Mitigative Effects of PFF-A Isolated from Ecklonia cava on Pigmentation in a Zebrafish Model and Melanogenesis in B16F10 Cells.

Melanin synthesis is a defense mechanism that prevents skin damage, but excessive accumulation of melanin occurs in the skin in various reactions such as pigmentation, lentigines, and freckles. Although anti-melanogenic effects have been demonstrated for various naturally occurring marine products that inhibit and control tyrosinase activity, most studies have not been extended to in vivo applications. Phlorofucofuroeckol-A (PFF-A, 12.5-100 µM) isolated from Ecklonia cava has previously been shown to have tyrosinase-mitigative effects in B16F10 cells, but it has not been evaluated in an in vivo model, and its underlying mechanism for anti-melanogenic effects has not been studied. In the present study, we evaluated the safety and efficacy of PFF-A for anti-melanogenic effects in an in vivo model. We selected low doses of PFF-A (1.5-15 nM) and investigated their mitigative effects on pigmentation stimulated by α-MSH in vivo and their related-mechanism in an in vitro model. The findings suggest that low-dose PFF-A derived from E. cava suppresses pigmentation in vivo and melanogenesis in vitro. Therefore, this study presents the possibility that PFF-A could be utilized as a new anti-melanogenic agent in the cosmeceutical industries.

Sorafenib induces pigmentation via the regulation of ß-catenin signalling pathway in melanoma cells.

We conducted large-scale screening test on drugs that were already approved for other diseases to find pigmentation-modulating agents. Among drugs with potential for pigmentation control, we selected sorafenib and further investigated the effect on pigmentation using HM3KO melanoma cells. As a result of treating melanoma cells with sorafenib, pigmentation was promoted in terms of melanin content and tyrosinase activity. Sorafenib increased mRNA and protein levels of pigmentation-related genes such as MITF, tyrosinase and TRP1. To uncover the action mechanism, we investigated the effect of sorafenib on the intracellular signalling pathways. Sorafenib reduced phosphorylation of AKT and ERK, suggesting that sorafenib induces pigmentation through inhibition of the AKT and ERK pathways. In addition, sorafenib significantly increased the level of active ß-catenin, together with activation of ß-catenin signalling. Mechanistic study revealed that sorafenib decreased phosphorylation of serine 9 (S9) of GSK3ß, while it increased phosphorylation of tyrosine 216 (Y216) of GSK3ß. These results suggest that sorafenib activates the ß-catenin signalling through the regulation of GSK3ß phosphorylation, thereby affecting the pigmentation process.

Iron Supplement-Enhanced Growth and Development of Hydrangea macrophylla In Vitro under Normal and High pH.

Hydrangea macrophylla is a popular perennial ornamental shrub commercially grown as potted plants, landscape plants, and cut flowers. In the process of reproduction and production of ornamental plants, the absorption of nutrients directly determines the value of the ornamental plants. Hydrangea macrophylla is very sensitive to the content and absorption of the micronutrient iron (Fe) that affects growth of its shoots. However, the physiological activity of Fe as affected by deficiency or supplementation is unknown. This work aimed at preliminary exploring the relationship between Fe and photosynthesis, and also to find the most favorable iron source and level of pH for the growth of H. macrophylla. Two Fe sources, non-chelated iron sulfate (FeSO4) and iron ethylenediaminetetraacetic acid (Fe-EDTA), were supplemented to the multipurpose medium with a final Fe concentration of 2.78 mg·L-1. The medium without any Fe supplementation was used as the control. The pH of the agar-solidified medium was adjusted to either 4.70, 5.70, or 6.70, before autoclaving. The experiment was conducted in a culture room for 60 days with 25/18 °C day and night temperatures, and a 16-hour photoperiod provided at a light intensity of 50 mmol·m-2·s-1 photosynthetic photon flux density (PPFD) from white light-emitting diodes. Supplementary Fe increased the tissue Fe content, and leaves were greener with the medium pH of 4.70, regardless of the Fe source. Compared to the control, the number of leaves for plantlets treated with FeSO4 and Fe-EDTA were 2.0 and 1.5 times greater, respectively. The chlorophyll, macronutrient, and micronutrient contents were the greatest with Fe-EDTA at pH 4.70. Furthermore, the Fe in the leaf affected the photosynthesis by regulating stomata development, pigment content, and antioxidant system, and also by adjusting the expression of genes related to Fe absorption, transport, and redistribution. Supplementation of Fe in a form chelated with EDTA along with a medium pH of 4.70 was found to be the best for the growth and development of H. macrophylla plantlets cultured in vitro.

Kaempferol, the melanogenic component of Sanguisorba officinalis, enhances dendricity and melanosome maturation/transport in melanocytes.

Kaempferol, a representative flavonoid constituent of Sanguisorba officinalis, promotes melanogenesis, but the underlying mechanisms remain unknown. Here, we evaluated the effects of kaempferol on melanocytes morphology and behavior and determined the mechanisms regulating kaempferol-induced pigmentation. We observed that kaempferol increased melanin contents and dendritic length and stimulated melanocyte migration both in vitro and vivo. It significantly enhanced the expression of microphthalmia-associated transcription factor (MITF) and downstream enzymes of melanin biosynthesis-tyrosinase (TYR), tyrosinase-related protein (TRP-1), and dopachrome tautomerase (DCT). It also induced melanosome maturation (increased stage III and IV melanosomes) and melanin transfer to dendritic tips; this was evidenced as follows: kaempferol-treated melanocytes exhibited the perimembranous accumulation of HMB45-positive melanosomes and increased the expression of Rab27A, RhoA, and Cdc42, which improved melanosome transport to perimembranous actin filaments. These results jointly indicated that kaempferol promotes melanogenesis and melanocyte growth. Additionally, kaempferol stimulated the phosphorylation of P38/ERK MAPK and downregulated p-PI3K, p-AKT, and p-P70s6K expression. Pre-incubation with P38 (SB203580) and ERK (PD98059) signaling inhibitors reversed the melanogenic and dendritic effects and MITF expression. PI3K/AKT inhibitor augmented kaempferol-induced melanin content and dendrite length. In summary, kaempferol regulated melanocytes' dendritic growth and melanosome quantity, maturation, and transport via P38/ERK MAPK and PI3K/AKT signaling pathways.

Effect of Fish Bone Bioactive Peptides on Oxidative, Inflammatory and Pigmentation Processes Triggered by UVB Irradiation in Skin Cells.

In the present study, we evaluated for the first time the photoprotective effect of fish bone bioactive peptides (FBBP) preparation isolated from silver carp (Hypophthalmichthys molitrix) discarded tissue using in vitro experimental models of skin cells exposed to ultraviolet B (UVB) irradiation and stressing agents. FBBP preparation was obtained by papain treatment of minced bones and centrifugal ultrafiltration, and the molecular weight (MW) distribution was characterized by size exclusion and reversed-phase high performance liquid chromatography (RP-HPLC). In vitro assessment of the effect of FBBP pretreatment in UVB-irradiated L929 fibroblasts and HaCaT keratinocytes revealed their cytoprotective activity. Their capacity to efficiently reduce reactive oxygen species (ROS) production and lipid peroxidation varied in a dose-dependent manner, and it was greater in fibroblasts. A decrease of proinflammatory cytokines secretion, in particular of tumor necrosis factor alpha (TNF-α), was found after FBBP pretreatment of THP-1-derived inflamed macrophages. Melanin production and tyrosinase activity investigated in UVB-irradiated Mel-Juso cells were lowered in direct relation to FBBP concentrations. FBBP fractions with high radical scavenging activity were separated by ion exchange chromatography, and two collagenic sequences were identified. All these results offer new scientific data on aquaculture fish bone-derived peptides confirming their ability to control the antioxidant, anti-inflammatory and pigmentation processes developed during UV irradiation of skin cells and recommend their use as valuable natural ingredients of photoprotective cosmeceutical products.

P-Glycoprotein Inhibitor Tariquidar Plays an Important Regulatory Role in Pigmentation in Larval Zebrafish.

Zebrafish has emerged as a powerful model in studies dealing with pigment development and pathobiology of pigment diseases. Due to its conserved pigment pattern with established genetic background, the zebrafish is used for screening of active compounds influencing melanophore, iridophore, and xanthophore development and differentiation. In our study, zebrafish embryos and larvae were used to investigate the influence of third-generation noncompetitive P-glycoprotein inhibitor, tariquidar (TQR), on pigmentation, including phenotype effects and changes in gene expression of chosen chromatophore differentiation markers. Five-day exposure to increasing TQR concentrations (1 µM, 10 µM, and 50 µM) resulted in a dose-dependent augmentation of the area covered with melanophores but a reduction in the area covered by iridophores. The observations were performed in three distinct regions-the eye, dorsal head, and tail. Moreover, TQR enhanced melanophore renewal after depigmentation caused by 0.2 mM 1-phenyl-2-thiourea (PTU) treatment. qPCR analysis performed in 56-h post-fertilization (hpf) embryos demonstrated differential expression patterns of genes related to pigment development and differentiation. The most substantial findings include those indicating that TQR had no significant influence on leukocyte tyrosine kinase, GTP cyclohydrolase 2, tyrosinase-related protein 1, and forkhead box D3, however, markedly upregulated tyrosinase, dopachrome tautomerase and melanocyte inducing transcription factor, and downregulated purine nucleoside phosphorylase 4a. The present study suggests that TQR is an agent with multidirectional properties toward pigment cell formation and distribution in the zebrafish larvae and therefore points to the involvement of P-glycoprotein in this process.

Melatonin application improves berry coloration, sucrose synthesis, and nutrient absorption in 'Summer Black' grape.

In this study, we investigated the effects of melatonin application on berry coloration, sugar accumulation, and nutrient absorption in 'Summer Black' grapes. Melatonin spraying at 100 µmol L-1 on grapes during veraison induced skin coloration earlier than that in controls, as well as higher transcript abundance of anthocyanin biosynthesis-related genes and transcription factors MYBA1 and MYBA2. Melatonin treatment increased the soluble sugar content, especially of sucrose, by promoting the activity of sucrose phosphate synthase, and also increased endogenous melatonin content and the concentrations of mineral nutrients N, K, Cu, Fe, and Zn in grape berries. Correlation analysis suggested that high sugar content promoted anthocyanin synthesis. These findings provide a sound theoretical basis for the development of techniques aimed to achieve optimum coloration of grapes in hot and rainy regions.

Flavonoids increase melanin production and reduce proliferation, migration and invasion of melanoma cells by blocking endolysosomal/melanosomal TPC2.

Two-pore channel 2 (TPC2) resides in endolysosomal membranes but also in lysosome-related organelles such as the melanin producing melanosomes. Gain-of-function polymorphisms in hTPC2 are associated with decreased melanin production and blond hair color. Vice versa genetic ablation of TPC2 increases melanin production. We show here an inverse correlation between melanin production and melanoma proliferation, migration, and invasion due to the dual activity of TPC2 in endolysosomes and melanosomes. Our results are supported by both genetic ablation and pharmacological inhibition of TPC2. Mechanistically, our data show that loss/block of TPC2 results in reduced protein levels of MITF, a major regulator of melanoma progression, but an increased activity of the melanin-generating enzyme tyrosinase. TPC2 inhibition thus provides a twofold benefit in melanoma prevention and treatment by increasing, through interference with tyrosinase activity, the synthesis of UV blocking melanin in melanosomes and by decreasing MITF-driven melanoma progression by increased GSK3ß-mediated MITF degradation.

Antioxidant Activity Evaluation of FlexirubinType Pigment from Chryseobacterium artocarpi CECT 8497 and Related Docking Study.

The current research is focused on studying the biological efficacy of flexirubin, a pigment extracted from Chryseobacterium artocarpi CECT 8497.Different methods such as DPPH, H2O2, NO•, O2•-, •OH, lipid peroxidation inhibition by FTC and TBA, ferric reducing and ferrous chelating activity were carried out to evaluate the antioxidant activity of flexirubin. Molecular docking was also carried out, seeking the molecular interactions of flexirubin and a standard antioxidant compound with SOD enzyme to figure out the possible flexirubin activity mechanism. The new findings revealed that the highest level of flexirubin exhibited similar antioxidant activity as that of the standard compound according to the H2O2, •OH, O2•-, FTC and TBA methods. On the other hand, flexirubin at the highest level has shown lower antioxidant activity than the positive control according to the DPPH and NO• and even much lower when measured by the FRAP method. Molecular docking showed that the interaction of flexirubin was in the binding cavity of the SOD enzyme and did not affect its metal-binding site. These results revealed that flexirubin has antioxidant properties and can be a useful therapeutic compound in preventing or treating free radical-related diseases.

The small molecule ZY-214-4 may reduce the virulence of Staphylococcus aureus by inhibiting pigment production.

BACKGROUND: In recent years, clinical Staphylococcus aureus isolates have become highly resistant to antibiotics, which has raised concerns about the ability to control infections by these organisms. The aim of this study was to clarify the effect of a new small molecule, ZY-214-4 (C19H11BrNO4), on S. aureus pigment production. RESULTS: At the concentration of 4 µg/mL, ZY-214-4 exerted a significant inhibitory effect on S. aureus pigment synthesis, without affecting its growth or inducing a toxic effect on the silkworm. An oxidant sensitivity test and a whole-blood killing test indicated that the S. aureus survival rate decreased significantly with ZY-214-4 treatment. Additionally, ZY-214-4 administration significantly reduced the expression of a pigment synthesis-related gene (crtM) and the superoxide dismutase genes (sodA) as determined by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. ZY-214-4 treatment also improved the survival rate of S. aureus-infected silkworm larvae. CONCLUSIONS: The small molecule ZY-214-4 has potential for the prevention of S. aureus infections by reducing the virulence associated with this bacterium.

Astaxanthin Z-isomer-rich diets enhance egg yolk pigmentation in laying hens compared to that in all-E-isomer-rich diets.

The effects of feeding diets containing astaxanthin with different Z-isomer ratios to laying hens on egg qualities, such as astaxanthin concentration in egg yolk and yolk color, were investigated. As the astaxanthin source, a natural microorganism Paracoccus carotinifaciens was used. Astaxanthin with different Z-isomer ratios was prepared by thermal treatment with different conditions and then added to the basal diet at a final astaxanthin concentration of 8 mg/kg. We found that, as the Z-isomer ratios of astaxanthin in the diet increased, the astaxanthin concentration in egg yolk and the yolk color fan score also increased significantly. Importantly, feeding a 50.6% Z-isomer ratio diet increased astaxanthin concentration in egg yolk by approximately fivefold and the color fan score by approximately 2 compared to that in hens fed an all-E-isomer-rich diet. Moreover, we showed that feeding Z-isomer-rich astaxanthin to laying hens increased plasma astaxanthin concentration by more than five times in comparison to that in hens fed an all-E-isomer-rich diet. These results indicate that Z-isomers of astaxanthin have higher bioavailability than that of the all-E-isomer and thus they exhibit greater egg yolk-accumulation efficiency.

Reciprocal Changes in miRNA Expression with Pigmentation and Decreased Proliferation Induced in Mouse B16F1 Melanoma Cells by L-Tyrosine and 5-Bromo-2'-Deoxyuridine.

Background: Many microRNAs have been identified as critical mediators in the progression of melanoma through its regulation of genes involved in different cellular processes such as melanogenesis, cell cycle control, and senescence. However, microRNAs' concurrent participation in syngeneic mouse B16F1 melanoma cells simultaneously induced decreased proliferation and differential pigmentation by exposure to 5-Brd-2'-dU (5'Bromo-2-deoxyuridine) and L-Tyr (L-Tyrosine) respectively, is poorly understood. Aim: To evaluate changes in the expression of microRNAs and identify which miRNAs in-network may contribute to the functional bases of phenotypes of differential pigmentation and reduction of proliferation in B16F1 melanoma cells exposed to 5-Brd-2'-dU and L-Tyr. Methods: Small RNAseq evaluation of the expression profiles of miRNAs in B16F1 melanoma cells exposed to 5-Brd-2'-dU (2.5 µg/mL) and L-Tyr (5 mM), as well as the expression by qRT-PCR of some molecular targets related to melanogenesis, cell cycle, and senescence. By bioinformatic analysis, we constructed network models of regulation and co-expression of microRNAs. Results: We confirmed that stimulation or repression of melanogenesis with L-Tyr or 5-Brd-2'-dU, respectively, generated changes in melanin concentration, reduction in proliferation, and changes in expression of microRNAs 470-3p, 470-5p, 30d-5p, 129-5p, 148b-3p, 27b-3p, and 211-5p, which presented patterns of coordinated and reciprocal co-expression, related to changes in melanogenesis through their putative targets Mitf, Tyr and Tyrp1, and control of cell cycle and senescence: Cyclin D1, Cdk2, Cdk4, p21, and p27. Conclusions: These findings provide insights into the molecular biology of melanoma of the way miRNAs are coordinated and reciprocal expression that may operate in a network as molecular bases for understanding changes in pigmentation and decreased proliferation induced in B16F1 melanoma cells exposed to L-Tyr and 5-Brd-2'-dU.

Expression and function analysis of crustacyanin gene family involved in resistance to heavy metal stress and body color formation in Exopalaemon carinicauda.

Crustacyanin has the function of binding astaxanthin which is the best antioxidant, and plays an important role in the body color variation of crustaceans. To investigate the causes of body color variation of the ridgetail white prawn, Exopalaemon carinicauda, the present study obtained four subtypes of crustacyanin gene: C1, C2, A1, and A2. Based on fluorescence quantitative polymerase chain reaction, lipocalin-C1 is mainly expressed in the eyestalk, lipocalin-C2 is in the ventral nerve cord, and lipocalin-A1 and lipocalin-A2 are in subcutaneous adipose tissues. Under the inhibiting effect of Cd2+ stress, the expression of four subtypes first increases and then decreases within 24 h, and reaches the maximum at 6 or 12 h. RNA interference experiments showed a decrease in the expression of lipocalin genes in subcutaneous adipose tissue for each subtype, with the body color changing from transparent to red, and the dark red spots on the epidermis changing to bright red. Moreover, the blue protein in the subcutaneous adipose tissue largely disappeared, based on the light micrographs. In view of these findings, the crustacyanin gene appears to fulfill some function in the resistance to heavy metal stress and body color formation of E. carinicauda.

Male zebra finches exposed to lead (Pb) during development have reduced volume of song nuclei, altered sexual traits, and received less attention from females as adults.

Lead (Pb) is a pervasive global contaminant that interferes with sensitive windows for neurological development and causes oxidative damage to tissues. The effects of moderate and high exposure to Pb have been well-studied in birds, but whether low-level early-life exposure to Pb influences adult phenotype remains unclear. Female songbirds use a male's song and coloration to discriminate between high- and low-quality males. Therefore, if early-life exposure to Pb disrupts song learning ability or shifts the allocation of antioxidant pigments away from colorful secondary sexual traits, male birds exposed to Pb may be less attractive to females. We exposed developing zebra finches (Taeniopygia guttata) to Pb-contaminated drinking water (100 or 1000 parts per billion [ppb]) after hatching (days 0-100). Once male finches reached adulthood (120-150 days post hatch), we measured song learning ability, coloration of bill and cheek patches, and volume of song nuclei in the brain. We also measured female preference for Pb-exposed males relative to control males. Finally, we measured motoric and spatial cognitive performance in male and female finches to assess whether cognitive traits differed in their sensitivity to Pb exposure. Male zebra finches exposed to 1000 ppb Pb had impaired song learning ability, reduced volume of song nuclei, bills with less redness and received less attention from females. Additionally, Pb exposure impaired motoric performance in both male and female finches but did not affect performance in a spatial cognitive task. Adult finches exposed to Pb-contaminated water had higher blood-Pb levels, though in all cases blood-Pb levels were below 7.0 µg dL-1. This study suggests that low-level exposure to Pb contributes to cognitive deficits that persist into adulthood and may indirectly influence fitness by altering secondary sexual traits and reducing male attractiveness.

Carotenoid deprivation and beta-carotene's effects on male and female turtle color.

Carotenoid-colored integuments commonly function as sexually selected honest signals because carotenoid pigments can be costly to obtain, ingest, absorb, metabolize or transport before being deposited into the integument. As such, carotenoid pigmentation is often sexually dichromatic, with males being more colorful than females. Sexual dichromatism may also occur in ultraviolet (UV) wavelengths, which is visible to organisms who possess UV-sensitive photoreceptors. The stripes and spots of painted turtles (Chrysemys picta) are carotenoid-based and reflect UV wavelengths. This research describes UV sexual dichromatism in painted turtles and shows how carotenoid deprivation changes spot and stripe color in male and female painted turtles. Adult turtles were fed a diet that was supplemented with carotenoids (i.e., C diet) or deprived of carotenoids (C-). Stripe and spot color were measured with UV-vis spectrometry, and blood was drawn from all turtles before and after the dietary treatment. HPLC analysis revealed five carotenoids (4 xanthophylls and beta-carotene) circulating in turtle blood. C-diet reduced yellow chroma and increased brightness of yellow and red stripes or spots, relative to the C diet, but there was no sexually dimorphic effect of carotenoid deprivation on color, nor did carotenoid deprivation affect UV reflectance. Carotenoid deprivation reduced all circulating carotenoids, but beta-carotene was the only pigment with a significant effect on post-experimental carotenoids, implying that changes in color were due in part to reduction in circulating levels of beta-carotene. Color generation appears to be complex in turtles and have dietary as well as non-dietary components.

Identification of the ceRNA networks in α-MSH-induced melanogenesis of melanocytes.

α-MSH is known for melanogenesis stimulation, and ceRNA is a new method involved in physiological regulation. However, whether ceRNA participates in α-MSH-induced melanogenesis remains unknown. We used ceRNA array to detect the expression profiles of lncRNAs, circRNAs, and mRNAs in melanocytes after α-MSH treatment. Moreover, the melanogenesis-related ceRNA regulatory networks were screened and validated. The expression profile analysis showed that 20 lncRNAs and 49 circRNAs changed five-fold after α-MSH treatment, while 933 mRNAs changed two-fold. Based on differentially expressed genes, GO and KEGG analysis were conducted and revealed that 14 genes were enriched in melanogenesis. Then, multiple lncRNA or circRNA-miRNA-mRNA ceRNA networks and lncRNA/circRNA-miRNA-mRNA quaternary ceRNA networks were identified. Thereinto, ENST00000606533, circ_0091223, and TYR expression were upregulated in α-MSH-treated melanocytes, while their complementary miR-1291 was decreased. Dual-luciferase reporter assay further verified that ENST00000606533 and circ_0091223 could bind to miR-1291. ENST00000606533 and circ_0091223 siRNAs decreased circ_0091223, ENST00000606533, and TYR expression, but increased miR-1291 expression. Conversely, miR-1291 mimics inhibited ENST00000606533, circ_0091223, and TYR expression. Moreover, miR-1291 inhibitor could reverse the inhibitory effect of the two siRNAs on TYR expression. Hence, the "ENST00000606533/circ_0091223-miR-1291-TYR" ceRNA network is involved in α-MSH-induced melanogenesis, and ceRNA networks may be potential therapeutic targets for skin pigmentation disorders.

Citric acid promoted melanin synthesis in B16F10 mouse melanoma cells, but inhibited it in human epidermal melanocytes and HMV-II melanoma cells via the GSK3ß/ß-catenin signaling pathway.

Melanin, a pigment synthesized by melanocytes in the skin, resists the damage caused by ultraviolet rays to cells. Citric acid, a well-known food additive, is commonly used as an antioxidant and is an important part of the tricarboxylic acid (TCA) cycle for energy production during cellular metabolism. Here, we aimed to investigate whether the addition of excess citric acid regulates melanin synthesis, and to delineate the underlying mechanism. First, we observed that citric acid exerts opposite redox effects on mouse and human cells. Interestingly, treatment with excess citric acid increased the melanin content in mouse cells but decreased it in human cells. Furthermore, the expression of factors important for melanin synthesis, such as microphthalmia-associated transcription factor (MITF), was also regulated by citric acid treatment-it was promoted in mouse cells and suppressed in human cells. Citric acid also impacted the upstream regulators of MITF, glycogen synthase kinase 3ß (GSK3ß), and ß-catenin. Second, we determined the importance of GSK3ß in the citric acid-mediated regulation of melanin synthesis, using a GSK3ß inhibitor (BIO). To the best of our knowledge, this is the first study to show that citric acid regulates melanin synthesis via the GSK3ß/ß-catenin signaling pathway, and that equal amounts of exogenous citric acid exert opposing effects on mouse and human cells.

PorA, a conserved C-terminal domain-containing protein, impacts the PorXY-SigP signaling of the type IX secretion system.

Porphyromonas gingivalis, a periodontal pathogen, translocates many virulence factors including the cysteine proteases referred to as gingipains to the cell surface via the type IX secretion system (T9SS). Expression of the T9SS component proteins is regulated by the tandem signaling of the PorXY two-component system and the ECF sigma factor SigP. However, the details of this regulatory pathway are still unknown. We found that one of the T9SS conserved C-terminal domain-containing proteins, PGN_0123, which we have designated PorA, is involved in regulating expression of genes encoding T9SS structural proteins and that PorA can be translocated onto the cell surface without the T9SS translocation machinery. X-ray crystallography revealed that PorA has a domain similar to the mannose-binding domain of Escherichia coli FimH, the tip protein of Type 1 pilus. Mutations in the cytoplasmic domain of the sensor kinase PorY conferred phenotypic recovery on the ΔporA mutant. The SigP sigma factor, which is activated by the PorXY two-component system, markedly decreased in the ΔporA mutant. These results strongly support a potential role for PorA in relaying a signal from the cell surface to the PorXY-SigP signaling pathway.

A Multi-Omics Approach Identifies Key Regulatory Pathways Induced by Long-Term Zinc Supplementation in Human Primary Retinal Pigment Epithelium.

In age-related macular degeneration (AMD), both systemic and local zinc levels decline. Elevation of zinc in clinical studies delayed the progression to end-stage AMD. However, the molecular pathways underpinning this beneficial effect are not yet identified. In this study, we used differentiated primary human fetal retinal pigment epithelium (RPE) cultures and long-term zinc supplementation to carry out a combined transcriptome, proteome and secretome analysis from three genetically different human donors. After combining significant differences, we identified the complex molecular networks using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). The cell cultures from the three donors showed extensive pigmentation, development of microvilli and basal infoldings and responded to zinc supplementation with an increase in transepithelial electrical resistance (TEER) (apical supplementation: 443.2 ± 79.3%, basal supplementation: 424.9 ± 116.8%, compared to control: 317.5 ± 98.2%). Significant changes were observed in the expression of 1044 genes, 151 cellular proteins and 124 secreted proteins. Gene set enrichment analysis revealed changes in specific molecular pathways related to cell adhesion/polarity, extracellular matrix organization, protein processing/transport, and oxidative stress response by zinc and identified a key upstream regulator effect similar to that of TGFB1.