MAX: a simple, affordable, and rapid tissue clearing reagent for 3D imaging of wide variety of biological specimens.

Transparency of biological specimens is crucial to obtaining detailed 3-dimensional images and understanding the structure and function of biological specimens. This transparency or tissue clearing can be achieved by adjusting the refractive index (RI) with embedding media and removing light barriers such as lipids, inorganic deposits, and pigments. Many currently available protocols consist of multiple steps to achieve sufficient transparency, making the process complex and time-consuming. Thus, in this study, we tailored the recipe for RI adjustment media named MAX based on the recently reported MACS protocol to achieve a single-step procedure, especially for ECM-rich tissues. This was achieved by the improvement of the tissue penetrability of the RI-matching reagent by combining MXDA with sucrose or iodixanol. While this was sufficient for the 3D imaging in many applications, MAX can also be combined with modular processes for de-lipidation, de-coloration, and de-calcification to further maximize the transparency depending on the special features of the tissues. Our approach provides an easy alternative for tissue clearing and 3D imaging.

Whole structural reconstruction and quantification of epidermal innervation through the suction blister method and skin-clearing technique.

Three-dimensional (3-D) analysis of intraepidermal nerve fibers (IENFs) is conducted to advance assessment methods for peripheral neuropathies and pruritic skin disorders. The skin-clearing technique was proven to be a reliable method for 3-D imaging of IENFs. Nonetheless, it still requires further improvement in the imaging process. The aim of this study was to standardize the 3-D evaluation method of IENFs and to suggest promising 3-D biomarkers for clinical application. A total of nine healthy individuals were prospectively enrolled. The newly adopted suction blister method was combined with the tissue-clearing technique. The detailed structure of the IENFs was reconstructed and quantified using the neuron tracing software. The suction blister method showed improved linear integrity of IENFs compared with those obtained from the previously used salt-split skin test. The 3-D parameters most significantly related to natural aging were the convex hull two-dimensional perimeter and the total length (both p = 0.020). The meaningful correlations were followed by total volume (p = 0.025), ends (p = 0.026), convex hull 3-D surface, and complexity (both p = 0.030). Thus, the 3-D parameters could be utilized as possible biomarkers to identify ambiguous pathologies of peripheral neuropathies and pruritic skin disorders.

Impact of COVID-19 on Clinicopathological Spectrum of Pityriasis Rosea in Korea.

BACKGROUND: Pityriasis rosea (PR) is a papulosquamous eruption with generally unknown origin but suspected to be related to viral etiologies. The clinicopathological spectrum of several disorders with viral etiologies has been altered after the coronavirus disease 2019 (COVID-19) pandemic. The author group could experience coherent histological alterations in PR after the COVID-19 pandemic. This study aimed to investigate how the clinicopathological findings of PR were changed after the COVID-19 pandemic. METHODS: Patients (n = 11) diagnosed with PR based on the clinical manifestations and skin biopsies between February 2018 and October 2019 and 11 patients in February 2020 and October 2021 were retrospectively analyzed by investigating the medical records. RESULTS: The patients with PR during the COVID-19 pandemic demonstrated statistically significant histopathological alterations from classic brisk and dense infiltration pattern to dormant and sparse infiltration and psoriasiform-dominant patterns (P = 0.019). PR was associated with more frequent pruritus during the pandemic period (P = 0.027). CONCLUSION: In conclusion, PR demonstrated a significant histopathological alteration with more frequent pruritus during the COVID-19 pandemic. The comparative results about clinicopathological findings of PR will provide a useful reference for dermatologists in the diagnostic process of PR in the COVID-19 pandemic.

Three-dimensional imaging for the analysis of human epidermal melanocytes.

Three-dimensional (3-D) analysis of human epidermal melanocytes is required for deeper understanding of melanocytic disorders. The purpose of this study was to standardize 3-D imaging and quantification for the evaluation of epidermal melanocytes. The epidermal specimen was obtained using the suction blister method from a patient with melanocytic nevus on the forearm skin. Cutaneous ACT-PRESTO, the tissue-clearing and labeling technique, was subsequently performed. With the 3-D image analysis program, morphological reconstruction and quantification of selected perilesional and melanocytic nevus areas were possible. The region of melanocytic nevus showed higher numbers of total melanocytic dendrites and similar numbers of cell bodies compared with perilesional area. In addition, the mean area and volume of cell bodies increased in the melanocytic nevus area compared with the results in the perilesional area. The 3-D evaluation method of human epidermal melanocytes can be applied to investigate novel pathologies related to hyper- or hypo-pigmentary disorders.

Monitoring Wound Healing with Topically Applied Optical NanoFlare mRNA Nanosensors.

An effective wound management strategy needs accurate assessment of wound status throughout the whole healing process. This can be achieved by examining molecular biomarkers including proteins, DNAs, and RNAs. However, existing methods for quantifying these biomarkers such as immunohistochemistry and quantitative polymerase chain reaction are usually laborious, resource-intensive, and disruptive. This article reports the development and utilization of mRNA nanosensors (i.e., NanoFlare) that are topically applied on cutaneous wounds to reveal the healing status through targeted and semi-quantitative examination of the mRNA biomarkers in skin cells. In 2D and 3D in vitro models, the efficacy and efficiency of these nanosensors are demonstrated in revealing the dynamic changes of mRNA biomarkers for different stages of wound development. In mouse models, this platform permits the tracking and identification of wound healing stages and a normal and diabetic wound healing process by wound healing index in real time.

What type of face mask should we choose in coronavirus disease 2019 pandemic considering photoprotective effectiveness?

BACKGROUND: Wearing a face mask is one of the most effective personal protective strategies to diminish the spread of coronavirus disease 2019 (COVID-19). Several dermatological outcomes were reported because of the prolonged use of face masks, especially due to the constant mask-on policy, but the photoprotective effect of face masks has received less attention. OBJECTIVE: The aim of this study was to provide guidance in the use of face masks by comparing the photoprotective effects of routinely used masks. METHODS: A total of 12 frequently used face masks were tested, including certified respirators, Korea filter (KF)94, KF-anti-droplet (KF-AD), and KF80. The amount of light that penetrates each face mask was measured using a light sensor that can quantify Ultraviolet A (UVA), visible light (VL), and infrared A (IR-A) rays. RESULTS: Black-colored KF94 masks and surgical masks reduced penetration of UVA, VL, and IR-A by approximately 100%. The UVA penetration decreased on average by 95.51%, 90.97%, 85.06%, and 86.41% with white-colored KF94, KF-AD, KF80, and surgical masks, respectively. The VL and IR-A were blocked by approximately 75.58%, 66.16%, 59.18%, and 64.48% with white-colored KF94, KF-AD, KF80, and surgical masks, respectively. CONCLUSION: In conclusion, the different photoprotective effectiveness of face masks was mainly determined by colors, and therefore, black-colored, multi-layered respirators can be recommended in terms of photoprotection in the COVID-19 pandemic. The quantified comparative results will be helpful to the person with pre-existing photo-aggravated dermatosis, especially in the season of the high intensity of sunlight.

Complications of Nasopharyngeal Swabs and Safe Procedures for COVID-19 Testing Based on Anatomical Knowledge.

Nasopharyngeal swabs have been widely to prevent the spread of coronavirus disease 2019 (COVID-19). Nasopharyngeal COVID-19 testing is a generally safe and well-tolerated procedure, but numerous complications have been reported in the media. Therefore, the present study aimed to review and document adverse events and suggest procedural references to minimize preventable but often underestimated risks. A total of 27 articles were selected for the review of 842 related documents in PubMed, Embase, and KoreaMed. The complications related to nasopharyngeal COVID-19 testing were reported to be rarely happened, ranging from 0.0012 to 0.026%. Frequently documented adverse events were retained swabs, epistaxis, and cerebrospinal fluid leakage, often associated with high-risk factors, including severe septal deviations, pre-existing skull base defects, and previous sinus or transsphenoidal pituitary surgery. Appropriate techniques based on sufficient anatomical knowledge are mandatory for clinicians to perform nasopharyngeal COVID-19 testing. The nasal floor can be predicted by the line between the nostril and external ear canal. For safe testing, the angle of swab insertion in the nasal passage should remain within 30° of the nasal floor. The swab was gently inserted along the nasal septum just above the nasal floor to the nasopharynx and remained on the nasopharynx for several seconds before removal. Forceful insertion should be attempted, and alternative examinations should be considered, especially in vulnerable patients. In conclusion, patients and clinicians should be aware of rare but possible complications and associated high-risk factors. The suggested procedural pearls enable more comfortable and safe nasopharyngeal COVID-19 testing for both clinicians and patients.

Co-flow injection for serial crystallography at X-ray free-electron lasers.

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro-molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.

Continuous organelle separation in an insulator-based dielectrophoretic device.

Heterogeneity in organelle size has been associated with devastating human maladies such as neurodegenerative diseases or cancer. Therefore, assessing the size-based subpopulation of organelles is imperative to understand the biomolecular foundations of these diseases. Here, we demonstrated a ratchet migration mechanism using insulator-based dielectrophoresis in conjunction with a continuous flow component that allows the size-based separation of submicrometer particles. The ratchet mechanism was realized in a microfluidic device exhibiting an array of insulating posts, tailoring electrokinetic and dielectrophoretic transport. A numerical model was developed to elucidate the particle migration and the size-based separation in various conditions. Experimentally, the size-based separation of a mixture of polystyrene beads (0.28 and 0.87 µ$\umu $ m) was accomplished demonstrating good agreement with the numerical model. Furthermore, the size-based separation of mitochondria was investigated using a mitochondria mixture isolated from HepG2 cells and HepG2 cells carrying the gene Mfn-1 knocked out, indicating distinct size-related migration behavior. With the presented continuous flow separation device, larger amounts of fractionated organelles can be collected in the future allowing access to the biomolecular signature of mitochondria subpopulations differing in size.

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Cholic Acid (MT921) after a Subcutaneous Injection in the Submental Area to Humans.

This study aimed to explore pharmacokinetics, pharmacodynamics, and safety/tolerability of MT921, an injectable cholic acid, after a single subcutaneous administration to healthy volunteers. A randomized, double-blinded, placebo-controlled, single dose-ascending phase 1 study enrolled 24 subjects who were assigned to three groups (60 mg, 120 mg, and 150 mg) of MT921. Blood samples were obtained for a 24-h period before and after injecting MT921 to the submental fat area. Plasma concentrations of cholic acid and deoxycholic acid were determined for pharmacokinetic analysis. Levels of free fatty acid, triglyceride, and total cholesterol were measured for pharmacodynamic analysis. Safety and tolerability were assessed until 21 days post-dose. While systemic exposure to cholic acid tended to increase as the MT921 dose increased, pharmacokinetic profiles of deoxycholic acid were similar among dose groups without showing significant changes. Pharmacodynamic profiles were comparable when measured at baseline and post-dose. The most frequent adverse events were injection site pain and edema. All adverse drug reactions resolved without treatment. MT921 appeared to be well-tolerated after an injection to the submental area at a dose up to 150 mg. Systemic exposure to cholic acid increased as the dose increased. Blood lipid profiles and deoxycholic acid levels were not affected by MT921 treatment.

An assessment method for dermal structures using cross-polarized light imaging with a green light-emitting diode.

BACKGROUND: Several experimental methods for evaluating dermal structures exist; however, most of these are not used in dermatology clinics because of cost and functional limitations. OBJECTIVE: To propose a simple, non-invasive method for dermal structure evaluation using a green light-emitting diode (LED) with cross-polarized light (CPL) imaging and compare the quality of the images taken using either green or white LED. MATERIALS AND METHODS: Skin specimens were taken from fifteen cadavers. Images were captured using CPL photography with a green or white LED. The Commission International d'Eclairage L*a*b* (CIELAB) values were calculated for each image. The skin specimens were processed and stained with Masson's trichrome to visualize collagen fibers with major image scattering. The images were histologically analyzed, and correlational and regression analyses were performed to determine the relationship between the L* values and histologic measurements. RESULTS: The L* values for the green images were positively correlated with collagen fiber density, reticular collagen bundle diameter, and dermal thickness. They were effective for estimating dermal properties. The L* values for the white images were positively correlated with reticular collagen bundle diameter and dermal thickness. Correlational coefficients for white images were lower than those for green images. In regression analysis, green images showed a higher coefficient of determination (R2 ) for predicting reticular collagen bundle diameter than white images (0.1128 vs. 0.0827). CONCLUSION: Cross-polarized light imaging with a green LED is a simple, non-invasive method for evaluating dermal structures. The use of a green LED was also more effective for image analysis.

A simple morphometric analysis method for dermal microstructure using color thresholding and moments.

BACKGROUND: Proper assessment of dermal collagen fibers by dermatologists and researchers is essential. Histologic evaluation methods have limitations. We present a simple method for measurement of collagen fibers in human skin using Masson's trichrome staining. MATERIALS AND METHODS: Normal skin specimens from a cadaver were processed with Masson's trichrome, which can effectively stain collagen fibers blue with aniline dye. Optical photomicrographs of these slides were analyzed using ImageJ software. Color image processing, a histogram-based function of ImageJ for image segmentation, was performed with color moments thresholding technique. We selected blue areas by adjusting the blue channel to include specific values. The selected areas were highlighted and evaluated. We divided the image into layers of 0.09-mm2 areas from the top to bottom of the dermis. Each area was cropped and evaluated. RESULTS: Quantitative assessment yielded the quantitative size occupied by collagen fibers in an area of 0.09 mm2 . Calculation of the percentage in each area can be used to determine the density of collagen fibers. CONCLUSION: Measurements obtained with our method can be applied to research on dermal collagen fibers. We present a convenient quantitative assessment method for the dermal constituents in Masson's trichrome-stained slides.

Tissue-Clearing Technique and Cutaneous Nerve Biopsies: Quantification of the Intraepidermal Nerve-Fiber Density Using Active Clarity Technique-Pressure Related Efficient and Stable Transfer of Macromolecules Into Organs.

BACKGROUND AND PURPOSE: Cutaneous nerve biopsies based on two-dimensional analysis have been regarded as a creditable assessment tool for diagnosing peripheral neuropathies. However, advancements in methodological imaging are required for the analysis of intact structures of peripheral nerve fibers. A tissue-clearing and labeling technique facilitates three-dimensional imaging of internal structures in unsectioned, whole biological tissues without excessive time or labor costs. We sought to establish whether a tissue-clearing and labeling technique could be used for the diagnostic evaluation of peripheral neuropathies. METHODS: Five healthy individuals and four patients with small-fiber neuropathy (SFN) and postherpetic neuralgia (PHN) were prospectively enrolled. The conventional methods of indirect immunofluorescence (IF) and bright-field immunohistochemistry (IHC) were adopted in addition to the tissue-clearing and labeling method called active clarity technique-pressure related efficient and stable transfer of macromolecules into organs (ACT-PRESTO) to quantify the intraepidermal nerve-fiber density (IENFD). RESULTS: The mean IENFD values obtained by IF, bright-field IHC, and ACT-PRESTO in the healthy control group were 6.54, 6.44, and 90.19 fibers/mm², respectively; the corresponding values in the patients with SFN were 1.99, 2.32, and 48.12 fibers/mm², respectively, and 3.06, 2.87, and 47.21 fibers/mm², respectively, in the patients with PHN. CONCLUSIONS: This study has shown that a tissue-clearing method provided not only rapid and highly reproducible three-dimensional images of cutaneous nerve fibers but also yielded reliable quantitative IENFD data. Quantification of the IENFD using a tissue-clearing and labeling technique is a promising way to improve conventional cutaneous nerve biopsies.

Quantification of intraepidermal nerve fiber density using three-dimensional microscopy.

Three-dimensional microscopy provides more extended depth of penetration compared with conventional light microscopy and is known to be useful in clinical evaluation of thick biological specimens. Skin nerve biopsy together with the quantification of intraepidermal nerve fibers in multiple thick sections has been widely adopted for evaluating peripheral neuropathies. The aim of the present study was to evaluate the effectivity of three-dimensional microscopy in reducing the required time and inter-rater discrepancies, especially in the case of personnel not familiar with the quantification methods. A total of six cryo-sectioned specimens were analyzed for the study and the skin samples were collected from one patient with postherpetic neuralgia who voluntarily participated in the study. Two investigators, a physician and non-physician assessed the intraepidermal nerve fiber densities and required analysis time using three different methods including direct visualization of tissue slides, and analysis with two- and three-dimensional images. Three-dimensional microscopy could produce images that enabled reliable evaluation of intraepidermal nerve fibers; the accuracy of analysis was statistically comparable between the physician and non-physician (p > .05). Three-dimensional microscopy also enabled the non-physician to proceed meaningfully faster evaluation compared with the direct visualization method (p = .03). Three-dimensional microscopy could be one of the useful methods to improve accuracy and convenience of the analysis of intraepidermal nerve fibers especially appropriate for unaccustomed physician or non-physician. RESEARCH HIGHLIGHTS: Three-dimensional microscopy is capable of producing images with more extended depth of penetration compared with conventional light microscopy and has been known to be suitable for clinical evaluation of thick biological specimens. Cutaneous nerve biopsy and the quantification of nerve fibers in thick sections has been widely adopted for evaluating peripheral neuropathies. Three-dimensional microscopy could be especially appropriate for unaccustomed physician or non-physician to improve accuracy and convenience of the analysis of intraepidermal nerve fibers.

High-Performance Acellular Tissue Scaffold Combined with Hydrogel Polymers for Regenerative Medicine.

Decellularization of tissues provides extracellular matrix (ECM) scaffolds for regeneration therapy and an experimental model to understand ECM and cellular interactions. However, decellularization often causes microstructure disintegration and reduction of physical strength, which greatly limits the use of this technique in soft organs or in applications that require maintenance of physical strength. Here, we present a new tissue decellularization procedure, namely CASPER (Clinically and Experimentally Applicable Acellular Tissue Scaffold Production for Tissue Engineering and Regenerative Medicine), which includes infusion and hydrogel polymerization steps prior to robust chemical decellularization treatments. Polymerized hydrogels serve to prevent excessive damage to the ECM while maintaining the sophisticated structures and biological activities of ECM components in various organs, including soft tissues such as brains and embryos. CASPERized tissues were successfully recellularized to stimulate a tissue-regeneration-like process after implantation without signs of pathological inflammation or fibrosis in vivo, suggesting that CASPERized tissues can be used for monitoring cell-ECM interactions and for surrogate organ transplantation.

Relationship between skin color and solar elastosis in aged Asian skin: A colorimetric-pathologic correlation.

Aged skin is reported to be associated with unattractive skin color changes and solar elastosis. However, comparative studies have not documented the possible correlation between the two factors. This study investigated the plausible relationship between the facial skin color of elderly Asians and solar elastosis. A total of 22 skin specimens were collected from 22 Korean patients who underwent cheek skin biopsies. Skin color was quantitatively measured using colorimetric photography techniques to produce CIE L*a*b* values; the degree of solar elastosis was quantifiably assessed using a histologic grading scale. These values were used to investigate a correlation between the CIE L*a*b* coordinates and solar elastosis grade. The solar elastosis grade increased according to patient age (r = 0.67, p = .0006). However, the extent of solar elastosis was not statistically correlated with the CIE L*a*b* values, including L*, a*, and b* (r = 0.02, p = .95; r = 0.15, p = 0.50; r = -0.07, p = 0.76, respectively). The results showed that the solar elastosis grade increased, according to patient age, because of cumulative actinic damage. However, colorimetric skin color data did not correlate with the degree of solar elastosis. Therefore, cutaneous color changes and solar elastosis are separate, age-related phenomena. Physicians should be aware of the possible histologic changes in actinically damaged facial skin, regardless of the skin color.

Skin microrelief profiles as a cutaneous aging index.

An objective measurement of cutaneous topographical information is important for quantifying the degree of skin aging. Our aim was to improve methods for measuring microrelief patterns using a three-dimensional analysis based on silicone replicas and scanning electron microscope (SEM). Another objective was to compare the results with those obtained using a two-dimensional analysis method based on dermoscopy. Silicone replicas were obtained from forearms, dorsum of the hands and fingers of 51 volunteers. Cutaneous profiles obtained by SEM with silicone replicas showed more consistent correlations with age than data obtained by dermoscopy. This indicates the advantage of three-dimensional topography analysis using silicone replicas and SEM over the widely used dermoscopic assessment. The cutaneous age was calculated using stepwise linear regression, and the result was 57.40-9.47 × (number of furrows on dorsum of the hand) × (width of furrows on dorsum of the hand).

Characteristics and Clinical Manifestations of Pigmented Purpuric Dermatosis.

BACKGROUND: Pigmented purpuric dermatoses (PPD) are a spectrum of disorders characterized by a distinct purpuric rash. Although PPD can be easily diagnosed, the disease entity remains an enigma and a therapeutic challenge. OBJECTIVE: The purpose of this study was to investigate the characteristics and clinical manifestations of PPD and to elucidate the relationship between assumed etiologic factors and the clinical manifestations of PPD and treatment responses. METHODS: Retrograde analyses were performed to identify appropriate PPD patients who visited Korea University Medical Center Anam Hospital from 2002 to 2012. RESULTS: Information on 113 patients with PPD was analyzed, and 38 subjects with skin biopsy were included for this study. Schamberg's disease was the most frequent clinical type (60.5%). Concomitant diseases included hypertension (15.8%), diabetes (10.5%), and others. Associated medication histories included statins (13.2%), beta blockers (10.5%), and others. Possibly associated etiologic factors were recent upper respiratory infection (5.3%), high orthostatic pressure due to prolonged standing (2.6%), and strenuous exercise (2.6%). A total of 36 patients (94.7%) were treated with one or more treatment methods, including oral antihistamines, pentoxifylline, topical steroids, and/or phototherapy. There was no significant difference in disease progress according to underlying diseases, medications, or association factors (p>0.05). CONCLUSION: Our overall results were grossly consistent with the existing literature, excluding several findings. Although a possible relationship between PPD and cardiovascular disease or cardiovascular medication was proposed at the beginning of the study, no statistically significant correlations were found according to the specific clinical types and treatment responses (p>0.05).