Assessment of inflammatory suppression and fibroblast infiltration in tissue remodelling by supercritical CO2 acellular dermal matrix (scADM) utilizing Sprague Dawley models.

Human skin-derived ECM aids cell functions but can trigger immune reactions; therefore it is addressed through decellularization. Acellular dermal matrices (ADMs), known for their regenerative properties, are used in tissue and organ regeneration. ADMs now play a key role in plastic and reconstructive surgery, enhancing aesthetics and reducing capsular contracture risk. Innovative decellularization with supercritical carbon dioxide preserves ECM quality for clinical use. The study investigated the cytotoxicity, biocompatibility, and anti-inflammatory properties of supercritical CO2 acellular dermal matrix (scADM) in vivo based on Sprague Dawley rat models. Initial experiments in vitro with fibroblast cells confirmed the non-toxic nature of scADM and demonstrated cell infiltration into scADMs after incubation. Subsequent tests in vitro revealed the ability of scADM to suppress inflammation induced by lipopolysaccharides (LPS) presenting by the reduction of pro-inflammatory cytokines TNF-α, IL-6, IL-1ß, and MCP-1. In the in vivo model, histological assessment of implanted scADMs in 6 months revealed a decrease in inflammatory cells, confirmed further by the biomarkers of inflammation in immunofluorescence staining. Besides, an increase in fibroblast infiltration and collagen formation was observed in histological staining, which was supported by various biomarkers of fibroblasts. Moreover, the study demonstrated vascularization and macrophage polarization, depicting increased endothelial cell formation. Alteration of matrix metalloproteinases (MMPs) was analyzed by RT-PCR, indicating the reduction of MMP2, MMP3, and MMP9 levels over time. Simultaneously, an increase in collagen deposition of collagen I and collagen III was observed, verified in immunofluorescent staining, RT-PCR, and western blotting. Overall, the findings suggested that scADMs offer significant benefits in improving outcomes in implant-based procedures as well as soft tissue substitution.

Effects on keratinocytes of the traditional combination of herb extract (Royal Oji Complex) implicated the improvement of young children's skin moisture and barrier.

BACKGROUND: Natural products are often friendly and can be used on children's skin after systematic and careful research. Therefore, in this study, the Royal Oji Complex (ROC), a product with natural ingredients, was used to study their effectiveness on keratinocytes taken from the skin of children from 0 to 3 years old. METHOD: Normal human epidermal keratinocytes and tissue-isolated keratinocytes (TIKC) from young donors were treated with three different concentrations of ROC: 0.1, 1, and 10 ppm. The mRNA expression of the epidermal barrier's essential genes, such as hyaluronic acid synthase 3 (Has3), involucrin (IVL), loricrin (LOR), and claudin-1 (CLD1) was investigated using qRT-PCR. Ceramide content was measured by ELISA, with retinoic acid (R.A.) and amarogentin (AMA) serving as positive controls. RESULTS: ROC significantly elevated HAS3 gene expression in HEKn cells, especially at 10 ppm, indicating potential advantages for skin hydration in young infants. IVL increased at first but decreased as ROC concentrations increased. LOR was upregulated at lower ROC concentrations but reduced at higher doses. CLD1 gene expression increased considerably in HEKn but reduced with increasing ROC doses. Ceramide concentration increased somewhat but not significantly at 10 ppm. CONCLUSION: ROC shows potential in altering keratinocyte gene expression, with unique responses in HEKn and TIKC from young donors. While changes in ceramide content were insignificant, these results help to comprehend ROC's multiple effects on young children's skin.

Evaluation of the antimalarial activity of SAM13-2HCl with morpholine amide (SKM13 derivative) against antimalarial drug-resistant Plasmodium falciparum and Plasmodium berghei infected ICR mice.

Antimalarial drugs are an urgently need and crucial tool in the campaign against malaria, which can threaten public health. In this study, we examined the cytotoxicity of the 9 antimalarial compounds chemically synthesized using SKM13-2HCl. Except for SKM13-2HCl, the 5 newly synthesized compounds had a 50% cytotoxic concentration (CC50) > 100 µM, indicating that they would be less cytotoxic than SKM13-2HCl. Among the 5 compounds, only SAM13-2HCl outperformed SKM13-2HCl for antimalarial activity, showing a 3- and 1.3-fold greater selective index (SI) (CC50/IC50) than SKM13-2HCl in vitro against both chloroquine-sensitive (3D7) and chloroquine -resistant (K1) Plasmodium falciparum strains, respectively. Thus, the presence of morpholine amide may help to effectively suppress human-infectious P. falciparum parasites. However, the antimalarial activity of SAM13-2HCl was inferior to that of the SKM13-2HCl template compound in the P. berghei NK65-infected mouse model, possibly because SAM13-2HCl had a lower polarity and less efficient pharmacokinetics than SKM13-2HCl. SAM13-2HCl was more toxic in the rodent model. Consequently, SAM13-2HCl containing morpholine was selected from screening a combination of pharmacologically significant structures as being the most effective in vitro against human-infectious P. falciparum but was less efficient in vivo in a P. berghei-infected animal model when compared with SKM13-2HCl. Therefore, SAM13-2HCl containing morpholine could be considered a promising compound to treat chloroquine-resistant P. falciparum infections, although further optimization is crucial to maintain antimalarial activity while reducing toxicity in animals.

Effect of Silicone Patch Containing Metal-organic Framework on Hypertrophic Scar Suppression.

BACKGROUND/AIM: Hypertrophic scars (HS) are an abnormal cutaneous condition of wound healing characterized by excessive fibrosis and disrupted collagen deposition. This study assessed the potential of a silicone patch embedded with chemically stable zirconium-based metal-organic frameworks (MOF)-808 structures to mitigate HS formation using a rabbit ear model. MATERIALS AND METHODS: A silicone patch was strategically engineered by incorporating Zr-MOF-808, a composite structure comprising metal ions and organic ligands. Structural integrity of the Zr-MOF-808 silicone patch was validated using scanning electron microscopy and X-ray diffraction analysis. The animals were divided into three groups: a control, no treatment group (Group 1), a silicone patch treatment group (Group 2), and a group treated with a 0.2% loaded Zr-MOF-808 silicone patch (Group 3). HS suppression effects were quantified using scar elevation index (SEI), dorsal skin thickness measurements, and myofibroblast protein expression. RESULTS: Histopathological examination of post-treatment HS samples revealed substantial reductions in SEI (34.6%) and epidermal thickness (49.5%) in Group 3. Scar hyperplasia was significantly diminished by 53.5% (p<0.05), while collagen density declined by 15.7% in Group 3 compared to Group 1. Western blot analysis of protein markers, including TGF-ß1, collagen-1, and α-SMA, exhibited diminished levels by 8.8%, 12%, and 21.3%, respectively, in Group 3, and substantially higher levels by 21.9%, 27%, and 39.9%, respectively, in Group 2. On the 35th day post-wound generation, Zr-MOF-808-treated models exhibited smoother, less conspicuous, and flatter scars. CONCLUSION: Zr-MOF-808-loaded silicone patch reduced HS formation in rabbit ear models by inducing the proliferation and remodeling of the wound healing process.

Identification of breeding habitats and kdr mutations in Anopheles spp. in South Korea.

BACKGROUND: Malaria is still endemic in South Korea. However, limited information is available on the current Anopheles breeding sites and the occurrence of insecticide resistance-associated genetic mutations and their distribution needed to control the malaria vector efficiently. METHODS: This study explored breeding sites of Anopheline adults in Gimpo-si, near the demilitarized zone (DMZ) in Gyeonggi-do province, South Korea, from 2022 to 2023. Genetic diversity was investigated based on the internal transcribed spacer (ITS2), cytochrome c oxidase subunit I (COI), and knockdown resistance (kdr) genes of Anopheles mosquitoes. A natural environment associated with the seasonal abundance of Anopheles larvae was characterized. RESULTS: Two breeding sites of Anopheles larvae and adults were found at a stream margin or shallow freshwater near the forest in Wolgot-myeon in Gimpo-si without cattle shed within 1 km and in Naega-myeon in Ganghwa-gun with cow shed within 100 m in 2022 and 2023, respectively. Both sites were located between the newly cultivated lands and the forest. Besides, both breeding sites were in the valley at a slight elevation of 60-70 m from ground lands and maintained the shadow all day. Overall, the Wolgot-myeon breeding site showed various Anopheles spp. larvae, including Anopheles sinensis. Naega-myeon, an additional breeding site found in 2023, had Anopheles sineroides larvae, and approximately 59.7% (89/149) of An. sinensis adults inhabited within a 100-m distance. The total collection, including larvae and adults, revealed that An. sinensis, Anopheles pullus, Anopheles kleini, An. sineroides, Anopheles belenrae, and Anopheles lindesayi accounted for 44.2% (118/267), 0.7% (2/267), 0.7% (2/267), 22.1% (59/267), 1.9% (5/267), and 30.3% (81/267), respectively. Furthermore, various kdr mutant genotypes (F/F, C/C, L/F, L/C and F/C) in An. sinensis, and the first kdr allele mutant (L/F1014) in An. belenrae were identified in South Korea. CONCLUSIONS: Two breeding sites of Anopheles larvae were studied in Wolgot-myeon and Naega-myeon. Various Anopheles spp. larvae were detected in both habitats, but overall, An. sinensis was the most prevalent adults in both study sites. The occurrence of kdr allele mutant of An. belenrae in South Korea was reported. Rigorous larvae monitoring of Anopheles spp., continuously updating information on Anopheles breeding sites, and understanding the environmental conditions of Anopheles habitats are required to develop an effective malaria control programme in South Korea.

Anti-malarial activity of HCl salt of SKM13 (SKM13-2HCl).

Malaria is among the most devastating and widespread tropical parasitic diseases in developing countries. To prevent a potential public health emergency, there is an urgent need for new antimalarial drugs, with single-dose cures, broad therapeutic potential, and novel mechanism of action. We synthesized HCl salt of SKM13 (SKM13-2HCl) based on the modification of SKM13 to improve solubility in water. The anti-malarial activity of the synthesized drug was evaluated in both in vitro and in vivo models. The selective index indicated that SKM13-2HCl showed the same effectiveness with SKM13 in Plasmodium falciparum in in-vitro. Even though, in vivo mouse study demonstrated that SKM13 (20 mg/kg) at single dose could not completely inhibit P. berghei growth in blood. The survival rate increased from 33 to 90% at 15 days after infection. However, SKM13-2HCl (20 mg/kg) at a single dose increased the survival rate up to 100% at the same duration. Ultra-High-Performance Liquid Chromatography (UHPLC) showed that solubility in water of SKM13 and SKM13-2HCL was 0.389 mg/mL and 417 mg/mL, respectively. Pharmacokinetics (PK) analysis corresponded to the increased solubility of SKM13-2HCl over SKM13. Haematological parameters [red blood cell (RBC) count, haemoglobin level, and haematocrit level] supported the comparable efficacy of SKM13 and SKM13-2HCl in a 4-day suppression test. One mode of these drugs was found to be activating phosphorylation of eIF2α, hallmark of ER-stress, to kill parasite. Novel salt derivative of SKM13 (SKM13-2HCl) have enhanced anti-malarial activity against P. falciparum with endoplasmic reticulum (ER)-stress and salt form of SKM13 is an excellent direction to develop anti-malarial drug candidate in mice model.

Development of a Rapid Fluorescent Diagnostic System for Early Detection of the Highly Pathogenic Avian Influenza H5 Clade 2.3.4.4 Viruses in Chicken Stool.

Rapid diagnosis is essential for the control and prevention of H5 highly pathogenic avian influenza viruses (HPAIVs). However, highly sensitive and rapid diagnostic systems have shown limited performance due to specific antibody scarcity. In this study, two novel specific monoclonal antibodies (mAbs) for clade 2.3.4.4 H5Nx viruses were developed by using an immunogen from a reversed genetic influenza virus (RGV). These mAbs were combined with fluorescence europium nanoparticles and an optimized lysis buffer, which were further used for developing a fluorescent immunochromatographic rapid strip test (FICT) for early detection of H5Nx influenza viruses on chicken stool samples. The result indicates that the limit of detection (LoD) of the developed FICT was 40 HAU/mL for detection of HPAIV H5 clade 2.3.4.4b in spiked chicken stool samples, which corresponded to 4.78 × 104 RNA copies as obtained from real-time polymerase chain reaction (RT-PCR). An experimental challenge of chicken with H5N6 HPAIV is lethal for chicken three days post-infection (DPI). Interestingly, our FICT could detect H5N6 in stool samples at 2 DPI earlier, with 100% relative sensitivity in comparison with RT-PCR, and it showed 50% higher sensitivity than the traditional colloidal gold-based rapid diagnostic test using the same mAbs pair. In conclusion, our rapid diagnostic method can be utilized for the early detection of H5Nx 2.3.4.4 HPAIVs in avian fecal samples from poultry farms or for influenza surveillance in wild migratory birds.

Molecular Characterization and Pathogenesis of H6N6 Low Pathogenic Avian Influenza Viruses Isolated from Mallard Ducks (Anas platyrhynchos) in South Korea.

The subtype H6N6 has been identified worldwide following the increasing frequency of avian influenza viruses (AIVs). These AIVs also have the ability to bind to human-like receptors, thereby increasing the risk of animal-human transmission. In September 2019, an H6N6 avian influenza virus-KNU2019-48 (A/Mallard (Anas platyrhynchos)/South Korea/KNU 2019-48/2019(H6N6))-was isolated from Anas platyrhynchos in South Korea. Phylogenetic analysis results revealed that the hemagglutinin (HA) gene of this strain belongs to the Korean lineage, whereas the neuraminidase (NA) and polymerase basic protein 1 (PB1) genes belong to the Chinese lineage. Outstanding internal proteins such as PB2, polymerase acidic protein, nucleoprotein, matrix protein, and non-structural protein belong to the Vietnamese lineage. Additionally, a monobasic amino acid (PRIETR↓GLF) at the HA cleavage site; non-deletion of the stalk region (residue 59-69) in the NA gene; and E627 in the PB2 gene indicate that the KNU2019-48 isolate is a typical low-pathogenic avian influenza (LPAI) virus. The nucleotide sequence similarity analysis of HA revealed that the highest homology (97.18%) of this isolate is to that of A/duck/Jiangxi/01.14 NCJD125-P/2015(H6N6), and the amino acid sequence of NA (97.38%) is closely related to that of A/duck/Fujian/10.11_FZHX1045-C/2016 (H6N6). An in vitro analysis of the KNU2019-48 virus shows a virus titer of not more than 2.8 Log10 TCID 50/mL until 72 h post-infection, whereas in the lungs, the virus is detected at 3 dpi (days post-infection). The isolated KNU2019-48 (H6N6) strain is the first reported AIV in Korea, and the H6 subtype virus has co-circulated in China, Vietnam, and Korea for half a decade. Overall, our study demonstrates that Korean H6N6 strain PB1-S375N, PA-A404S, and S409N mutations are infectious in humans and might contribute to the enhanced pathogenicity of this strain. Therefore, we emphasize the importance of continuous and intensive surveillance of the H6N6 virus not only in Korea but also worldwide.

In Vitro Evaluation of Two Novel Antimalarial Derivatives of SKM13: SKM13-MeO and SKM13-F.

Antimalarial drugs play an important role in the control and treatment of malaria, a deadly disease caused by the protozoan parasite Plasmodium spp. The development of novel antimalarial agents effective against drug-resistant malarial parasites is urgently needed. The novel derivatives, SKM13-MeO and SKM13-F, were designed based on an SKM13 template by replacing the phenyl group with electron-donating (-OMe) or electron-withdrawing groups (-F), respectively, to reverse the electron density. A colorimetric assay was used to quantify cytotoxicity, and in vitro inhibition assays were performed on 3 different blood stages (ring, trophozoite, and schizonts) of P. falciparum 3D7 and the ring/mixed stage of D6 strain after synchronization. The in vitro cytotoxicity analysis showed that 2 new SKM13 derivatives reduced the cytotoxicity of the SKM13 template. SKM13 maintained the IC50 at the ring and trophozoite stages but not at the schizont stage. The IC50 values for both the trophozoite stage of P. falciparum 3D7 and ring/mixed stages of D6 demonstrated that 2 SKM13 derivatives had decreased antimalarial efficacy, particularly for the SKM13-F derivative. SKM13 may be comparably effective in ring and trophozoite, and electron-donating groups (-OMe) may be better maintain the antimalarial activity than electron-withdrawing groups (-F) in SKM13 modification.

Genetic Characterization and Pathogenesis of Avian Influenza Virus H7N3 Isolated from Spot-Billed Ducks in South Korea, Early 2019.

Low-pathogenicity avian influenza viruses (LPAIV) introduced by migratory birds circulate in wild birds and can be transmitted to poultry. These viruses can mutate to become highly pathogenic avian influenza viruses causing severe disease and death in poultry. In March 2019, an H7N3 avian influenza virus-A/Spot-billed duck/South Korea/WKU2019-1/2019 (H7N3)-was isolated from spot-billed ducks in South Korea. This study aimed to evaluate the phylogenetic and mutational analysis of this isolate. Molecular analysis revealed that the genes for HA (hemagglutinin) and NA (neuraminidase) of this strain belonged to the Central Asian lineage, whereas genes for other internal proteins such as polymerase basic protein 1 (PB1), PB2, nucleoprotein, polymerase acidic protein, matrix protein, and non-structural protein belonged to that of the Korean lineage. In addition, a monobasic amino acid (PQIEPR/GLF) at the HA cleavage site, and the non-deletion of the stalk region in the NA gene indicated that this isolate was a typical LPAIV. Nucleotide sequence similarity analysis of HA revealed that the highest homology (99.51%) of this isolate is to that of A/common teal/Shanghai/CM1216/2017 (H7N7), and amino acid sequence of NA (99.48%) was closely related to that of A/teal/Egypt/MB-D-487OP/2016 (H7N3). An in vitro propagation of the A/Spot-billed duck/South Korea/WKU2019-1/2019 (H7N3) virus showed highest (7.38 Log10 TCID50/mL) virus titer at 60 h post-infection, and in experimental mouse lungs, the virus was detected at six days' post-infection. Our study characterizes genetic mutations, as well as pathogenesis in both in vitro and in vivo model of a new Korea H7N3 viruses in 2019, carrying multiple potential mutations to become highly pathogenic and develop an ability to infect humans; thus, emphasizing the need for routine surveillance of avian influenza viruses in wild birds.

Emergence of Novel Reassortant H1N1 Avian Influenza Viruses in Korean Wild Ducks in 2018 and 2019.

Influenza A virus subtype H1N1 has caused global pandemics like the "Spanish flu" in 1918 and the 2009 H1N1 pandemic several times. H1N1 remains in circulation and survives in multiple animal sources, including wild birds. Surveillance during the winter of 2018-2019 in Korea revealed two H1N1 isolates in samples collected from wild bird feces: KNU18-64 (A/Greater white-fronted goose/South Korea/KNU18-64/2018(H1N1) and WKU19-4 (A/wild bird/South Korea/WKU19-4/2019(H1N1). Phylogenetic analysis indicated that M gene of KNU18-64(H1N1) isolate resembles that of the Alaskan avian influenza virus, whereas WKU19-4(H1N1) appears to be closer to the Mongolian virus. Molecular characterization revealed that they harbor the amino acid sequence PSIQRSGLF and are low-pathogenicity influenza viruses. In particular, the two isolates harbored three different mutation sites, indicating that they have different virulence characteristics. The mutations in the PB1-F2 and PA protein of WKU19-4(H1N1) indicate increasing polymerase activity. These results corroborate the kinetic growth data for WKU19-4 in MDCK cells: a dramatic increase in the viral titer after 12 h post-inoculation compared with that in the control group H1N1 (CA/04/09(pdm09)). The KNU18-64(H1N1) isolate carries mutations indicating an increase in mammal adaptation; this characterization was confirmed by the animal study in mice. The KNU18-64(H1N1) group showed the presence of viruses in the lungs at days 3 and 6 post-infection, with titers of 2.71 ± 0.16 and 3.71 ± 0.25 log10(TCID50/mL), respectively, whereas the virus was only detected in the WKU19-4(H1N1) group at day 6 post-infection, with a lower titer of 2.75 ± 0.51 log10(TCID50/mL). The present study supports the theory that there is a relationship between Korea and America with regard to reassortment to produce novel viral strains. Therefore, there is a need for increased surveillance of influenza virus circulation in free-flying and wild land-based birds in Korea, particularly with regard to Alaskan and Asian strains.

Fluorescent Immunosorbent Assay for Chikungunya Virus Detection.

BACKGROUND: When infected with the chikungunya virus (CHIKV), 3% to 28% of CHIKV-infected individuals remain asymptomatic, necessitating the development of improved high-throughput screening methods to overcome the limitations of molecular diagnostics or enzyme-linked immunosorbent assays (ELISAs). OBJECTIVE: In this study, two novel monoclonal antibodies (mAbs) targeting envelope 1 (E1) of CHIKV were developed and applied in a fluorescence-linked immunosorbent assay (FLISA) using coumarin-derived dendrimer as the fluorophore. METHODS: The performance of the FLISA was compared with that of ELISA. RESULTS: Using the two novel mAbs (2B5 and 2C8), FLISA could detect 1 × 105 PFU/mL of CHIKV, exhibiting a 2-fold lower limit of detection (LOD) compared to ELISA. The LOD of FICT corresponded to a comparative threshold value of 23.95 and 4 × 106 of RNA copy number/µL. In the presence of human sera and blood, virus detection by FLISA was 3-fold better than ELISA, with an LOD of 2 × 105 PFU/mL. Sera and blood interfered with the ELISA, resulting in 6 × 105 PFU/mL as the LOD. CONCLUSIONS: FLISA using two novel mAbs and coumarin-derived dendrimer is a superior diagnostic assay for detecting CHIKV in human sera and blood, compared to conventional ELISA.