits and skin moisturizing effect. These effects involve reducing hyaluronidase, elastase, collagenase, and MMP-1 activities, as well as inhibition of melanin production and tyrosinase activities. Therefore, the antioxidant E-AP-SM2001 may serve as a predictable functional ingredient.

Epidemiological and Genetic Characterization of Coxsackievirus A6-Associated Hand, Foot, and Mouth Disease in Gwangju, South Korea, in 2022.

Coxsackievirus A6 (CV-A6) has emerged as the predominant causative agent of hand, foot, and mouth disease (HFMD) in young children. Since the declaration of coronavirus disease 2019 (COVID-19) as a global pandemic, the incidence of infectious diseases, including HFMD, has decreased markedly. When social mitigation was relaxed during the COVID-19 pandemic in 2022, the re-emergence of HFMD was observed in Gwangju, South Korea, and seasonal characteristics of the disease appeared to have changed. To investigate the molecular characteristics of enterovirus (EV) associated with HFMD during 2022, 277 specimens were collected. Children aged younger than 5 years accounted for the majority of affected individuals. EV detection and genotyping were performed using real-time RT-PCR and nested RT-PCR followed by sequence analysis. The EV detection rate was found to be 82.3%, and the main genotype identified was CV-A6. Sixteen CV-A6 samples were selected for whole genome sequencing. According to phylogenetic analysis, all CV-A6 strains from this study belonged to the sub-genotype D3 clade based on VP1 sequences. Analysis of 3D polymerase phylogeny showed that only the recombinant RF-A group was identified. In conclusion, circulating EV types should be continuously monitored to understand pathogen emergence and evolution during the post-pandemic era.

Unique contribution of the cell wall-binding endoglucanase G to the cellulolytic complex in Clostridium cellulovorans.

The cellulosomes produced by Clostridium cellulovorans are organized by the specific interactions between the cohesins in the scaffolding proteins and the dockerins of the catalytic components. Using a cohesin biomarker, we identified a cellulosomal enzyme which belongs to the glycosyl hydrolase family 5 and has a domain of unknown function 291 (DUF291) with functions similar to those of the surface layer homology domain in C. cellulovorans. The purified endoglucanase G (EngG) had the highest synergistic degree with exoglucanase (ExgS) in the hydrolysis of crystalline cellulose (EngG/ExgS ratio = 3:1; 1.71-fold). To measure the binding affinity of the dockerins in EngG for the cohesins of the main scaffolding protein, a competitive enzyme-linked interaction assay was performed. Competitors, such as ExgS, reduced the percentage of EngG that were bound to the cohesins to less than 20%; the results demonstrated that the cohesins prefer to bind to the common cellulosomal enzymes rather than to EngG. Additionally, in surface plasmon resonance analysis, the dockerin in EngG had a relatively weak affinity (30- to 123-fold) for cohesins compared with the other cellulosomal enzymes. In the cell wall affinity assay, EngG anchored to the cell surfaces of C. cellulovorans using its DUF291 domain. Immunofluorescence microscopy confirmed the cell surface display of the EngG complex. These results indicated that in C. cellulovorans, EngG assemble into both the cellulolytic complex and the cell wall complex to aid in the hydrolysis of cellulose substrates.

Treatment outcomes and compliance according to obesity in patients with obstructive sleep apnea.

The objective of this study was to assess treatment outcomes and compliance according to obesity among groups of patients with obstructive sleep apnea (OSA) receiving different treatments. A total of 297 patients with OSA treated between 2006 and 2009 underwent pre- and post-treatment polysomnography. One hundred and fifty-one patients were treated with continuous airway positive pressure (CPAP), 76 with mandible advancement device (MAD), and 70 with oropharyngeal surgery. All patients were classified according to obesity. Treatment success rate and compliance of CPAP were analyzed according to obesity. For each treatment modality, the overall treatment success rate was not significantly different between obese and non-obese patients. However, the oxygen desaturation index was different in patients who were treated with MAD and surgery. Additionally, obese patients with severe OSA showed an unfavorable response to CPAP treatment. For CPAP compliance, obese patients showed a tendency to be highly compliant with CPAP treatment at 12 months than non-obese patients. This study showed that obesity might be a factor in determining the success or failure of treatment. Additionally, obesity may be a predictive factor to determine CPAP compliance.

Endogenous Endophthalmitis From a Klebsiella pneumoniae Liver Abscess: The Incidence, Risk Factors, and Utility of Imaging.

PURPOSE: We investigated the rate of ophthalmologic examinations to detect endogenous endophthalmitis in patients with pyogenic liver abscesses (PLAs) and the incidence and risk factors of endophthalmitis from a PLA caused by Klebsiella pneumoniae (PLA-K). DESIGN: Retrospective case series. METHODS: A total of 536 patients admitted to a university hospital in Korea to treat PLAs during 2012-2022 were included. The proportion of patients who were referred for ophthalmologic examinations was investigated and the incidence of endophthalmitis in 248 patients with PLA-K was calculated. Univariate and multivariate logistic regression analyses were performed to define risk factors including demographic characteristics, underlying diseases, radiologic findings, and systemic conditions. RESULTS: A comprehensive ophthalmologic examination was performed in 73.7% of all patients with PLAs, and the incidence of endophthalmitis from a PLA-K was 7.3%. A liver abscess >5 cm increased the incidence of endogenous endophthalmitis 4-fold compared with smaller abscesses (odds ratio [OR] = 4.01 [95% confidence interval {CI}, 1.02-15.78], P = .047) and portal or hepatic vein thrombophlebitis increased the incidence approximately 4-fold (OR = 4.04 [95% CI, 1.10-14.83], P = .036). Acute cholangitis was approximately 8-fold (OR = 8.33 [95% CI, 1.25-55.71], P = .029), and disseminated intravascular coagulation was approximately 6-fold (OR = 5.76 [95% CI, 1.22-27.21], P = .027) more related to prevalence of endophthalmitis. Other extrahepatic infections increased the incidence approximately 43-fold (OR = 43.06 [95% CI, 10.14-182.90], P < .001). CONCLUSIONS: Clinicians should consider the risk of endogenous endophthalmitis when PLA-K patients have large liver abscesses (>5 cm), acute cholangitis, portal or hepatic vein thrombophlebitis, disseminated intravascular coagulation, or other extrahepatic infections.

Optimization of HPLCCAD method for simultaneous analysis of different lipids in lipid nanoparticles with analytical QbD.

Since lipid nanoparticles (LNP) have emerged as a potent drug delivery system, the objective of this study was to develop and optimize a robust high-performance liquid chromatography with charged aerosol detectors (HPLCCAD) method to simultaneously quantify different lipids in LNPs using the analytical quality by design (AQbD) approach. After defining analytical target profile (ATP), critical method attributes (CMAs) were established as a resolution between the closely eluting lipid peaks and the total analysis time. Thus, potential high-risk method parameters were identified through the initial risk assessment. These parameters were screened using Plackett-Burman design, and three critical method parameters (CMPs)-MeOH ratio, flow rate, and column temperature-were selected for further optimization. Box-Behnken design was employed to develop the quadratic models that explain the relationship between the CMPs and CMAs and to determine the optimal operating conditions. Moreover, to ensure the robustness of the developed method, a method operable design region (MODR) was established using the Monte Carlo simulation. The MODR was identified within the probability map, where the risk of failure to achieve the desired CMAs was less than 1%. The optimized method was validated according to the ICH guidelines (linearity: R2 > 0.995, accuracy: 97.15-100.48% recovery, precision: RSD < 5%) and successfully applied for the analysis of the lipid in the LNP samples. The development of the analytical method to quantify the lipids is essential for the formulation development and quality control of LNP-based drugs since the potency of LNPs is significantly dependent on the compositions and contents of the lipids in the formation.

Peripheral Neuropathy and Decreased Locomotion of a RAB40B Mutation in Human and Model Animals.

Rab40 proteins are an atypical subgroup of Rab GTPases containing a unique suppressor of the cytokine signaling (SOCS) domain that is recruited to assemble the CRL5 E3 ligase complex for proteolytic regulation in various biological processes. A nonsense mutation deleting the C-terminal SOCS box in the RAB40B gene was identified in a family with axonal peripheral neuropathy (Charcot-Marie-Tooth disease type 2), and pathogenicity of the mutation was assessed in model organisms of zebrafish and Drosophila. Compared to control fish, zebrafish larvae transformed by the human mutant hRAB40B-Y83X showed a defective swimming pattern of stalling with restricted localization and slower motility. We were consistently able to observe reduced labeling of synaptic markers along neuromuscular junctions of the transformed larvae. In addition to the neurodevelopmental phenotypes, compared to normal hRAB40B expression, we further examined ectopic expression of hRAB40B-Y83X in Drosophila to show a progressive decline of locomotion ability. Decreased ability of locomotion by ubiquitous expression of the human mutation was reproduced not with GAL4 drivers for neuron-specific expression but only when a pan-glial GAL4 driver was applied. Using the ectopic expression model of Drosophila, we identified a genetic interaction in which Cul5 down regulation exacerbated the defective motor performance, showing a consistent loss of SOCS box of the pathogenic RAB40B. Taken together, we could assess the possible gain-of-function of the human RAB40B mutation by comparing behavioral phenotypes in animal models; our results suggest that the mutant phenotypes may be associated with CRL5-mediated proteolytic regulation.

Long-term sentinel surveillance of enteroviruses in Gwangju, South Korea, 2011-2020.

Human enteroviruses (EVs) are associated with a broad spectrum of diseases. To understand EV epidemiology, we present longitudinal data reflecting changing EV prevalence patterns in South Korea. We collected 7160 specimens from patients with suspected EV infections in ten hospitals in Gwangju, Korea during 2011-2020. RNA extraction and real-time reverse transcription polymerase chain reaction using EV-specific probes and primers were performed. EV genotyping and phylogenetic analysis were performed; EVs were detected in 3076 samples (43.0%), and the annual EV detection rate varied. EV infection rates did not differ with sex, and children aged ≤ 4 years were the most prone to EV infection; this trend did not change over time. Overall, 35 different EV types belonging to four distinctive species and rhinoviruses were identified. Although serotype distribution changed annually, the most frequently observed EVs were EV-A71 (13.1% of the cases), CVA6 (8.3%), CVB5 (7.6%), CVA16 (7.6%), CVA10 (7.5%), E18 (7.5%), E30 (7.0%), and E11 (5.0%) during 2011-2020. The predominant EV genotypes by clinical manifestation were CVB5 for aseptic meningitis; EV-A71 for hand, foot, and mouth disease cases; and CVA10 for herpangina. These results will aid the development of vaccines against EV infection and allow comprehensive disease control.