Drug delivery and antibiofilm efficacy of nano silver fluoride sustained release orthodontic elastomerics against Streptococcus mutans.

We aimed to investigate the chemical and physical properties of nano silver fluoride sustained release orthodontic elastomerics (NSF-RE) and determine their antimicrobial and antibiofilm formation activities against Streptococcus mutans. Orthodontic elastomerics were dip-coated with NSF solution in ethyl cellulose (EC) and polyethylene glycol 6000 (PEG). The studied groups included NSF (no EC/PEG), NSF-E (EC), NSF-EP1 (EC:PEG, 4:1), and NSF-EP2 (EC:PEG, 2:1). The cumulative release of silver nanoparticles (AgNPs) and fluoride, along with the compatibility of the tensile force with orthodontic brackets, was evaluated. The antimicrobial activity was evaluated using an agar diffusion test. The inhibition of biofilm formation was evaluated using colony-forming units (CFUs), biofilm thickness, and the live/dead cell ratio. NSF-RE containing EC sustained the release of AgNPs and fluoride for > 7 days. Tensile forces were not significantly different among the groups. The inhibition zone was 2.64- and 1.31-fold larger with NSF-EP2 than that with NSF and NSF-E, respectively. NSF-EP2 was the most effective in inhibiting biofilm formation with significant reductions in CFUs, biofilm thickness, and live/dead cell ratio by 57, 86, and 96%, respectively, as compared to those in the control group. Overall, sustained release of AgNPs and fluoride by NSF-RE provides antimicrobial and antibiofilm effects against S. mutans.

Highly sensitive and label-free protein immunoassay-based biosensor comprising infrared metamaterial absorber inducing strong coupling.

A mid-infrared label-free immunoassay-based biosensor is an effective device to help identify and quantify biomolecules. This biosensor employs a surface-enhanced infrared absorption spectroscopy, which is a highly potent sensing technique for detecting minute quantities of analytes. In this study, a biosensor was constructed using a metamaterial absorber, which facilitated strong coupling effects. For maximum coupling effect, it is necessary to enhance the near-field intensity and the spatial and spectral overlap between the optical cavity resonance and the vibrational mode of the analyte. Due to significant peak splitting, conventional baseline correction methods fail to adequately analyze such a coupling system. Therefore, we employed a coupled harmonic oscillation model to analyze the spectral distortion resulting from the peak splitting induced by the strong coupling effect. The proposed biosensor with a thrombin-binding aptamer-based immunoassay could achieve a limit of detection of 267.4 pM, paving the way for more efficient protein detection in clinical practice.

Design of High-Payload Ascorbyl Palmitate Nanosuspensions for Enhanced Skin Delivery.

A high-payload ascorbyl palmitate (AP) nanosuspension (NS) was designed to improve skin delivery following topical application. The AP-loaded NS systems were prepared using the bead-milling technique, and softly thickened into NS-loaded gel (NS-G) using hydrophilic polymers. The optimized NS-G system consisted of up to 75 mg/mL of AP, 0.5% w/v of polyoxyl-40 hydrogenated castor oil (Kolliphor® RH40) as the suspending agent, and 1.0% w/v of sodium carboxymethyl cellulose (Na.CMC 700 K) as the thickening agent, in citrate buffer (pH 4.5). The NS-G system was embodied as follows: long and flaky nanocrystals, 493.2 nm in size, -48.7 mV in zeta potential, and 2.3 cP of viscosity with a shear rate of 100 s-1. Both NS and NS-G provided rapid dissolution of the poorly water-soluble antioxidant, which was comparable to that of the microemulsion gel (ME-G) containing AP in solubilized form. In an ex vivo skin absorption study using the Franz diffusion cell mounted on porcine skin, NS-G exhibited faster absorption in skin, providing approximately 4, 3, and 1.4 times larger accumulation than that of ME-G at 3, 6, and 12 h, respectively. Therefore, the high-payload NS makes it a promising platform for skin delivery of the lipid derivative of ascorbic acid.

R-loop-derived cytoplasmic RNA-DNA hybrids activate an immune response.

R-loops are RNA-DNA-hybrid-containing nucleic acids with important cellular roles. Deregulation of R-loop dynamics can lead to DNA damage and genome instability1, which has been linked to the action of endonucleases such as XPG2-4. However, the mechanisms and cellular consequences of such processing have remained unclear. Here we identify a new population of RNA-DNA hybrids in the cytoplasm that are R-loop-processing products. When nuclear R-loops were perturbed by depleting the RNA-DNA helicase senataxin (SETX) or the breast cancer gene BRCA1 (refs. 5-7), we observed XPG- and XPF-dependent cytoplasmic hybrid formation. We identify their source as a subset of stable, overlapping nuclear hybrids with a specific nucleotide signature. Cytoplasmic hybrids bind to the pattern recognition receptors cGAS and TLR3 (ref. 8), activating IRF3 and inducing apoptosis. Excised hybrids and an R-loop-induced innate immune response were also observed in SETX-mutated cells from patients with ataxia oculomotor apraxia type 2 (ref. 9) and in BRCA1-mutated cancer cells10. These findings establish RNA-DNA hybrids as immunogenic species that aberrantly accumulate in the cytoplasm after R-loop processing, linking R-loop accumulation to cell death through the innate immune response. Aberrant R-loop processing and subsequent innate immune activation may contribute to many diseases, such as neurodegeneration and cancer.

Match activity profile of professional female soccer players during a season.

It is necessary to understand movement characteristics of elite female soccer players during a match to develop effective training program. The purpose of this study is to analyze the physical demands of Korean female professional soccer players during a competitive season. Twenty-four female professional players (age: 27.8±3.9 years; height: 165.7± 5.1 cm) from a team, belonging to the women's professional soccer league in South Korea participated in the study. The players participated in 11 home matches and 10 away matches from April 26 to November 9 of the 2011 season. Body weight and body mass index decreased during the first half-season compared to the preseason (P<0.05), and the decreased values were maintained until the second half-season. Total distance covered by the players was more than 9.5 km per match. No difference was found in the total distance, movement distance by exercise intensities, number of sprints and accelerations, and maximum speed between the first half- and second half-seasons. Midfielders covered the most distance during a match compared to other positions (P<0.05). Wing forward covered the most distance of high-intensity exercise. Maximum speed during the match was higher among wing forward and forward than in other positions (P<0.05). There were no significant differences between home and away matches in all variables (P>0.05). In conclusion, in order to improve the performance of female soccer players and prevent injuries, a training program should be constructed that considers the characteristics of each player and playing position rather than uniform training in a team.

Design of Montelukast Nanocrystalline Suspension for Parenteral Prolonged Delivery.

Background: Montelukast (MTK), a representative leukotriene receptor antagonist, is currently being investigated as a potential candidate for treating Alzheimer's disease. For potent and effective dosing in elderly patients, a parenteral prolonged delivery system is favored, with improved medication adherence with reduced dosage frequency. Purpose: This study aimed to design a nanocrystalline suspension (NS)-based MTK prolonged delivery system and evaluate its pharmacokinetics profile and local tolerability following subcutaneous administration. Methods: To decelerate the dissolution rate, the amorphous MTK raw material was transformed into a crystalline state using a solvent-mediated transformation method and subsequently formulated into NS using a bead-milling technique. The MTK NSs were characterized by morphology, particle size, crystallinity, and in vitro dissolution profiles. The pharmacokinetic profile and local tolerability at the injection site following subcutaneous injection of MTK suspension were evaluated in rats. Results: Microscopic and physical characterization revealed that the amorphous MTK powder was lucratively transformed into a crystalline form in acidic media (pH 4). MTK crystalline suspensions with different diameters (200 nm, 500 nm, and 3 µm) were uniformly prepared using bead-milling technology, employing polysorbate 80 as suspending agent. Prepared crystalline suspensions exhibited analogous crystallinity (melting point, 150°C) and size-dependent in vitro dissolution profiles. MTK NSs with particle sizes of 200 nm and 500 nm provided a protracted pharmacokinetic profile for up to 4 weeks in rats, with a higher maximum drug concentration in plasma than the 3 µm-sized injectable suspensions. Histopathological examination revealed that MTK NS caused chronic granulomatous inflammation at the injection site, which resolved after 4 weeks. Conclusion: The MTK parenteral NS delivery system is expected to be a valuable tool for treating Alzheimer's disease with extended dose intervals.

Anti-biofilm activity of chlorhexidine-releasing elastomerics against dental microcosm biofilms.

OBJECTIVE: To evaluate the anti-biofilm activity of chlorhexidine-releasing elastomerics (CRE) developed to prevent biofilm-related diseases in orthodontic patients, using dental microcosm biofilms. METHODS: Elastomerics coated with one of two solutions (CRE 1 and 2) were attached to bovine enamel specimens. Uncoated elastomerics were used for negative (distilled water [DW]) and positive (0.1% chlorhexidine [CHX]) control groups. After saliva inoculation on the surface of the specimen for biofilm formation, DW and CRE groups were treated with DW, and the positive control group was treated with CHX twice a day for 5 min. After 7 days of biofilm formation, colony-forming units (CFUs, total and aciduric bacteria), red/green (R/G) ratio, biofilm thickness, live/dead cell ratio, and bacterial morphology in the biofilms were evaluated. Enamel demineralization was evaluated by fluorescence loss (ΔF). RESULTS: The CFUs of total and aciduric bacteria and R/G ratios in the CRE groups were significantly lower than those in the DW group with a reduction by 13%, 13%, and 19%, respectively (p < 0.05). The CFUs of total bacteria was significantly lower in the CRE groups than in the 0.1% CHX group (p < 0.05). Among the CRE groups, only CRE 1 exhibited a significantly reduced biofilm thickness of 54% compared to the DW group (p < 0.05) and apparent changes in bacterial morphology. ΔF in the CRE groups was significantly higher by 36% compared to that in the DW group (p < 0.05). CONCLUSIONS: CREs exhibited anti-biofilm and demineralization-inhibiting effect. Particularly, CRE 1 using dichloromethane as the solvent was most effective against biofilms. CLINICAL SIGNIFICANCE: Chlorhexidine-releasing elastomerics exhibited increased anti-biofilm and demineralization-inhibiting effect compared to 0.1% chlorhexidine mouthwash. Therefore, it is possible to prevent biofilm-related diseases simply and effectively by applying chlorhexidine-releasing elastomerics to orthodontic patients.

TREX1 degrades the 3' end of the small DNA oligonucleotide products of nucleotide excision repair in human cells.

The nucleotide excision repair (NER) machinery removes UV photoproducts from DNA in the form of small, excised damage-containing DNA oligonucleotides (sedDNAs) ∼30 nt in length. How cells process and degrade these byproducts of DNA repair is not known. Using a small scale RNA interference screen in UV-irradiated human cells, we identified TREX1 as a major regulator of sedDNA abundance. Knockdown of TREX1 increased the level of sedDNAs containing the two major UV photoproducts and their association with the NER proteins TFIIH and RPA. Overexpression of wild-type but not nuclease-inactive TREX1 significantly diminished sedDNA levels, and studies with purified recombinant TREX1 showed that the enzyme efficiently degrades DNA located 3' of the UV photoproduct in the sedDNA. Knockdown or overexpression of TREX1 did not impact the overall rate of UV photoproduct removal from genomic DNA or cell survival, which indicates that TREX1 function in sedDNA degradation does not impact NER efficiency. Taken together, these results indicate a previously unknown role for TREX1 in promoting the degradation of the sedDNA products of the repair reaction. Because TREX1 mutations and inefficient DNA degradation impact inflammatory and immune signaling pathways, the regulation of sedDNA degradation by TREX1 may contribute to photosensitive skin disorders.

Active DNA damage eviction by HLTF stimulates nucleotide excision repair.

Nucleotide excision repair (NER) counteracts the onset of cancer and aging by removing helix-distorting DNA lesions via a "cut-and-patch"-type reaction. The regulatory mechanisms that drive NER through its successive damage recognition, verification, incision, and gap restoration reaction steps remain elusive. Here, we show that the RAD5-related translocase HLTF facilitates repair through active eviction of incised damaged DNA together with associated repair proteins. Our data show a dual-incision-dependent recruitment of HLTF to the NER incision complex, which is mediated by HLTF's HIRAN domain that binds 3'-OH single-stranded DNA ends. HLTF's translocase motor subsequently promotes the dissociation of the stably damage-bound incision complex together with the incised oligonucleotide, allowing for an efficient PCNA loading and initiation of repair synthesis. Our findings uncover HLTF as an important NER factor that actively evicts DNA damage, thereby providing additional quality control by coordinating the transition between the excision and DNA synthesis steps to safeguard genome integrity.

XPA is susceptible to proteolytic cleavage by cathepsin L during lysis of quiescent cells.

The xeroderma pigmentosum group A (XPA) protein plays an essential role in the removal of UV photoproducts and other bulky lesions from DNA as a component of the nucleotide excision repair (NER) machinery. Using cell lysates prepared from confluent cultures of human cells and from human skin epidermis, we observed an additional XPA antibody-reactive band on immunoblots that was approximately 3-4 kDa smaller than the native, full-length XPA protein. Biochemical studies revealed this smaller molecular weight XPA species to be due to proteolysis at the C-terminus of the protein, which negatively impacted the ability of XPA to interact with the NER protein TFIIH. Further work identified the endopeptidase cathepsin L, which is expressed at higher levels in quiescent cells, as the protease responsible for cleaving XPA during cell lysis. These results suggest that supplementation of lysis buffers with inhibitors of cathepsin L is important to prevent cleavage of XPA during lysis of confluent cells.

Ultrasensitive Molecule Detection Based on Infrared Metamaterial Absorber with Vertical Nanogap.

Surface-enhanced infrared absorption (SEIRA) spectroscopy is a powerful methodology for sensing and identifying small quantities of analyte molecules via coupling between molecular vibrations and an enhanced near-field induced in engineered structures. A metamaterial absorber (MA) is proposed as an efficient SEIRA platform; however, its efficiency is limited because it requires the appropriate insulator thickness and has a limited accessible area for sensing. SEIRA spectroscopy is proposed using an MA with a 10 nm thick vertical nanogap, and a record-high reflection difference SEIRA signal of 36% is experimentally achieved using a 1-octadecanethiol monolayer target molecule. Theoretical and experimental comparative studies are conducted using MAs with three different vertical nanogaps. The MAs with a vertical nanogap are processed using nanoimprint lithography and isotropic dry etching, which allow cost-effective large-area patterning and mass production. The proposed structure may provide promising routes for ultrasensitive sensing and detection applications.

High-Payload Nanosuspension of Centella asiatica Extract for Improved Skin Delivery with No Irritation.

BACKGROUND: The titrated extract of Centella asiatica (CA) has received much attention as a cosmeceutical ingredient owing to its anti-wrinkle effect. However, due to the low solubility and high molecular weight of pharmacologically active constituents, including asiatic acid (AA), madecassic acid (MA), and asiaticoside (AS), it is challenging to fabricate high-payload topical preparations of CA with satisfactory skin absorption profiles. PURPOSE: This study aimed to design a high-payload topical preparation of CA using nanocrystallization technique and to evaluate its skin absorption profile and local tolerability. METHODS: High-payload nanocrystal suspensions (NSs) were prepared using lab-scale bead-milling technology, by adjusting the type and amount of suspending agent, CA content, type of vehicle, and milling speed. CA-loaded NSs were characterized in terms of morphology, particle size, crystallinity, and in vitro dissolution pattern. Skin absorption of CA nanocrystals was evaluated using a vertical Franz diffusion cell mounted with porcine skin. In vivo skin irritation following topical application of high-payload NS was assessed in normal rats. RESULTS: The optimized NS system, composed of 10% (w/v) CA, 0.5% polyvinylpyrrolidone (PVP) K30 as steric stabilizer, and 89.5% of distilled water, was characterized as follows: spherical or elliptical in shape, 200 nm in size, with low crystallinity. The in vitro dissolution of AA or MA from NSs was markedly faster compared to raw material, under sink condition. Penetration of AA, MA, and AS in the porcine skin was markedly elevated using the high-payload NS formula, providing 5-, 4-, and 4.5-fold higher accumulation in skin layer, compared to that of the marketed cream formula (CA 1%, Madeca cream). Moreover, topical application of high-payload NS was tolerable, showing neither erythema nor oedema in normal rats. CONCLUSION: The novel NS system is expected to be a virtuous approach for offering a better skin absorption of CA, without using an excess quantity of solubilizers.

Multi-Domain Convolutional Neural Networks for Lower-Limb Motor Imagery Using Dry vs. Wet Electrodes.

Motor imagery (MI) brain-computer interfaces (BCIs) have been used for a wide variety of applications due to their intuitive matching between the user's intentions and the performance of tasks. Applying dry electroencephalography (EEG) electrodes to MI BCI applications can resolve many constraints and achieve practicality. In this study, we propose a multi-domain convolutional neural networks (MD-CNN) model that learns subject-specific and electrode-dependent EEG features using a multi-domain structure to improve the classification accuracy of dry electrode MI BCIs. The proposed MD-CNN model is composed of learning layers for three domain representations (time, spatial, and phase). We first evaluated the proposed MD-CNN model using a public dataset to confirm 78.96% classification accuracy for multi-class classification (chance level accuracy: 30%). After that, 10 healthy subjects participated and performed three classes of MI tasks related to lower-limb movement (gait, sitting down, and resting) over two sessions (dry and wet electrodes). Consequently, the proposed MD-CNN model achieved the highest classification accuracy (dry: 58.44%; wet: 58.66%; chance level accuracy: 43.33%) with a three-class classifier and the lowest difference in accuracy between the two electrode types (0.22%, d = 0.0292) compared with the conventional classifiers (FBCSP, EEGNet, ShallowConvNet, and DeepConvNet) that used only a single domain. We expect that the proposed MD-CNN model could be applied for developing robust MI BCI systems with dry electrodes.

Inducible Prmt1 ablation in adult vascular smooth muscle leads to contractile dysfunction and aortic dissection.

Vascular smooth muscle cells (VSMCs) have remarkable plasticity in response to diverse environmental cues. Although these cells are versatile, chronic stress can trigger VSMC dysfunction, which ultimately leads to vascular diseases such as aortic aneurysm and atherosclerosis. Protein arginine methyltransferase 1 (Prmt1) is a major enzyme catalyzing asymmetric arginine dimethylation of proteins that are sources of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase. Although a potential role of Prmt1 in vascular pathogenesis has been proposed, its role in vascular function has yet to be clarified. Here, we investigated the role and underlying mechanism of Prmt1 in vascular smooth muscle contractility and function. The expression of PRMT1 and contractile-related genes was significantly decreased in the aortas of elderly humans and patients with aortic aneurysms. Mice with VSMC-specific Prmt1 ablation (smKO) exhibited partial lethality, low blood pressure and aortic dilation. The Prmt1-ablated aortas showed aortic dissection with elastic fiber degeneration and cell death. Ex vivo and in vitro analyses indicated that Prmt1 ablation significantly decreased the contractility of the aorta and traction forces of VSMCs. Prmt1 ablation downregulated the expression of contractile genes such as myocardin while upregulating the expression of synthetic genes, thus causing the contractile to synthetic phenotypic switch of VSMCs. In addition, mechanistic studies demonstrated that Prmt1 directly regulates myocardin gene activation by modulating epigenetic histone modifications in the myocardin promoter region. Thus, our study demonstrates that VSMC Prmt1 is essential for vascular homeostasis and that its ablation causes aortic dilation/dissection through impaired myocardin expression.

Probing TGF-ß1-induced cytoskeletal rearrangement by fluorescent-labeled silica nanoparticle uptake assay.

Cytoskeletal proteins are essential in maintaining cell morphology, proliferation, and viability as well as internalizing molecules in phagocytic and non-phagocytic cells. Orderly aligned cytoskeletons are disturbed by a range of biological processes, such as the epithelial-mesenchymal transition, which is observed in cancer metastasis. Although many biological methods have been developed to detect cytoskeletal rearrangement, simple and quantitative in vitro approaches are still in great demand. Herein, we applied a flow cytometry-based nanoparticle uptake assay to measure the degree of cytoskeletal rearrangement induced by transforming growth factor ß1 (TGF-ß1). For the assay, silica nanoparticles, selected for their high biocompatibility, were fluorescent-labeled to facilitate quantification with flow cytometry. Human keratinocyte HaCaT cells were treated with different concentrations of TGF-ß1 and then exposed to FITC-labeled silica nanoparticles. Increasing concentrations of TGF-ß1 induced gradual changes in cytoskeletal rearrangement, as confirmed by conventional assays. The level of nanoparticle uptake increased by TGF-ß1 treatment in a dose-dependent manner, indicating that our nanoparticle uptake assay can be used as a quick and non-destructive approach to measure cytoskeletal rearrangement.

Enhancement of refractive index sensing for an infrared plasmonic metamaterial absorber with a nanogap.

An infrared plasmonic metamaterial absorber with a nanogap was numerically and experimentally investigated as a refractive index sensor. We experimentally demonstrated large enhancements of both sensitivity (approximately 1091 nm/refractive index unit) and figure of merit (FOM*; approximately 273) owing to the nanogap formation in the metamaterial absorber to achieve perfect absorption (99%). The refractive index sensing platform was fabricated by producible nanoimprint lithography and isotropic dry etching processes to have a large area and low cost while providing a practical solution for high-performance plasmonic biosensors.

Preparation of multilayer periodic nanopatterned WO3-based photoanode by reverse nanoimprinting for water splitting.

Photoelectrochemical (PEC) water splitting has been studied extensively as an environmentally friendly technology for hydrogen production using solar energy. WO3is considered a promising semiconducting material for photoanodes due to its high electron mobility, good hole diffusion length, and chemical stability. Periodic nanostructures of WO3have been investigated for enhancing the PEC performance of WO3-based photoanodes. In this study, facile fabrication of periodic nanostructures of WO3was achieved using reverse nanoimprint lithography, and the multilayer stacking of nanopatterned WO3film was also confirmed. The multilayer nanopatterned WO3films were used as photoanodes for PEC water splitting. The performance of the fabricated photoanode in PEC was 2 times higher than that of planar WO3film due to its higher light absorbance and lower charge transfer resistance.

Lin28a attenuates TGF-ß-induced renal fibrosis.

Lin28a has diverse functions including regulation of cancer, reprogramming and regeneration, but whether it promotes injury or is a protective reaction to renal injury is unknown. We studied how Lin28a acts in unilateral ureteral obstruction (UUO)-induced renal fibrosis following unilateral ureteral obstruction, in a mouse model. We further defined the role of Lin28a in transforming growth factor (TGF)-signaling pathways in renal fibrosis through in vitro study using human tubular epithelium-like HK-2 cells. In the mouse unilateral ureteral obstruction model, obstruction markedly decreased the expression of Lin28a, increased the expression of renal fibrotic markers such as type I collagen, α-SMA, vimentin and fibronectin. In TGF-ß-stimulated HK-2 cells, the expression of Lin28a was reduced and the expression of renal fibrotic markers such as type I collagen, α-SMA, vimentin and fibronectin was increased. Adenovirus-mediated overexpression of Lin28a inhibited the expression of TGF-ß-stimulated type I collagen, α-SMA, vimentin and fibronectin. Lin28a inhibited TGF-ß-stimulated SMAD3 activity, via inhibition of SMAD3 phosphorylation, but not the MAPK pathway ERK, JNK or p38. Lin28a attenuates renal fibrosis in obstructive nephropathy, making its mechanism a possible therapeutic target for chronic kidney disease. [BMB Reports 2020; 53(11): 594-599].

Detection of the small oligonucleotide products of nucleotide excision repair in UVB-irradiated human skin.

UVB radiation results in the formation of potentially mutagenic photoproducts in the DNA of epidermal skin cells. In vitro approaches have demonstrated that the nucleotide excision repair (NER) machinery removes UV photoproducts from DNA in the form of small (∼30-nt-long), excised, damage-containing DNA oligonucleotides (sedDNAs). Though this process presumably takes place in human skin exposed to UVB radiation, sedDNAs have not previously been detected in human skin. Using surgically discarded human skin, we have optimized the detection of the sedDNA products of NER from small amounts of human epidermal tissue ex vivo within minutes of UVB exposure and after UVB doses that normally lead to minimal erythema. Moreover, sedDNA generation was inhibited by treatment of skin explants with spironolactone, which depletes the epidermis of the essential NER protein XPB to mimic the skin of xeroderma pigmentosum patients. Time course experiments revealed that a partially degraded form of the sedDNAs could be readily detected even 12 hours following UVB exposure, which indicates that these repair products are relatively stable in human skin epidermis. Together, these data suggest that sedDNA detection may be a useful assay for determining how genetic, environmental, and other factors influence NER activity in human skin epidermis and whether abnormal sedDNA processing contributes to photosensitive skin disorders.