Extracellular Vesicles Derived from Senescent Fibroblasts Attenuate the Dermal Effect on Keratinocyte Differentiation.

The skin is a multilayered and primary defensive organ. Intimate intercellular communication in the skin is necessary to ensure effective surveillance. Extracellular vesicles (EVs) are being explored for their involvement in intercellular skin communication. The aim of this study was to evaluate how human dermal fibroblasts (HDFs) accelerate EV production during senescence and the effects of senescence-associated EVs on epidermal homeostasis. Replicative senescent HDFs were assessed with senescence-associated ß-galactosidase staining and the expression of senescence-related markers. Isolated EVs were characterized by dynamic light scattering and EV marker expression. EVs secreted from untreated young or senescent HDFs, or from those treated with a nSMase inhibitor, antioxidant, and lysosomal activity regulators, were determined by sandwich ELISA for CD81. Human epidermal keratinocytes were treated with young- and senescent HDF-derived EVs. Compared to young HDFs, senescent HDFs produced relatively high levels of EVs due to the increased nSMase activity, oxidative stress, and altered lysosomal activity. The nSMase inhibitor, antioxidant, and agents that recovered lysosomal activity reduced EV secretion in senescent HDFs. Relative to young HDF-derived EVs, senescent HDF-derived EVs were less supportive in keratinocyte differentiation and barrier function but increased proinflammatory cytokine IL-6 levels. Our study suggests that dermis-derived EVs may regulate epidermal homeostasis by reflecting cellular status, which provides insight as to how the dermis communicates with the epidermis and influences skin senescence.

Comparative Lipidomic Analysis of Extracellular Vesicles Derived from Lactobacillus plantarum APsulloc 331261 Living in Green Tea Leaves Using Liquid Chromatography-Mass Spectrometry.

Lactobacillus plantarum is a popular probiotic species due to its safe and beneficial effects on humans; therefore, novel L. plantarum strains have been isolated and identified from various dietary products. Given that bacteria-derived extracellular vesicles (EVs) have been considered as efficient carriers of bioactive materials and shown to evoke cellular responses effectively, L. plantarum-derived EVs are expected to efficiently elicit health benefits. Herein, we identified L. plantarum APsulloc 331261 living in green tea leaves and isolated EVs from the culture medium. We performed quantitative lipidomic analysis of L. plantarum APsulloc 331261 derived EVs (LEVs) using liquid chromatography-mass spectrometry. In comparison to L. plantarum APsulloc 331261, in LEVs, 67 of 320 identified lipid species were significantly increased and 19 species were decreased. In particular, lysophosphatidylserine(18:4) and phosphatidylcholine(32:2) were critically increased, showing over 21-fold enrichment in LEVs. In addition, there was a notable difference between LEVs and the parent cells in the composition of phospholipids. Our results suggest that the lipidomic profile of bacteria-derived EVs is different from that of the parent cells in phospholipid content and composition. Given that lipids are important components of EVs, quantitative and comparative analyses of EV lipids may improve our understanding of vesicle biogenesis and lipid-mediated intercellular communication within or between living organisms.

Staphylococcus aureus-derived extracellular vesicles induce monocyte recruitment by activating human dermal microvascular endothelial cells in vitro.

BACKGROUND: Atopic dermatitis (AD) represents the most common inflammatory skin disorder in children showing massive infiltration of immune cells. The colonization of AD-afflicted skin by Staphylococcus aureus and S. aureus-derived extracellular vesicles (SEVs) has been associated with AD pathogenesis; however, the molecular mechanism underlying SEV-mediated inflammatory responses remains unclear. OBJECTIVE: We investigated how SEVs can mediate inflammatory responses in AD pathogenesis by examining the effect of SEVs on human dermal microvascular endothelia cells (HDMECs). METHODS: HDMECs were treated with SEVs, and the expression of cell adhesion molecules or cytokines was assessed using RT-qPCR, Western blot or cytokine array analyses. The receptor for SEVs and related signalling molecules in HDMECs were addressed and verified via gene knockdown or inhibitor experiments. The recruitment assay of human THP-1 monocytic cells on HDMECs was performed after SEV treatment in the presence or absence of the verified receptor or signalling molecule. RESULTS: SEVs, but not other gram-positive bacteria-derived extracellular vesicles, directly activated HDMECs by increasing the expression of cell adhesion molecules (E-selectin, VCAM1 and ICAM1) and that of IL-6, the inflammatory cytokine; consequently, they enhanced the recruitment of THP-1 monocytic cells to HDMECs. The SEV-induced HDMEC activation was dependent on Toll-like receptor 4 and the NF-κB signalling pathway, which was rapidly activated within 1 hour post-treatment and followed by an upregulation of cell adhesion molecules and IL-6 at later time-points. Moreover, SEV-mediated HDMEC responses were more rapid and intense than those induced by the same protein concentrations of S. aureus extracts. CONCLUSIONS & CLINICAL RELEVANCE: SEVs as proinflammatory factors could mediate immune cell infiltration in AD by efficiently inducing endothelial cell activation and monocyte recruitment, which may provide insights into alleviating the S. aureus-mediated onset or progression of AD and its phenotypes.

TIMP3 is a CLOCK-dependent diurnal gene that inhibits the expression of UVB-induced inflammatory cytokines in human keratinocytes.

As the outermost physical barrier of an organism, the skin is diurnally exposed to UV radiation (UVR). Recent studies have revealed that the skin exhibits a circadian rhythm in various functions, and this oscillation is disturbed and reset via a strong environmental cue, the UVR. However, a molecular link between circadian perturbation by UVR and UVR-induced cellular responses has not been investigated. We identified tissue inhibitor of metalloproteinase ( TIMP)- 3 as a novel circadian locomotor output cycles kaput (CLOCK)-dependent diurnal gene by using a CLOCK-knockdown strategy in human keratinocytes. Among dozens of identified transcripts down-regulated by CLOCK knockdown, TIMP3 displayed a rhythmic expression in a CLOCK-dependent manner, in which the expression of matrix metalloproteinase (MMP)-1 and inflammatory cytokines, such as TNF-α, chemokine (C-X-C motif) ligand (CXCL)-1, and IL-8, were inversely regulated. Upon UVB exposure, the expression of CLOCK and TIMP3 was down-regulated, which led to an up-regulation of secretion of MMP1 and TNF-α proteins and in the transcription of CXCL1 and IL-8 via CCAAT-enhancer binding protein (C/EBP)-α. UVB-induced TNF-α secretion increased further or decreased by knockdown or overexpression of TIMP3, respectively, as well as by CLOCK. As a novel CLOCK-dependent diurnal gene, TIMP3 inhibits the expression of inflammatory cytokines that are up-regulated by UV irradiation in human keratinocytes. Thus, our work suggests a molecular link between circadian perturbation by UVR and UVR-induced inflammation.-Park, S., Kim, K., Bae, I.-H., Lee, S. H., Jung, J., Lee, T. R., Cho, E.-G. TIMP3 is a CLOCK-dependent diurnal gene that inhibits the expression of UVB-induced inflammatory cytokines in human keratinocytes.

Kojyl Cinnamate Ester Derivatives Increase Adiponectin Expression and Stimulate Adiponectin-Induced Hair Growth Factors in Human Dermal Papilla Cells.

Adiponectin (APN), released mainly from adipose tissue, is a well-known homeostatic factor for regulating glucose levels, lipid metabolism, and insulin sensitivity. A recent study showed that human hair follicles express APN receptors and the presence of APN-mediated hair growth signaling, thereby suggesting that APN is a potent hair growth-promoting adipokine. Previously, kojyl cinnamate ester derivatives (KCEDs) were synthesized in our institute as new anti-aging or adiponectin-/adipogenesis-inducing compounds. Here, we tested the activity of these derivatives to induce endogenous APN secretion. Among the derivatives, KCED-1 and KCED-2 showed improved activity in inducing APN mRNA expression, secretion of APN protein, and adipogenesis in human subcutaneous fat cells (hSCFs) when compared with the effects of Seletinoid G, a verified APN inducer. When human follicular dermal papilla cells were treated with the culture supernatant of KCED-1- or KCED-2-treated hSCFs, the mRNA expression of APN-induced hair growth factors such as insulin-like growth factor, hepatocyte growth factor, and vascular endothelial growth factor was upregulated compared with that in the control. Taken together, our study shows that among kojyl cinnamate ester derivatives, KCED-1, KCED-2, as well as Seletinoid G are effective inducers of endogenous APN production in subcutaneous fat tissues, which may in turn contribute to the promotion of hair growth in the human scalp.

Circadian Expression of TIMP3 Is Disrupted by UVB Irradiation and Recovered by Green Tea Extracts.

The human skin is the outermost physical barrier and has its own circadian machinery that works either cooperatively with the central clock, or autonomously. Circadian rhythms have been observed in many functions related to epidermal homeostasis including hydration and inflammation, and this functional oscillation is disturbed by ultraviolet radiation (UVR), which is a strong environmental cue. Among the genes estimated to show circadian expression in the skin, metalloproteinase inhibitor 3 (TIMP3), has a rhythmic expression in synchronized human keratinocytes similar to that of the core clock gene PER1 and an epidermal circadian regulatory gene, aquaporin 3 (AQP3) but was antiphase to the core clock gene BMAL1. Tumor necrosis factor-α (TNF-α), the regulatory target of TIMP3 via a disintegrin and metalloproteinase domain 17 (ADAM17), was inversely regulated when TIMP3 expression was downregulated by ultraviolet B (UVB) treatment. When synthetic TIMP3 peptides were applied to the cells, the secretion of TNF-α did not increase following the UVB treatment. Similar to TIMP3 peptides, Camellia sinensis leaf-derived extracts showed a distinguishing efficacy in recovering TIMP3 expression, downregulated by UVB treatment. Together, our results suggest that TIMP3 reversely mediates UVR-induced inflammation by being highly expressed during the daytime; therefore, recovering the circadian expression of TIMP3 using synthetic TIMP3 peptides or bioactive natural ingredients could at least in part inhibit the UVR-induced cellular phenomena.

Basic amino-acid side chains regulate transmembrane integrin signalling.

Side chains of Lys/Arg near transmembrane domain (TMD) membrane-water interfaces can 'snorkel', placing their positive charge near negatively charged phospholipid head groups; however, snorkelling's functional effects are obscure. Integrin ß TMDs have such conserved basic amino acids. Here we use NMR spectroscopy to show that integrin ß(3)(Lys 716) helps determine ß(3) TMD topography. The α(ΙΙb)ß(3) TMD structure indicates that precise ß(3) TMD crossing angles enable the assembly of outer and inner membrane 'clasps' that hold the αß TMD together to limit transmembrane signalling. Mutation of ß(3)(Lys 716) caused dissociation of α(ΙΙb)ß(3) TMDs and integrin activation. To confirm that altered topography of ß(3)(Lys 716) mutants activated α(ΙΙb)ß(3), we used directed evolution of ß(3)(K716A) to identify substitutions restoring default state. Introduction of Pro(711) at the midpoint of ß(3) TMD (A711P) increased α(ΙΙb)ß(3) TMD association and inactivated integrin α(ΙΙb)ß(3)(A711P,K716A). ß(3)(Pro 711) introduced a TMD kink of 30 ± 1° precisely at the border of the outer and inner membrane clasps, thereby decoupling the tilt between these segments. Thus, widely occurring snorkelling residues in TMDs can help maintain TMD topography and membrane-embedding, thereby regulating transmembrane signalling.

ΔNp63 intronic miR-944 is implicated in the ΔNp63-mediated induction of epidermal differentiation.

ΔNp63 is required for both the proliferation and differentiation of keratinocytes, but its role in the differentiation of these cells is poorly understood. The corresponding gene, TP63, harbors the MIR944 sequence within its intron. However, the mechanism of biogenesis and the function of miR-944 are unknown. We found that miR-944 is highly expressed in keratinocytes, in a manner that is concordant with that of ΔNp63 mRNA, but the regulation of miR-944 expression under various conditions did not correspond with that of ΔNp63. Bioinformatics analysis and functional studies demonstrated that MIR944 has its own promoter. We demonstrate here that MIR944 is a target of ΔNp63. Promoter analysis revealed that the activity of the MIR944 promoter was markedly enhanced by the binding of ΔNp63, which was maintained by the supportive action of AP-2 during keratinocyte differentiation. Our results indicated that miR-944 biogenesis is dependent on ΔNp63 protein, even though it is generated from ΔNp63 mRNA-independent transcripts. We also demonstrated that miR-944 induces keratin 1 and keratin 10 expression by inhibiting ERK signaling and upregulating p53 expression. Our findings suggested that miR-944, as an intronic miRNA and a direct target of ΔNp63, contributes to the function of ΔNp63 in the induction of epidermal differentiation.

S100A7 (psoriasin) inhibits human epidermal differentiation by enhanced IL-6 secretion through IκB/NF-κB signalling.

Psoriasin (S100A7), a member of the S100 protein family, is a well-known antimicrobial peptide and a signalling molecule which regulates cellular function and is highly expressed in hyperproliferative skin conditions such as atopic dermatitis (AD) and psoriasis with disrupted skin barrier function. However, its role in epidermal differentiation remains unknown. We examined the effect of S100A7 on epidermal differentiation in normal human keratinocytes (NHKs) and on a reconstituted human epidermis model. When NHKs were exposed to disruptive stimuli such as Staphylococcus aureus, ultraviolet irradiation and retinoic acid, the secretion of S100A7 into the culture medium increased and the expression of epidermal differentiation markers decreased. Treatment of NHKs with S100A7 significantly inhibited epidermal differentiation by reducing the expression of keratin 1, keratin 10, involucrin and loricrin and by increasing the expression of abnormal differentiation markers (keratin 6 and keratin 16). We verified that the MyD88-IκB/NF-κB signal cascade was activated via RAGE after S100A7 treatment, resulting in the upregulation of interleukin-6. Finally, we confirmed that S100A7 is a negative regulator of epidermal differentiation using a reconstituted human epidermis model. This study suggests that S100A7-related signalling molecules could be potent targets for recovering skin barrier function in AD and psoriasis where S100A7 is accumulated excessively.

The Development of Sugar-Based Anti-Melanogenic Agents.

The regulation of melanin production is important for managing skin darkness and hyperpigmentary disorders. Numerous anti-melanogenic agents that target tyrosinase activity/stability, melanosome maturation/transfer, or melanogenesis-related signaling pathways have been developed. As a rate-limiting enzyme in melanogenesis, tyrosinase has been the most attractive target, but tyrosinase-targeted treatments still pose serious potential risks, indicating the necessity of developing lower-risk anti-melanogenic agents. Sugars are ubiquitous natural compounds found in humans and other organisms. Here, we review the recent advances in research on the roles of sugars and sugar-related agents in melanogenesis and in the development of sugar-based anti-melanogenic agents. The proposed mechanisms of action of these agents include: (a) (natural sugars) disturbing proper melanosome maturation by inducing osmotic stress and inhibiting the PI3 kinase pathway and (b) (sugar derivatives) inhibiting tyrosinase maturation by blocking N-glycosylation. Finally, we propose an alternative strategy for developing anti-melanogenic sugars that theoretically reduce melanosomal pH by inhibiting a sucrose transporter and reduce tyrosinase activity by inhibiting copper incorporation into an active site. These studies provide evidence of the utility of sugar-based anti-melanogenic agents in managing skin darkness and curing pigmentary disorders and suggest a future direction for the development of physiologically favorable anti-melanogenic agents.

Synaptic protein α1-takusan mitigates amyloid-ß-induced synaptic loss via interaction with tau and postsynaptic density-95 at postsynaptic sites.

The synaptic toxicity of soluble amyloid-ß (Aß) oligomers plays a critical role in the pathophysiology of Alzheimer's disease (AD). Here we report that overexpressed α1-takusan, which we previously identified as a protein that enhances synaptic activity via interaction with PSD-95, mitigates oligomeric Aß-induced synaptic loss. In contrast, takusan knockdown results in enhanced synaptic damage. α1-Takusan interacts with tau either directly or indirectly, and prevents Aß-induced tau hyperphosphorylation and mitochondrial fragmentation. Deletion analysis identified the second domain (D2) within the takusan protein that is required for PSD-95 clustering and synaptic protection from Aß. A 51 aa sequence linking D2 to the PDZ-binding C terminus was found to be as effective as full-length takusan in protecting synapses from Aß-induced damage. Moreover, a sequence containing the D2 from the human protein discs large homolog 5, when linked to a C-terminal PDZ-binding motif, can also increase the clustering of PSD-95 in cortical dendrites. In summary, α1-takusan protects synapses from Aß-induced insult via interaction with PSD-95 and tau. Thus, takusan-based protein sequences from either mouse or human may be of potential therapeutic benefit in AD.

Regulation of pigmentation by substrate elasticity in normal human melanocytes and melanotic MNT1 human melanoma cells.

The elasticity of the cellular microenvironment is a key regulator of cellular physiology in many cell types. To investigate the effects of substrate stiffness on the pigmentation process, we cultured normal human melanocytes (NHM) and MNT1 melanoma cells on laminin-coated polydimethylsiloxane (PDMS) substrates of different stiffness. The dendricity of NHM and MNT1 cells was reduced as the substrate stiffness decreased, and the degree of melanosome transfer from NHM or MNT1 cells to normal human keratinocytes was decreased on softer substrates with the reduced dendricity. Gene and protein expressions of MITF, tyrosinase, TRP2, and gp100/PMEL17 exhibited a consistent decreasing trend with the decreasing stiffness. Because the stiffness sensing is mediated by focal adhesion complex through integrin receptors, we checked laminin specific integrin alpha 6 and p-FAK for MNT1 cells to observe that the substrate adhesion was weakened as the substrate stiffness decreased. Weaker adhesion on a softer substrate was accompanied by dynamic shape changes in MNT1 cells with higher speed and larger scattering. Dendritic MNT1 cells cultured on a stiffer substrate exhibited lower migration with smaller root mean squared displacement. These results demonstrate the possibility that skin pigmentation can be influenced by mechanical properties of the cellular microenvironment and can increase when the skin becomes stiff.

Novel inhibitory effect of N-(2-hydroxycyclohexyl)valiolamine on melanin production in a human skin model.

Hyper-pigmentation causes skin darkness and medical disorders, such as post-inflammatory melanoderma and melasma. Therefore, the development of anti-melanogenic agents is important for treating these conditions and for cosmetic production. In our previous paper, we demonstrated that the anti-diabetic drug voglibose, a valiolamine derivative, is a potent anti-melanogenic agent. In addition, we proposed an alternative screening strategy to identify valiolamine derivatives with high skin permeability that act as anti-melanogenic agents when applied topically. In this study, we synthesized several valiolamine derivatives with enhanced lipophilicity and examined their inhibitory effects in a human skin model. N-(2-hydroxycyclohexyl)valiolamine (HV) possesses a stronger inhibitory effect on melanin production than voglibose in a human skin model, suggesting that HV is a more potent anti-melanogenic agent for the skin.

Producing highly effective extracellular vesicles using IBAR and talin F3 domain fusion.

Extracellular vesicles (EVs), transporting diverse cellular components, play a crucial role in intercellular communication in numerous physiological and pathological processes. EVs have also been recognized as a drug delivery platform for therapeutic purposes and cell-free regenerative medicine. While various approaches have focused on increasing EV production for efficient use therapeutic use of EVs, enhancing the quality of EVs, such as ensuring efficient uptake by their target cells, has not been widely explored. In this study, we linked a negative membrane curvature-forming inverse BAR (IBAR) domain with an integrin ß tail-binding talin F3 domain to create the IBAR-F3 fusion protein. We observed that IBAR-F3 can trigger filopodia-like membrane protrusions and attract integrins to those protrusion-rich regions, when expressed in Chinese hamster ovary cells expressing integrin αIIbß3. Surprisingly, the expression of IBAR-F3 also induced a robust production of EVs, which were then efficiently taken up by nearby cells in an integrin-dependent manner. Moreover, IBAR triggered integrin activation, presumably by inducing negative membrane curvature that likely disrupts the interaction between the integrin α and ß transmembrane domain. Therefore, we suggest that IBAR-F3 should be utilized to promote both EV production and efficient uptake mediated by integrins. Furthermore, the negative curvature-inducing integrin activation suggests that integrins on EVs can be activated by the nanoscale change in the curvature of the EV without the need for conventional machinery to activate integrin inside the EVs.

Isolation, genomic analysis and functional characterization of Enterococcus rotai CMTB-CA6, a putative probiotic strain isolated from a medicinal plant Centella asiatica.

Probiotics and their derivatives offer significant health benefits by supporting digestive health, boosting the immune system, and regulating the microbiomes not only of the internal gastrointestinal track but also of the skin. To be effective, probiotics and their derivatives must exhibit robust antimicrobial activity, resilience to adverse conditions, and colonization capabilities in host tissues. As an alternative to animal-derived probiotics, plant-derived lactic acid bacteria (LAB) present promising advantages, including enhanced diversity and tolerance to challenging environments. Our study focuses on exploring the potential of plant-derived LAB, particularly from the medicinal plant Centella asiatica, in improving skin conditions. Through a bacterial isolation procedure from C. asiatica leaves, Enterococcus rotai CMTB-CA6 was identified via 16S rRNA sequencing, whole genome sequencing, and bioinformatic analyses. Based on genomic analysis, antimicrobial-resistance and virulence genes were not detected. Additionally, the potential functions of E. rotai CMTB-CA6 were characterized by its lysates' ability to regulate skin microbes, such as stimulating the growth of Staphylococcus epidermidis while inhibiting that of Cutibacterium acnes, to restore the viability of human dermal fibroblasts under inflammatory conditions, and to demonstrate effective antioxidant activities both in a cell-free system and in human dermal fibroblasts. Our investigation revealed the efficacy of E. rotai CMTB-CA6 lysates in improving skin conditions, suggesting its potential use as a probiotic-derived agent for skin care products. Considering the ecological relationship between plant-inhabited bacteria and their host plants, we suggest that the utilization of E. rotai CMTB-CA6 strain for fermenting its host plant, C. asiatica, could be a novel approach to efficiently enriching bioactive molecules for human health benefits.

High-frequency hippocampal oscillations activated by optogenetic stimulation of transplanted human ESC-derived neurons.

After transplantation, individual stem cell-derived neurons can functionally integrate into the host CNS; however, evidence that neurons derived from transplanted human embryonic stem cells (hESCs) can drive endogenous neuronal network activity in CNS tissue is still lacking. Here, using multielectrode array recordings, we report activation of high-frequency oscillations in the ß and γ ranges (10-100 Hz) in the host hippocampal network via targeted optogenetic stimulation of transplanted hESC-derived neurons.

Epithin/PRSS14 proteolytically regulates angiopoietin receptor Tie2 during transendothelial migration.

Epithin/PRSS14, a type II transmembrane serine protease, is involved in normal epithelial development and tumor progression. Here we report, as an interacting substrate of epithin, a receptor tyrosine kinase Tie2 that is well known for important roles in the vessel stability. Epithin interacts with and degrades the Tie2 extracellular portion that contains the ligand-binding domain. Epithin is located in the neighbor of Tie2-expressing vessels in normal tissue. Furthermore, epithin can cleave and degrade Tie2 not only in the same cell but also from neighboring cells nearby, resulting in the degradation of the Tie2 ectodomain. The remaining Tie2 fragment was highly phosphorylated and was able to recruit a downstream effector, phosphatidylinositol 3-kinase. Knocking down epithin expression using short hairpin RNA in thymoma cell severely impaired the migration through endothelial cells that show the actin rearrangement during the process. The diminution of epithin protein expression in 4T1 breast cancer cells caused the significant decrease in the number of transendothelial migrating cells in vitro as well as in those of metastasizing tumor nodules in vivo, Therefore, we propose that epithin, which regulates endothelial Tie2 functions, plays a critical role in the fine tuning of transendothelial migration for normal and cancer cells.

Novel inhibitory effect of the antidiabetic drug voglibose on melanogenesis.

Overproduction of melanin can lead to medical disorders such as postinflammatory melanoderma and melasma. Therefore, developing antimelanogenic agents is important for both medical and cosmetic purposes. In this report, we demonstrated for the first time that the antidiabetic drug voglibose is a potent antimelanogenic agent. Voglibose is a representative antidiabetic drug possessing inhibitory activity towards human α-glucosidase; it blocked the proper N-glycan modification of tyrosinase, resulting in a dramatic reduction of the tyrosinase protein level by altering its stability and subsequently decreasing melanin production. Acarbose, another antihyperglycaemic drug that has a lower inhibitory effect on human intracellular α-glucosidase compared with voglibose, did not cause any changes in either the N-glycan modification of tyrosinase or the tyrosinase protein level, indicating that voglibose was the most efficient antimelanogenic agent among the widely used antihyperglycaemic agents. Considering that voglibose was originally selected from the valiolamine derivatives in a screen for an oral antidiabetic drug with a strong inhibitory activity towards intestinal α-glucosidase and low cell permeability, we propose an alternative strategy for screening compounds from valiolamine derivatives that show high inhibitory activity towards human intracellular α-glucosidases and high cell permeability, with the goal of obtaining antimelanogenic agents that are effective inside the cells.

Human Probiotic Lactobacillus paracasei-Derived Extracellular Vesicles Improve Tumor Necrosis Factor-α-Induced Inflammatory Phenotypes in Human Skin.

Lactic acid bacteria (LAB), a probiotic, provide various health benefits. We recently isolated a new Lactobacillus paracasei strain with strong anti-inflammatory effects under lipopolysaccharide-induced conditions and proposed a new mode of action-augmenting the endoplasmic reticulum stress pathway for anti-inflammatory functions in host cells. The beneficial effects of the L. paracasei strains on the skin have been described; however, the effects of L. paracasei-derived extracellular vesicles (LpEVs) on the skin are poorly understood. Herein, we investigated whether LpEVs can improve inflammation-mediated skin phenotypes by determining their effects on primary human skin cells and a three-dimensional (3D) full-thickness human skin equivalent under tumor necrosis factor (TNF)-α-challenged inflammatory conditions. LpEVs were efficiently taken up by the human skin cells and were much less cytotoxic to host cells than bacterial lysates. Furthermore, low LpEV concentrations efficiently restored TNF-α-induced cellular phenotypes, resulting in increased cell proliferation and collagen synthesis, but decreased inflammatory factor levels (matrix metalloproteinase 1, interleukin 6, and interleukin 8) in the human dermal fibroblasts, which was comparable to that of retinoic acid, a representative antiaging compound. The beneficial effects of LpEVs were validated in a 3D full-thickness human skin equivalent model. LpEV treatment remarkably restored the TNF-α-induced epidermal malformation, abnormal proliferation of keratinocytes in the basal layer, and reduction in dermal collagen synthesis. Additionally, LpEVs penetrated and reached the deepest dermal layer within 24 h when overlaid on top of a 3D full-thickness human skin equivalent. Furthermore, they possessed superior antioxidant capacity compared with the human cell-derived EVs. Taken together, the anti-inflammatory probiotic LpEVs can be attractive antiaging and antioxidant substances for improving inflammation-induced skin phenotypes and disorders.

Non pharmacological high-intensity ultrasound treatment of human dermal fibroblasts to accelerate wound healing.

Inspired by the effectiveness of low-intensity ultrasound on tissue regeneration, we investigated the potential effect of short-term high-intensity ultrasound treatment for acceleration of wound healing in an in vitro wound model and dermal equivalent, both comprising human dermal fibroblasts. Short-term ultrasound of various amplitudes significantly increased the proliferation and migration of fibroblasts and subsequently increased the production of the extracellular matrix components fibronectin and collagen type I, both of which are important for wound healing and are secreted by fibroblasts. In addition, ultrasound treatment increased the contraction of a fibroblast-embedded three-dimensional collagen matrix, and the effect was synergistically increased in the presence of TGF-ß. RNA-sequencing and bioinformatics analyses revealed changes in gene expression and p38 and ERK1/2 MAPK pathway activation in the ultrasound-stimulated fibroblasts. Our findings suggest that ultrasound as a mechanical stimulus can activate human dermal fibroblasts. Therefore, the activation of fibroblasts using ultrasound may improve the healing of various types of wounds and increase skin regeneration.