Mouse Ear Research Articles

Traditional Use, Chemical Constituents, and Pharmacological Activity of Maytenus elaeodendroides Stem Bark

Plants belonging to the genus Maytenus are members of the Celastraceae family. They have been widely used by different peoples as treatment for curing many diseases. The aim of this study was to explore the anti-inflammatory and antioxidant properties of Maytenus elaeodendroides stem bark extracts, an endemic Cuban plant. The antioxidant activity of four extracts (EtOH, EtOAc, n-BuOH, and diethyl ether/petroleum ether 1:1) was determined using DPPH and FRAP methods. Meanwhile, anti-inflammatory effects by the edema method were induced by croton oil in the mouse ear. The investigated extracts showed radical reduction capacity and prevented ear inflammation at doses of 4 mg/ear. In addition, FIA/ESI/IT/MSn was used to determine the qualitative chemical composition of the EtOAc extract and allowed the identification of five flavan-3-ol monomers, four dimers, and other proanthocyanidin oligomers. From this extract three flavan-3-ol compounds (elaeocyanidin and 4′-O-methylgallocatechin), one of them new (2′-hydroxy-4′-methoxy-epigallocatechin), and a proanthocyanidin dimer (afzelechin-(4β→8)-4′-O-methylepigallocatechin) were isolated and identified by the chromatographic method and spectroscopic techniques, mainly ESI-MS and NMR spectroscopic methods.

Large depth-of-field ultraviolet-visible photoacoustic histologic and microvascular imaging in vivo

Photoacoustic microscopy (PAM) can image diverse biological microstructures by harnessing characteristic optical absorption spectra of intrinsic nonfluorescent biomolecules. We incorporate ultraviolet (UV) and visible pulsed lasers into a PAM to develop an UV-visible PAM for label-free histologic and microvascular imaging, where the cell nuclei and blood vessels are specifically captured at high contrast relying on strong optical absorption of DNA/RNA and hemoglobin at 266 and 532 nm wavelengths, respectively. Moreover, two diffractive optical elements operating at UV and visible spectra are designed for engineering the excitation beams, significantly enlarging depths of field (DOFs > 200 μm). The UV-visible PAM demonstrates combined capabilities of dual imaging contrast, elongated DOFs, and micrometer-scale lateral resolution for delineating the spatial microarchitectures of both cell nuclei and blood vessels that are at different depth locations in the biological specimens with uneven surfaces. Longitudinal monitoring of the trauma is performed in mouse ear in vivo. Potentially, our UV-visible PAM could offer comprehensive histologic and microvascular information in a broad range of biomedical studies.

Antioxidant activity and whitening effect of vitamin C and superoxide dismutase from Rosa roxburghii Tratt

Abstract With growing concerns about the negative impact of environmental pollution on skin health, there is an increased demand for natural active ingredients to combat free radicals and excessive melanin production. We focus on the Rosa roxburghii Tratt (RR) as a potential source of skin-lightening compounds. Vitamin C (Vc) and superoxide dismutase (SOD)-rich extracts (RRVC and RRSOD) were extracted from RR using macroporous resin column chromatography and graded ammonium sulfate precipitation. The antioxidant and skin-lightening effects of these extracts were evaluated. RRVC and RRSOD showed strong inhibitory activity against various radicals and tyrosinase. When formulated together, their antioxidant and skin-lightening abilities were significantly enhanced, with an optimal ratio of 5:32 (RRVS). In vivo studies on C57BL/6J mice revealed that these two extracts and their formulations effectively lowered oxidative stress levels in a model of D-galactose-induced aging in mice. Furthermore, they inhibited melanogenesis, and tyrosinase activity in B16 cells and reduced UV (ultraviolet radiation)-induced melanin deposition in mouse ears. The combination of RRVC and RRSOD had a more potent melanin-scavenging effect compared with the individual extracts. These findings suggest that RRVC and RRSOD extracts, especially when combined in the optimal ratio, can whiten skin, both enhancing the value of RR products and offering natural alternatives for skin lightening.

Label-Free Non-Contact Vascular Imaging using Photon Absorption Remote Sensing.

Functional vascular imaging is a critical method for early detection and prevention of disease. Established non-contact vascular imaging techniques capture predominantly structural information. In this study, a novel non-contact label-free in vivo Photon Absorption Remote Sensing (PARS) microscope is developed for structural and functional vascular imaging. The presented in vivo PARS microscope captures the endogenous absorption of green (532nm) light to form a complete picture of vasculature and surrounding tissues. Imaging system repeatability is enhanced through robust transient absorption signal extraction, and state-of-the-art real-time alignment methods. Detailed imaging of vascular structure is demonstrated through in vivo microscopy of two established animal models: mouse ear and chicken embryo. Preliminary functional contrast is realized through video rate imaging of red blood cell dynamics in the capillary networks of chicken embryos. The presented in vivo PARS microscope successfully captures detailed structural and functional vascular contrast. This innovative non-contact label-free imaging technique holds promise as a tool for preventative medical care, as functional change often precedes structural change.

Spatial distribution and stability of Gd0.6Eu0.4VO4 nanoparticles injected in mouse ear pinnae

Spatial distribution and stability of Gd0.6Eu0.4VO4 nanoparticles injected in mouse ear pinnae

Position Matters: Effect of Nitro Group in Chalcones on Biological Activities and Correlation via Molecular Docking

A series of nine nitro group-containing chalcones were synthesized to investigate their anti-inflammatory and vasorelaxant activities via in vivo, ex vivo, and in silico studies. The anti-inflammatory effects of the compounds were evaluated via a TPA-induced mouse ear edema model, and the vasorelaxant effects were evaluated via an isolated organ model in addition to molecular docking studies. The compounds with the highest anti-inflammatory activity were 2 (71.17 ± 1.66%), 5 (80.77 ± 2.82%), and 9 (61.08 ± 2.06%), where the nitro group is located at the ortho position in both rings, as confirmed by molecular docking with COX-1 and COX-2. The compounds with the highest vasorelaxant activity were 1 (81.16 ± 7.55%), lacking a nitro group, and 7 (81.94 ± 2.50%), where the nitro group is in the para position of the B ring; both of these compounds interact with the eNOS enzyme during molecular docking. These results indicate that the position of the nitro group in the chalcone plays an important role in these anti-inflammatory and vasorelaxant activities.

3D Printed Microneedles for the Transdermal Delivery of NAD+ Precursor: Toward Personalization of Skin Delivery.

3D printing of microneedles (μNDs) for transdermal therapy has the potential to enable patient personalization based on the target disease, site of application, and dosage requirements. To convert this concept to reality, it is necessary that the 3D printing technology can deliver high resolution, an affordable cost, and large print volumes. With the introduction of benchtop 4K and 8K 3D printers, it is now possible to manufacture medical devices like μNDs at sufficient resolution and low cost. In this research, we systematically optimized the 3D printing design parameters such as resin viscosity, print angle, layer height, and curing time to generate customizable μNDs. We have also developed an innovative 3D coating microtank device to optimize the coating method. We have applied this to the development of novel μNDs to deliver an established NAD+ precursor molecule, nicotinamide mononucleotide (NMN). A methacrylate-based polymer photoresin (eSun resin) was diluted with methanol to adjust the resin viscosity. The 3D print layer height of 25 μm yielded a smooth surface, thus reducing edge-ridge mismatches. Printing μNDs at 90° to the print platform yielded 84.28 ± 2.158% (n = 5) of the input height thus increasing the tip sharpness (48.52 ± 10.43 μm, n = 5). The formulation containing fluorescein (model molecule), sucrose (viscosity modifier), and Tween-20 (surface tension modifier) was coated on the μNDs using the custom designed microtank setup, and the amount deposited was determined fluorescently. The dye-coated μND arrays inserted into human skin (in vitro) showed a fluorescence signal at a depth of 150 μm (n = 3) into the skin. After optimization of the 3D printing parameters and coating protocol using fluorescein, NMN was coated onto the μNDs, and its diffusion was assessed in full-thickness human skin in vitro using a Franz diffusion setup. Approximately 189 ± 34.5 μg (5× dipped coated μNDs) of NMN permeated through the skin and 41.2 ± 7.53 μg was left in the skin after 24 h. Multiphoton microscopy imaging of NMN-coated μND treated mouse ear skin ex vivo demonstrated significantly (p < 0.05) increased free-unbound NADPH and reduced fluorescence lifetime of NADPH, both of which are indicative of cellular metabolic rates. Our study demonstrates that low-cost benchtop 3D printers can be used to print high-fidelity μNDs with the ability to rapidly coat and release NMN which consequently caused changes in intracellular NAD+ levels.

Different allergen-induced asthma and anaphylaxis show distinctive IgE pathway dependence

BackgroundThe pathogenesis of allergic asthma is classically understood to involve allergens crosslinking Immunoglobulin E (IgE) bound to mast cells, leading to the release of histamine and other inflammatory mediators. ObjectiveThis study aims to explore whether different allergens have distinct IgE-dependent mechanisms in mouse models of both asthma and anaphylaxis. MethodsWild type (WT) and IgE knockout (IgE KO) mice were used to generate asthma mouse models. Lung inflammation was assessed by histological analysis and inflammatory cells in Bronchial Alveolar Lavage Fluids (BALF). Passive Cutaneous Anaphylaxis (PCA) was conducted to quantify IgE, and Toluidine Blue staining was used to visualize mast cell recruitment. ResultsAll allergen-treated mice exhibited significant airway inflammation. Compared to WT mice, HDM-treated IgE KO mice showed an attenuated inflammatory profile, particularly with reduced eosinophils. These mice had lower serum levels of HDM-specific IgE and IgG1 as well as reduced levels of Th2 cytokines interleukin 4 (IL-4) and IL-5 in BALF. CRE-treated IgE KO mice also displayed a reduced inflammation, with significant differences only in eosinophils. Interestingly, no significant differences in airway inflammation were observed between OVA-treated IgE KO mice and WT mice. Furthermore, both HDM and CRE-induced PCA model showed significantly less Evan's blue dye leakage and mast cell recruitment in the ears of IgE KO mice compared to WT mice, with no differences for OVA-induced PCA and mast cell recruitment. ConclusionThe results highlight the variability in IgE pathway dependence among different allergens, emphasizing the need for further research to delineate the underlying mechanisms.

Complete genome sequence of Pseudomonas aeruginosa strain NCTR 501 isolated from the inner ear of a laboratory mouse (Mus musculus) diagnosed with otitis media.

We report the circularized 6,427,509-bp genome of Pseudomonas aeruginosa isolated from the inner ear of a laboratory mouse (Mus musculus) diagnosed with otitis media. The genome of P. aeruginosa NCTR 501 has a circular chromosome of 6,420,288 bp and two plasmids of 4,042 and 3,179 bp, respectively.

Long-Term, Non-Invasive Detection of Low-Dose Ionizing Radiation Exposure

Ionizing radiation induces complex changes in cells and tissues. The conventional approach to biological dosimetry has been to integrate physical and clinical measurements to optimize dose assessment. Molecular biodosimetry is an effective strategy to monitor radiation exposure and hematologic, cytogenetic, protein and transcript-based approaches have been developed to increase dose estimation accuracy. However, these approaches are invasive, time-consuming, have limited effectiveness over time, and importantly do not accurately inform on low dose radiation exposures. Therefore, novel, non-invasive, biomarkers are required that can overcome these limitations. We developed a pipeline that employs Fourier transform infrared (FTIR) spectroscopy in the mid-infrared spectrum to identify a signature of low dose ionizing radiation exposure in mouse ear pinnae over time. Two cohorts of C57BL/6J and one cohort of BALB/c mice were followed for ninety days after total body X-ray exposures of 10, 50, 100 or 200 cGy. The stratum corneum layer of the ear pinnae were repeatedly measured with attenuated total reflectance - focal plane array (ATR-FPA)-FTIR at 5, 14, 21, 49 and 90 days after radiation exposure. We found statistically significant discriminative power for all doses and all tested time-points out to 90 days after exposure. Classification accuracy was maximized when testing 14 days after exposure (specificity > 0.9 with a sensitivity threshold of 0.9) and dropped by roughly 30% sensitivity at 90 days. Since only hundreds of samples were required to learn highly discriminative signatures, developing human-relevant diagnostic capabilities is likely feasible and this non-invasive procedure points toward future non-invasive biodosimetry applications at population scales.

A Near-Infrared-II Excitable Pyridinium Probe with 1000-Fold ON/OFF Ratio for γ-Glutamyltranspeptidase and Cancer Detection.

Activity-based detection of γ-Glutamyltranspeptidase (GGT) using near-infrared (NIR) fluorescent probes is a promising strategy for early cancer diagnosis. Although NIR pyridinium probes show high performance in biochemical analysis, the aggregation of both the probes and parental fluorochromes in biological environments is prone to result in a low signal-to-noise ratio (SBR), thus affecting their clinical applications. Here, we develop a GGT-activatable aggregate probe called OTBP-G for two-photon fluorescence imaging in various biological environments under 1040 nm excitation. By rationally tunning the hydrophilicity and donor-acceptor strength, we enable a synergistic effect between twisted intramolecular charge transfer and intersystem crossing processes and realize a perfect dark state for OTBP-G before activation. After the enzymatic reaction, the parental fluorochrome exhibits bright aggregation-induced emission peaking at 670 nm. The fluorochrome-to-probe transformation can induce 1000-fold fluorescence ON/OFF ratio, realizing in vitro GGT detection with an SBR > 900. Activation of OTBP-G occurs within 1 min in vivo, showing an SBR > 400 in mouse ear blood vessels. OTBP-G can further enable the early detection of pulmonary metastasis in breast cancer by topically spraying, outperforming the clinical standard hematoxylin and eosin staining. We anticipate that the in-depth study of OTBP-G can prompt the development of early cancer diagnosis and tumor-related physiological research. Moreover, this work highlights the crucial role of hydrophilicity and donor-acceptor strength in maximizing the ON/OFF ratio of the TICT probes and showcases the potential of OTBP as a versatile platform for activity-based sensing.

Design and Synthesis of sEH/HDAC6 Dual-Targeting Inhibitors for the Treatment of Inflammatory Pain.

Soluble epoxide hydrolase (sEH) and HDAC6 mediate the NF-κB pathway in inflammatory responses, and their inhibitors exhibit powerful anti-inflammatory and analgesic activities in treating both inflammation and pain. Therefore, a series of dual-targeting inhibitors containing urea or squaramide and hydroxamic acid moieties were designed and synthesized, and their role as a new sEH/HDAC6 dual-targeting inhibitor in inflammatory pain was evaluated in a formalin-induced mice model and a xylene-induced mouse ear swelling model. Among them, compounds 28g and 28j showed the best inhibitory and selectivity of sEH and HDAC6. Compound 28g had satisfactory pharmacokinetic characteristics in rats. Following administration at 30 mg/kg, compound 28g exhibited more effective analgesic activity than either an sEH inhibitor (GL-B437) or an HDAC6 inhibitor (Rocilinostat) alone and coadministration of both inhibitors. Thus, these novel sEH/HDAC6 dual-targeting inhibitors exhibited powerful analgesic activity in nociceptive behavior and are worthy of further development.

Deep Learning‐Enhanced Fiber‐Coupled Laser Diode‐Based Photoacoustic Microscopy for High‐Resolution Microvasculature Imaging

AbstractBlood vessel imaging provides crucial information for physiological monitoring and disease diagnosis. Optical‐resolution photoacoustic microscopy (PAM) is gaining intense attention in high‐resolution in vivo imaging of blood vessels. However, its applicability in clinical settings is hindered by the requirement of bulky and costly pulsed lasers as well as complex alignment procedures. Here, as a novel approach to high‐quality and cost‐effective microvasculature imaging, a dual‐wavelength fiber‐coupled laser diode‐based PAM (FC‐LD‐PAM) system is demonstrated on the mouse ear and brain in vivo. Furthermore, assisted with a deep learning method, the proposed technique effectively combines the advantages when using both small and large core‐size multimode fibers—successfully enhances the resolution of blurry mouse microvascular images obtained using a large core‐size fiber, revealing fine details that are comparable to those achieved using a small core‐size fiber; significantly reduces the overall time for data acquisition up to fourfold, improving the efficiency significantly in the imaging process. In addition, the proposed approach can achieve accurate and high‐resolution mapping of oxygen saturation in vivo, providing functional insight on living tissue. Therefore, the FC‐LD‐PAM can serve as a translational tool for high‐resolution imaging with enhanced accessibility and versatility in the biomedical field.

Daily Supplementation with Bifidobacterium longum KACC91563 Alleviates Allergic Contact Dermatitis in an Animal Model.

Allergic contact dermatitis (ACD) is the most common chronic inflammatory skin disease (or immune-mediated disease), causing disruption to our psychological condition and life quality. In this study, the therapeutic properties of probiotic Bifidobacterium longum (B. longum) was investigated by using an ACD-induced animal model. For ACD induction, BALB/c mice ear and dorsal skin were sensitized with 240 µL of 1% (w/v) 2,4-dinitrochlorobenzene (DNCB) twice (3-day intervals). After a week of the first induction, the mice were re-sensitized by painting on their dorsal skin and ear with 0.4% (w/v) DNCB for a further three times (once per week). Before the ACD induction of 2 weeks and throughout the trial period, the BALB/c mice were supplemented daily with 1 mL of 1.0 × 109 CFU or 5.0 × 109 CFU B. longum using an intragastric gavage method. The ACD-induced mice without B. longum supplementation were used as a control. Results show that B. longum supplementation significantly alleviated ACD symptoms (e.g., ear swelling, epidermal damage) and immune response (e.g., reduced immune cell recruitment, serum IgE level, and cytokine production). The therapeutic efficiency of B. longum increased as the supplementation dose increased. Thus, daily supplementation with 5.0 × 109 CFU probiotic B. longum could be an effective method for the prevention and treatment of ACD.

LPS exacerbates TRPV4-mediated itch through the intracellular TLR4-PI3K signalling.

Pruritus is often accompanied with bacterial infections, but the underlying mechanism is not fully understood. Although previous studies revealed that lipopolysaccharides (LPS) could directly activate TRPV4 channel and TRPV4 is involved in the generation of both acute itch and chronic itch, whether and how LPS affects TRPV4-mediated itch sensation remains unclear. Here, we showed that LPS-mediated TRPV4 sensitization exacerbated GSK101-induced scratching behaviour in mice. Moreover, this effect was compromised in TLR4-knockout mice, suggesting LPS acted through a TLR4-dependent mechanism. Mechanistically, LPS enhanced GSK101-evoked calcium influx in mouse ear skin cells and HEK293T cells transfected with TRPV4. Further, LPS sensitized TRPV4 channel through the intracellular TLR4-PI3K-AKT signalling. In summary, our study found a modulatory role of LPS in TRPV4 function and highlighted the TLR4-TRPV4 interaction in itch signal amplification.

A phylogenetically distant clade of Nostoc–like (Cyanobacteria) taxa with the description of Reofilinostoc matlalcueyense gen. et sp. nov. from an extreme environment

Although species of the order Nostocales have morphological similarities to each other, some Nostoc species have been considered cryptic and are classified in the families Nostocaceae, Nodulariaceae and Aphanizomenaceae. Since then, the phylogenetic and morphological evaluation of Nostoc-like organisms has led to the taxonomic revision of some genera in different families. In the present work a cyanobacterium morphologically identified as Nostoc was isolated from an extreme cold lotic environment, in a high mountain of the Transmexican Volcanic Belt, and was taxonomically and phylogenetically characterized based on a polyphasic approach. Analysis of the 16S rRNA gene sequence references: Reofilinostoc matlalcueyense gen. et sp. nov. Thus, establishing a new genus of the family Nodulariaceae. The morphology of Reofilinostoc in the wild is reminiscent of a mouse ear, it is of 0.4-1.2 cm in length and has a firm cartilaginous texture; colonies can be pale green, brown or dark green in colour. Through a microscopic inspection, the filaments resemble to Komarekiella atlantica, Desikacharya nostocoides and Minunostoc cylindricum. While, the phylogenetic approach yielded similar results and consistently showed that Reofilinostoc matlalcueyense F02 (OR724089) is closely related to D. nostocoides and M. cylindricum in the core clade of Nodulariaceae. This research revealed that some comparisons between genera of Nodulariaceae exhibited similarity values higher than 97%, such as Atlanticothrix vs. Goleter and Cyanocohniella vs. Anabaenopsis. Nevertheless, in comparison to these phylogenetically closer genera, a substantial difference in the length and structure of D1-D1’, Box-B and V3 helix was demostrated; where Reofilinostoc, Desikacharya, and Minunostoc are part of the Nodulariaceae family; leading to the establishment of Reofilinostoc gen. nov., with Reofilinostoc matlalcueyense sp. nov. as the type species.

Discovery of novel CXCR4 inhibitors for the treatment of inflammation by virtual screening and biological evaluation

C-X-C chemokine receptor type 4 (CXCR4) exerts considerable influence on the pathogenesis of inflammatory disorders and offers a potent avenue for drug intervention. This research utilizes a hybrid virtual screening methodology constructed using computer-aided drug design to discover novel CXCR4 inhibitors for the treatment of inflammation. First, a compound library was screened by Lipinski's five rules and adsorption, distribution, metabolism, excretion and toxicity properties. Second, the HypoGen algorithm was used in constructing a 3D-QSAR pharmacophore model and verify it layer by layer, and the obtained optimal pharmacophore 1 (Hypo 1) was used as a 3D query for compound screening. Then, hit compounds were obtained through molecular docking (Libdock and CDOCKER). The toxicity of the compounds to MDA-MB-231 cells was evaluated in vitro, and their binding affinity to the target was evaluated according to how they compete with 12G5 antibody for CXCR4 on the surfaces of the MDA-MB-231 cells. Compound Hit14 showed the strongest binding affinity among the hit compounds and inhibited cell migration and invasion in Matrigel invasion and wound healing assay at a concentration of 100 nM, demonstrating a better effect than AMD3100. Western Blot experiments further showed that Hit14 blocked the CXCR4/CXCL12-mediated phosphorylation of Akt. Meanwhile, cellular thermal displacement assay analysis showed that CXCR4 protein bound to Hit14 had high thermal stability. Finally, through in vivo experiments, we found that Hit14 inhibited mouse ear inflammation and reduced ear swelling and damage. Therefore, Hit14 is a promising drug for the further development of CXCR4 inhibitors for inflammation treatment.

Doxycycline monohydrate and azelaic acid Co-loaded nanoemulgel for the treatment of facial rosacea: In vitro and In vivo evaluation

Nanoemulgel have gained importance as topical delivery systems because of their potential to incorporate hydrophilic and lipophilic drugs simultaneously like emulsions and improved stability and spreadability like gels. Therefore, the current research was aimed to prepare, optimize, and evaluate the combined therapeutic efficacy of doxycycline monohydrate (DOX.H2O) and azelaic acid (AZL) loaded into a nanoemulgel (NE) against facial rosacea. The nanoemulsion was prepared via phase inversion low energy emulsification method by solubilizing DOX.H2O in peppermint oil and AZL in deionized water. The formulation was optimized using Design Expert 13. The optimized nanoemulsion has a particle size of 210.6 nm, a PDI of 0.234 and a zeta potential of −15.1 mV. SEM observations revealed the spherical morphology of oil globules, whereas FTIR results confirmed that no chemical interaction among the ingredients took place. The in vitro release study showed the burst release of 82.1 % AZL and 48.1 % release of DOX.H2O from the optimized DOX-AZL co-loaded NE in 6 h. The ex vivo permeation profile showed a minimum permeation of DOX.H2O and AZL across rat skin. Croton oil induced inflammation in mice ear confirmed the therapeutic efficacy of the prepared NE against facial rosacea. Considering the results obtained herein, the developed NE could enhance the safety and efficacy of the combination therapy against facial rosacea.

Genome-wide association study for age-related hearing loss in CFW mice.

Age-related hearing impairment is the most common cause of hearing loss and is one of the most prevalent conditions affecting the elderly globally. It is influenced by a combination of environmental and genetic factors. The mouse and human inner ears are functionally and genetically homologous. Investigating the genetic basis of age-related hearing loss (ARHL) in an outbred mouse model may lead to a better understanding of the molecular mechanisms of this condition. We used Carworth Farms White (CFW) outbred mice, because they are genetically diverse and exhibit variation in the onset and severity of ARHL. The goal of this study was to identify genetic loci involved in regulating ARHL. Hearing at a range of frequencies was measured using Auditory Brainstem Response (ABR) thresholds in 946 male and female CFW mice at the age of 1, 6, and 10 months. We obtained genotypes at 4.18 million single nucleotide polymorphisms (SNP) using low-coverage (mean coverage 0.27x) whole-genome sequencing followed by imputation using STITCH. To determine the accuracy of the genotypes we sequenced 8 samples at >30x coverage and used calls from those samples to estimate the discordance rate, which was 0.45%. We performed genetic analysis for the ABR thresholds for each frequency at each age, and for the time of onset of deafness for each frequency. The SNP heritability ranged from 0 to 42% for different traits. Genome-wide association analysis identified several regions associated with ARHL that contained potential candidate genes, including Dnah11, Rapgef5, Cpne4, Prkag2, and Nek11. We confirmed, using functional study, that Prkag2 deficiency causes age-related hearing loss at high frequency in mice; this makes Prkag2 a candidate gene for further studies. This work helps to identify genetic risk factors for ARHL and to define novel therapeutic targets for the treatment and prevention of ARHL.

Skin treatment with non-thermal plasma modulates the immune system through miR-223-3p and its target genes

ABSTRACT Non-thermal plasma, a partially ionized gas, holds significant potential for clinical applications, including wound-healing support, oral therapies, and anti-tumour treatments. While its applications showed promising outcomes, the underlying molecular mechanisms remain incompletely understood. We thus apply non-thermal plasma to mouse auricular skin and conducted non-coding RNA sequencing, as well as single-cell blood sequencing. In a time-series analysis (five timepoints spanning 2 hours), we compare the expression of microRNAs in the plasma-treated left ears to the unexposed right ears of the same mice as well as to the ears of unexposed control mice. Our findings indicate specific effects in the treated ears for a set of five miRNAs: mmu-miR-144-5p, mmu-miR-144-3p, mmu-miR-142a-5p, mmu-miR-223-3p, and mmu-miR-451a. Interestingly, mmu-miR-223-3p also exhibits an increase over time in the right non-treated ear of the exposed mice, suggesting systemic effects. Notably, this miRNA, along with mmu-miR-142a-5p and mmu-miR-144-3p, regulates genes and pathways associated with wound healing and tissue regeneration (namely ErbB, FoxO, Hippo, and PI3K-Akt signalling). This co-regulation is particularly remarkable considering the significant seed dissimilarities among the miRNAs. Finally, single-cell sequencing of PBMCs reveals the downregulation of 12 from 15 target genes in B-cells, Cd4+ and Cd8+ T-cells. Collectively, our data provide evidence for a systemic effect of non-thermal plasma.