The Eyelid Meibomian Gland Deficiency in Fucosyltransferase 1 Knockout Mice.

To investigate the effect of fucosyltransferase (FUT) 1-mediated fucosylation on meibomian glands (MG), we first confirmed that FUT1 and its fucosylated products were expressed in the eyelid, conjunctiva and skin in wild-type (WT) mice, whereas their mRNA and protein levels were downregulated in Fut1 knock-out (KO) mice. We then evaluated age-dependent changes in the total and acinar areas of MG, meibocyte differentiation, lipid synthesis, and eyelid inflammation and oxidative stress in Fut1 KO and WT mice. Results show that both the total and acinar areas of MG were smaller in Fut1 KO mice than in WT mice in all evaluated age groups. Meibocyte differentiation, lipid-producing capacities and the enzyme levels responsible for lipid synthesis were reduced in Fut1 KO mice, compared to WT controls. The levels of pro-inflammatory cytokines and oxidative-stress-related markers were elevated in the eyelids and MG of FUT1 KO mice. These findings demonstrate the physiologic function of FUT1-mediated fucosylation in MG development and function, and indicate its potential role in ocular surface homeostasis.

Increased Histone Acetylation and Decreased Expression of Specific Histone Deacetylases in Ultraviolet-Irradiated and Intrinsically Aged Human Skin In Vivo.

Histone deacetylases (HDACs) are conserved enzymes that remove acetyl groups from lysine side chains in histones and other proteins and play a crucial role in epigenetic regulation. Previously, we showed that histone acetylation is implicated in ultraviolet (UV)-induced inflammation and matrix impairment. To elucidate the histone acetylation status and specific HDACs involved in skin aging, we examined the changes in histone acetylation, global HDAC activity, and the expression of HDACs and sirtuins (SIRTs) in intrinsically aged and photoaged human skin as well as in UV-irradiated human skin in vivo. Following acute UV irradiation, the acetylated histone H3 (AcH3) level was increased, but HDAC activity and the expression levels of HDAC4, HDAC11, and SIRT4 were significantly decreased. In intrinsically aged skin, AcH3 levels were increased, but HDAC activity and the expression levels of HDAC4, HDAC5, HDAC10, HDAC11, SIRT6, and SIRT7 were significantly decreased. However, histone acetylation and HDAC expression in photoaged skin were not significantly different from those in intrinsically aged skin. Collectively, HDAC4 and HDAC11 were decreased in both UV-irradiated and intrinsically aged skin, suggesting that they may play a universal role in increased histone acetylation associated with skin aging.

Topical treatment with a cathepsin G inhibitor, ß-keto-phosphonic acid, blocks ultraviolet irradiation-induced basement membrane damage in hairless mouse skin.

BACKGROUND: Ultraviolet light (UV) exposure contributes various effects to skin including damage of the basement membrane. Cathepsin G (CTSG) belongs to serine protease family, and its upregulation is involved in wrinkle formation by chronic UV irradiation. However, the effect of CTSG on the basement membrane damage in skin remains unclear. PURPOSE: To investigate the effects of topical treatment with a CTSG inhibitor, ß-keto-phosphonic acid (KPA), on basement membrane damage in chronically UV-irradiated hairless mouse skin. METHODS: The dorsal skin of hairless mice was exposed to UV three times per week for 8 weeks. KPA was applied immediately after each session of UV irradiation. The basement membrane components, CTSG expression, and neutrophil infiltration were analyzed by immunofluorescence staining. The basement membrane structures were visualized by transmission electron microscope. CTSG and MMP-13 protein levels were analyzed by Western blotting. Assessment of wrinkle formation was examined using a skin replica assay. RESULTS: ß-keto-phosphonic acid prevented UV irradiation-induced decrease in type VII collagen, laminin 332, and perlecan at the basement membrane zone and prevented UV-induced breakage of lamina densa and UV-induced shortening of hemidesmosome. KPA prevented UV-induced CTSG and MMP-13 expressions in chronically UV-irradiated hairless mice. Increase in neutrophil infiltration by UV irradiation and UV-induced wrinkle formation was also prevented by KPA. CONCLUSION: Our present study showed the possible involvement of CTSG in UV-induced basement membrane damage in skin through topical treatment with a CTSG inhibitor, KPA. Thus, inhibition of CTSG may be a useful strategy for the prevention of UV-induced basement membrane damage and photoaging.

Construction of 3D multicellular microfluidic chip for an in vitro skin model.

Current in vitro skin models do not recapitulate the complex architecture and functions of the skin tissue. In particular, on-chip construction of an in vitro model comprising the epidermis and dermis layer with vascular structure for mass transport has not been reported yet. In this study, we aim to develop a microfluidic, three-dimensional (3D) skin chip with fluidic channels using PDMS and hydrogels. Mass transport within the collagen hydrogel matrix was verified with fluorescent model molecules, and a transport-reaction model of oxygen and glucose inside the skin chip was developed to aid the design of the microfluidic skin chip. Comparison of viabilities of dermal fibroblasts and HaCaT cultured in the chip with various culture conditions revealed that the presence of flow plays a crucial role in maintaining the viability, and both cells were viable after 10 days of air exposure culture. Our 3D skin chip with vascular structures can be a valuable in vitro model for reproducing the interaction between different components of the skin tissue, and thus work as a more physiologically realistic platform for testing skin reaction to cosmetic products and drugs.

UV Irradiation Increases Appetite and Prevents Body Weight Gain through the Upregulation of Norepinephrine in Mice.

UV irradiation of the human skin downregulates lipid synthesis and adipokine production in subcutaneous fat. Recent evidence has suggested that UV exposure limits body weight gain in mouse models of obesity. However, the relationship between norepinephrine and UV irradiation has not been previously reported. Chronic UV exposure stimulated food intake but prevented body weight gain. Leptin, an appetite-suppressing hormone, was significantly reduced in the serum of the UV-irradiated mice. In contrast, UV irradiation induced browning of subcutaneous white adipose tissues without increasing physical activity. Notably, UV irradiation significantly increased norepinephrine levels, and the inhibition of norepinephrine production reversed the effects of chronic UV irradiation on food intake and body weight gain. In conclusion, chronic UV irradiation induces norepinephrine release, resulting in the stimulation of food intake due to the downregulation of leptin levels, but it prevents weight gain by inducing the browning process and elevating energy expenditure.

Acute UV irradiation increases heparan sulfate proteoglycan levels in human skin.

Glycosaminoglycans are important structural components in the skin and exist as various proteoglycan forms, except hyaluronic acid. Heparan sulfate (HS), one of the glycosaminoglycans, is composed of repeated disaccharide units, which are glucuronic acids linked to an N-acetyl-glucosamine or its sulfated forms. To investigate acute ultraviolet (UV)-induced changes of HS and HS proteoglycans (HSPGs), changes in levels of HS and several HSPGs in male human buttock skin were examined by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR) after 2 minimal erythema doses (MED) of UV irradiation (each n = 4-7). HS staining revealed that 2 MED of UV irradiation increased its expression, and staining for perlecan, syndecan-1, syndecan-4, CD44v3, and CD44 showed that UV irradiation increased their protein levels. However, analysis by real-time qPCR showed that UV irradiation did not change mRNA levels of CD44 and agrin, and decreased perlecan and syndecan-4 mRNA levels, while increased syndecan-1 mRNA level. As HS-synthesizing or -degrading enzymes, exostosin-1 and heparanase mRNA levels were increased, but exostosin-2 was decreased by UV irradiation. UV-induced matrix metalloproteinase-1 expression was confirmed for proper experimental conditions. Acute UV irradiation increases HS and HSPG levels in human skin, but their increase may not be mediated through their transcriptional regulation.

Changes in glycosaminoglycans and related proteoglycans in intrinsically aged human skin in vivo.

Glycosaminoglycans (GAGs) and proteoglycans (PGs) are involved in various structural functions and physiological regulations in the skin. To investigate the intrinsic ageing-dependent GAG and PG changes and their gender-specific difference, immunohistochemical stains of several GAGs and PGs were performed in sun-protected buttock skin tissues of young and old, male and female (total n = 32) human skin. Stains of alcian blue, hyaluronic acid (HA) and heparan sulphate were reduced in aged skin in both genders, whereas chondroitin sulphate stain was decreased only in female. Stains of HA synthase-2, CD44, CD44v3, syndecan-1 and decorin were decreased in aged skin in both genders, whereas perlecan stain was reduced only in female, and syndecan-4 stain did not change. Versican stain was increased in male aged skin, but not in female. Age- and gender-related changes in GAGs and PGs in intrinsically aged buttock skin elucidated in this study may play important roles in intrinsic skin ageing process.

Transcriptional regulation of proteoglycans and glycosaminoglycan chain-synthesizing glycosyltransferases by UV irradiation in cultured human dermal fibroblasts.

Various kinds of glycosaminoglycans (GAGs) and proteoglycans (PGs) have been known to be involved in structural and space-filling functions, as well as many physiological regulations in skin. To investigate ultraviolet (UV) radiation-mediated regulation of GAGs and PGs in cultured human dermal fibroblasts, transcriptional changes of many types of PGs and GAG chain-synthesizing enzymes at 18 hr after 75 mJ/cm(2) of UV irradiation were examined using quantitative real-time polymerase chain reaction methods. Hyaluronic acid synthase (HAS)-1, -2, and -3 and hyaluronidase-2 mRNA expressions were significantly increased by UV irradiation. Expressions of lumican, fibromodulin, osteoglycin, syndecan-2, perlecan, agrin, versican, decorin, and biglycan were significantly decreased by UV irradiation, while syndecan-1 was increased. Expressions of GAG chain-synthesizing glycosyltransferases, xylosyltransferase-1, ß1,3-glucuronyltransferase-1, ß1,4-galactosyltransferase-2, -4, exostosin-1, chondroitin polymerizing factor, and chondroitin sulfate synthase-3 were significantly reduced, whereas those of ß1,3-galactosyltransferase-6, ß1,4-galactosyltransferase-3, -7, ß-1,3-N-acetylglucosaminyltran sferase-2, and -7 were increased by UV irradiation. Heparanase-1 mRNA expression was increased, but that of heparanase-2 was reduced by UV irradiation. Time-course investigation of representative genes showed consistent results. In conclusion, UV irradiation may increase hyaluronic acid production through HAS induction, and decrease other GAG productions through downregulation of PG core proteins and GAG chain-synthesizing glycosyltransferases in cultured human dermal fibroblasts.

Identifying molecular targets for reverse aging using integrated network analysis of transcriptomic and epigenomic changes during aging.

Aging is associated with widespread physiological changes, including skeletal muscle weakening, neuron system degeneration, hair loss, and skin wrinkling. Previous studies have identified numerous molecular biomarkers involved in these changes, but their regulatory mechanisms and functional repercussions remain elusive. In this study, we conducted next-generation sequencing of DNA methylation and RNA sequencing of blood samples from 51 healthy adults between 20 and 74 years of age and identified aging-related epigenetic and transcriptomic biomarkers. We also identified candidate molecular targets that can reversely regulate the transcriptomic biomarkers of aging by reconstructing a gene regulatory network model and performing signal flow analysis. For validation, we screened public experimental data including gene expression profiles in response to thousands of chemical perturbagens. Despite insufficient data on the binding targets of perturbagens and their modes of action, curcumin, which reversely regulated the biomarkers in the experimental dataset, was found to bind and inhibit JUN, which was identified as a candidate target via signal flow analysis. Collectively, our results demonstrate the utility of a network model for integrative analysis of omics data, which can help elucidate inter-omics regulatory mechanisms and develop therapeutic strategies against aging.

Discovery of a transdermally deliverable pentapeptide for activating AdipoR1 to promote hair growth.

Alopecia induced by aging or side effects of medications affects millions of people worldwide and impairs the quality of life; however, there is a limit to the current medications. Here, we identify a small transdermally deliverable 5-mer peptide (GLYYF; P5) that activates adiponectin receptor 1 (AdipoR1) and promotes hair growth. P5 sufficiently reproduces the biological effect of adiponectin protein via AMPK signaling pathway, increasing the expression of hair growth factors in the dermal papilla cells of human hair follicle. P5 accelerates hair growth ex vivo and induces anagen hair cycle in mice in vivo. Furthermore, we elucidate a key spot for the binding between AdipoR1 and adiponectin protein using docking simulation and mutagenesis studies. This study suggests that P5 could be used as a topical peptide drug for alleviating pathological conditions, which can be improved by adiponectin protein, such as alopecia.

Minichromosome maintenance protein expression according to the grade of atypism in actinic keratosis.

The minichromosome maintenance (MCM) family is a group of proteins that are key initiation factors for DNA replication and are expressed only in cycling cells. Recent studies on various cancerous conditions have shown that MCM proteins are better markers for malignant cells compared to other proliferative markers. It has been also proven that MCM proteins are independent prognostic factors. The aim of this study was to characterize the pattern and frequency of MCM 2 protein expression in actinic keratosis (AK) and determine whether the expression is correlated with the degree of histological atypism. Biopsy samples of 34 patients who had been diagnosed as AK were used in this study. Samples were divided into three groups (grade I, grade II, and grade III) according to the degree of atypism. Immunohistochemical staining for MCM 2 protein, Ki-67, and proliferating cell nuclear antigen was performed, and the number of positively staining cells per unit area (10⁻4 µm²) was calculated for evaluation of immunoreactivity. MCM 2 protein was expressed in atypical keratinocytes in AK. Mean numbers of immunoreactive cells positive for MCM 2 were 165.1 in grade I, 304.5 in grade II, and 513.3 in grade III. Moreover, the correlation between the immunoreactivity for MCM 2 protein and AK grade was significantly more positive than that for other markers. Thus, we suggest that MCM 2 protein is a reliable marker for diagnosing and grading AK and further could be hypothesized as an important prognostic factor.

Prevention of UV-induced skin damages by 11,14,17-eicosatrienoic acid in hairless mice in vivo.

Polyunsaturated fatty acids (PUFAs) are known to play important roles in various physiological and pathological processes. Recent studies have shown that some omega-3 (omega-3) PUFAs, such as eicosapentaenoic acid (EPA) and dodecahexaenoic acid (DHA), have protective effects on acute and chronic UV-induced changes. However, the effects of other omega-3 PUFAs including 11,14,17-eicosatrienoic acid (20:3) (ETA) on UV-induced skin damages are poorly understood. In this study, we investigated the cutaneous photoprotective effects of ETA in hairless mice in vivo. Female HR-1 hairless mice were topically treated with vehicle (ethanol:polyethylene glycol=30:70) only, 0.1% ETA, or 1% ETA once a day for 3 successive days after one time UV irradiation (200 mJ/cm(2)) on dorsal skins. Skin biopsy was carried out on the fourth day (72 hr after UV irradiation). We found that topical treatment with ETA attenuated UV-induced epidermal and dermal thickness and infiltration of inflammatory cells, and impairment of skin barrier function. In addition, ETA suppressed the expression of IL-1beta, COX-2, and MMP-13 induced by UV irradiation. Our results show that the topical application of ETA protects against UV-induced skin damage in hairless mice and suggest that ETA can be a potential agent for preventing and/or treating UV-induced inflammation and photoaging.

Time-variant reproductive number of COVID-19 in Seoul, Korea.

OBJECTIVES: To estimate time-variant reproductive number (Rt) of coronavirus disease 19 based on either number of daily confirmed cases or their onset date to monitor effectiveness of quarantine policies. METHODS: Using number of daily confirmed cases from January 23, 2020 to March 22, 2020 and their symptom onset date from the official website of the Seoul Metropolitan Government and the district office, we calculated Rt using program R's package "EpiEstim". For asymptomatic cases, their symptom onset date was considered as -2, -1, 0, +1, and +2 days of confirmed date. RESULTS: Based on the information of 313 confirmed cases, the epidemic curve was shaped like 'propagated epidemic curve'. The daily Rt based on Rt_c peaked to 2.6 on February 20, 2020, then showed decreased trend and became <1.0 from March 3, 2020. Comparing both Rt from Rt_c and from the number of daily onset cases, we found that the pattern of changes was similar, although the variation of Rt was greater when using Rt_c. When we changed assumed onset date for asymptotic cases (-2 days to +2 days of the confirmed date), the results were comparable. CONCLUSIONS: Rt can be estimated based on Rt_c which is available from daily report of the Korea Centers for Disease Control and Prevention. Estimation of Rt would be useful to continuously monitor the effectiveness of the quarantine policy at the city and province levels.

A novel role for the TRPV1 channel in UV-induced matrix metalloproteinase (MMP)-1 expression in HaCaT cells.

Transient receptor potential vanilloid type 1 (TRPV1) is a molecular sensor for detecting adverse stimuli, such as capsaicin, heat, and acid. TRPV1 has been localized in keratinocytes and is suggested to be a mediator of heat-induced matrix metalloproteinase-1 (MMP-1). With regard to the multimodal activation of TRPV1, we hypothesize that TRPV1 might also mediate UV-induced MMP-1 in keratinocytes. In HaCaT, a human keratinocyte cell line, we initially confirmed capsaicin-induced membrane current and Ca(2+) influx. UV irradiation induced slow and persistent calcium influx and increased membrane current, which was inhibited by TRPV1 inhibitors (capsazepine and ruthenium red). The UV-induced MMP-1 expression in HaCaT was also decreased by TRPV1 inhibitors and was facilitated by capsaicin. Knock-down of TRPV1 using siRNA transfection also decreased MMP-1 expression, as well as UV-induced Ca(2+) influx in HaCaT. UV failed to induce MMP-1 expression in HaCaT cells cultured in Ca(2+)-free media. Both the UV-induced increase in [Ca(2+)](i) and MMP-1 were suppressed by Gö6976 (a calcium-dependent PKC inhibitor), but not by rottlerin (a calcium-independent PKC inhibitor). In addition to a plausible role of TRPV1 in UV-induced MMP-1 expression, we showed that UV increased TRPV1 expression in both HaCaT cells and human skin in vivo. From these results, we suggest that UV-induced MMP-1 expression might be mediated in part by PKC-dependent activation of TRPV1 and subsequent Ca(2+)-influx in human keratinocytes. J. Cell. Physiol. 219: 766-775, 2009. (c) 2009 Wiley-Liss, Inc.

Increased expression of TRPV1 channel in intrinsically aged and photoaged human skin in vivo.

Transient receptor potential vanilloid type 1 (TRPV1) is activated by various stimuli including capsaicin, heat and acid. While TRPV1 has been localized in the epidermis, little is known about the physiological role of TRPV1 in the skin, especially in skin ageing. In this study, we investigated the effect of acute UV irradiation on TRPV1 expression in human skin and the changes in TRPV1 mRNA and protein in intrinsic ageing and photoageing using human sun-protected (upper inner arm) and sun-exposed (forearm) skin of young and elderly subjects. Western blot analysis of UV-irradiated young buttock skin revealed that the expression of TRPV1 protein was increased at 24 h (2.3-fold) and 48 h (2.4-fold) after UV irradiation. Real-time PCR analysis also showed that the mRNA level of TRPV1 was augmented by 2.4-fold at 4 h after UV irradiation. TRPV1 protein was expressed at higher levels by 2.6-fold in the sun-protected skin of the elderly subjects than in that of young people according to western blotting, real-time PCR analysis and immunohistochemical staining. In addition, the photoaged skin of elderly showed increased expression of TRPV1 mRNA and protein compared with that of the sun-protected skin of the same individuals. Also, we found increased expression of TRPV1 in nerve fibres of elderly persons using double staining of TRPV1 and nerve fibres. Based on the above results, our data suggest that the expression of TRPV1 is affected by both the intrinsic ageing and photoageing processes.

Modulation of elastin exon 26A mRNA and protein expression in human skin in vivo.

Photoaged skin contains elastotic materials in the upper reticular dermis, a result of a commonly known as solar elastosis. It is known that the primary transcript of elastin undergoes extensive alternative splicing and that this results in the translation of multiple heterogeneous protein isoforms. In this study, we found that UV irradiation and heat treatment increased the levels of elastin transcript containing exon 26A and its encoded elastin isoform in the epidermis of human skin in vivo and in cultured human keratinocytes in vitro. We also found that the elastin transcript containing exon 26A was upregulated in photoaged forearm skin compared with intrinsically aged buttock skin in the same elderly individuals. We observed that topical retinoic acid treatment to human skin did not increase the expression of exon 26A mRNA, but that tropoelastin mRNA expression was increased by this treatment. These findings suggest that the production of the elastin isoform containing exon 26A peptide is increased by UV exposure and heat treatment in human skin in vivo and that it may play an important role in the development of solar elastosis in photoaged human skin.

Effects of infrared radiation and heat on human skin aging in vivo.

Sunlight damages human skin, resulting in a wrinkled appearance. Since natural sunlight is polychromatic, its ultimate effects on the human skin are the result of not only the action of each wavelength separately, but also interactions among the many wavelengths, including UV, visible light, and infrared (IR). In direct sunlight, the temperature of human skin rises to about 40 degrees C following the conversion of absorbed IR into heat. So far, our knowledge of the effects of IR radiation or heat on skin aging is limited. Recent work demonstrates that IR and heat exposure each induces cutaneous angiogenesis and inflammatory cellular infiltration, disrupts the dermal extracellular matrix by inducing matrix metalloproteinases, and alters dermal structural proteins, thereby adding to premature skin aging. This review provides a summary of current research on the effects of IR radiation and heat on aging in human skin in vivo.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 15-19; doi:10.1038/jidsymp.2009.7.

Chromosome-scale assembly comparison of the Korean Reference Genome KOREF from PromethION and PacBio with Hi-C mapping information.

BACKGROUND: Long DNA reads produced by single-molecule and pore-based sequencers are more suitable for assembly and structural variation discovery than short-read DNA fragments. For de novo assembly, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) are the favorite options. However, PacBio's SMRT sequencing is expensive for a full human genome assembly and costs more than $40,000 US for 30× coverage as of 2019. ONT PromethION sequencing, on the other hand, is 1/12 the price of PacBio for the same coverage. This study aimed to compare the cost-effectiveness of ONT PromethION and PacBio's SMRT sequencing in relation to the quality. FINDINGS: We performed whole-genome de novo assemblies and comparison to construct an improved version of KOREF, the Korean reference genome, using sequencing data produced by PromethION and PacBio. With PromethION, an assembly using sequenced reads with 64× coverage (193 Gb, 3 flowcell sequencing) resulted in 3,725 contigs with N50s of 16.7 Mb and a total genome length of 2.8 Gb. It was comparable to a KOREF assembly constructed using PacBio at 62× coverage (188 Gb, 2,695 contigs, and N50s of 17.9 Mb). When we applied Hi-C-derived long-range mapping data, an even higher quality assembly for the 64× coverage was achieved, resulting in 3,179 scaffolds with an N50 of 56.4 Mb. CONCLUSION: The pore-based PromethION approach provided a high-quality chromosome-scale human genome assembly at a low cost with long maximum contig and scaffold lengths and was more cost-effective than PacBio at comparable quality measurements.