Femtosecond pulsed laser photodynamic therapy activates melanin and eradicates malignant melanoma.

Photodynamic therapy (PDT) relies on a series of photophysical and photochemical reactions leading to cell death. While effective for various cancers, PDT has been less successful in treating pigmented melanoma due to high light absorption by melanin. Here, this limitation is addressed by 2-photon excitation of the photosensitizer (2p-PDT) using ~100 fs pulses of near-infrared laser light. A critical role of melanin in enabling rather than hindering 2p-PDT is elucidated using pigmented and non-pigmented murine melanoma clonal cell lines in vitro. The photocytotoxicities were compared between a clinical photosensitizer (Visudyne) and a porphyrin dimer (Oxdime) with ~600-fold higher σ2p value. Unexpectedly, while the 1p-PDT responses are similar in both cell lines, 2p activation is much more effective in killing pigmented than non-pigmented cells, suggesting a dominant role of melanin 2p-PDT. The potential for clinical translational is demonstrated in a conjunctival melanoma model in vivo, where complete eradication of small tumors was achieved. This work elucidates the melanin contribution in multi-photon PDT enabling significant advancement of light-based treatments that have previously been considered unsuitable in pigmented tumors.

Adaptive cooling strategy via human hair: High optothermal conversion efficiency of solar radiation into thermal dissipation.

Natural species have developed complex nanostructures in a hierarchical pattern to control the absorption, reflection, or transmission of desired solar and infrared wavelengths. This bio-inspired structure is a promising method to manipulating solar energy and thermal management. In particular, human hair is used in this article to highlight the optothermal properties of bio-inspired structures. This study investigated how melanin, an effective solar absorber, and the structural morphology of aligned domains of keratin polymer chains, leading to a significant increase in solar path length, which effectively scatter and absorb solar radiation across the hair structure, as well as enhance thermal ramifications from solar absorption by fitting its radiative wavelength to atmospheric transmittance for high-yield radiative cooling with realistic human body thermal emission.

Exploratory Rearing Is Governed by Hypothalamic Melanin-Concentrating Hormone Neurons According to Locus Ceruleus.

Information seeking, such as standing on tiptoes to look around in humans, is observed across animals and helps survival. Its rodent analog-unsupported rearing on hind legs-was a classic model in deciphering neural signals of cognition and is of intense renewed interest in preclinical modeling of neuropsychiatric states. Neural signals and circuits controlling this dedicated decision to seek information remain largely unknown. While studying subsecond timing of spontaneous behavioral acts and activity of melanin-concentrating hormone (MCH) neurons (MNs) in behaving male and female mice, we observed large MN activity spikes that aligned to unsupported rears. Complementary causal, loss and gain of function, analyses revealed specific control of rear frequency and duration by MNs and MCHR1 receptors. Activity in a key stress center of the brain-the locus ceruleus noradrenaline cells-rapidly inhibited MNs and required functional MCH receptors for its endogenous modulation of rearing. By defining a neural module that both tracks and controls rearing, these findings may facilitate further insights into biology of information seeking.

Genetically Encoded Lizard Color Divergence for Camouflage and Thermoregulation.

Local adaptation is critical in speciation and evolution, yet comprehensive studies on proximate and ultimate causes of local adaptation are generally scarce. Here, we integrated field ecological experiments, genome sequencing, and genetic verification to demonstrate both driving forces and molecular mechanisms governing local adaptation of body coloration in a lizard from the Qinghai-Tibet Plateau. We found dark lizards from the cold meadow population had lower spectrum reflectance but higher melanin contents than light counterparts from the warm dune population. Additionally, the colorations of both dark and light lizards facilitated the camouflage and thermoregulation in their respective microhabitat simultaneously. More importantly, by genome resequencing analysis, we detected a novel mutation in Tyrp1 that underpinned this color adaptation. The allele frequencies at the site of SNP 459# in the gene of Tyrp1 are 22.22% G/C and 77.78% C/C in dark lizards and 100% G/G in light lizards. Model-predicted structure and catalytic activity showed that this mutation increased structure flexibility and catalytic activity in enzyme TYRP1, and thereby facilitated the generation of eumelanin in dark lizards. The function of the mutation in Tyrp1 was further verified by more melanin contents and darker coloration detected in the zebrafish injected with the genotype of Tyrp1 from dark lizards. Therefore, our study demonstrates that a novel mutation of a major melanin-generating gene underpins skin color variation co-selected by camouflage and thermoregulation in a lizard. The resulting strong selection may reinforce adaptive genetic divergence and enable the persistence of adjacent populations with distinct body coloration.

Metal-Loaded Synthetic Melanin via Oxidative Polymerization of Neurotransmitter Norepinephrine Exhibiting High Photothermal Conversion.

Polydopamine (PDA)-derived melanin-like materials exhibit significant photothermal conversion owing to their broad-spectrum light absorption. However, their low near-infrared (NIR) absorption and inadequate hydrophilicity compromise their utilization of solar energy. Herein, we developed metal-loaded poly(norepinephrine) nanoparticles (PNE NPs) by predoping metal ions (Fe3+, Mn3+, Co2+, Ca2+, Ga3+, and Mg2+) with norepinephrine, a neuron-derived biomimetic molecule, to address the limitations of PDA. The chelation between catechol and metal ions induces a ligand-to-metal charge transfer (LMCT) through the formation of donor-acceptor pairs, modulating the light absorption behavior and reducing the band gap. Under 1 sun illumination, the Fe-loaded PNE coated wood evaporator achieved a high seawater evaporation rate and efficiency of 1.75 kg m-2 h-1 and 92.4%, respectively, owing to the superior hydrophilicity and photothermal performance of PNE. Therefore, this study offers a comprehensive exploration of the role of metal ions in enhancing the photothermal properties of synthetic melanins.

Melanin granules morphology and distribution in human black hair investigated by focused ion beam scanning electron microscopy: Differences between Asian and Caucasian hair.

Melanin granules (melanosomes) in Asian and Caucasian black hairs were investigated by focused ion beam scanning electron microscopy (FIB-SEM). This technique facilitates a direct evaluation of the three-dimensional distribution and morphology of melanin granules without requiring their isolation from hair. Three-dimensional reconstructed images of melanin granule distribution in hair samples were obtained using serial SEM images observed by FIB-SEM. Melanin granules in black hair tended to be three-dimensionally dense in the outer periphery of the cortex. The morphometric parameters of melanin granules were calculated using the reconstructed three-dimensional images. The results confirmed that melanin granules in Caucasian black hair were much smaller those in Asian black hair. Moreover, it was indicated that the relative frequency distribution of the volume of melanin granules was significantly different between Asians and Caucasians.

Inhibitory actions of melanin-concentrating hormone in the lateral septum.

Melanin-concentrating hormone (MCH) neurons can co-express several neuropeptides or neurotransmitters and send widespread projections throughout the brain. Notably, there is a dense cluster of nerve terminals from MCH neurons in the lateral septum (LS) that innervate LS cells by glutamate release. The LS is also a key region integrating stress- and anxiety-like behaviours, which are also emerging roles of MCH neurons. However, it is not known if or where the MCH peptide acts within the LS. We analysed the projections from MCH neurons in male and female mice anteroposteriorly throughout the LS and found spatial overlap between the distribution pattern of MCH-immunoreactive (MCH-ir) fibres with MCH receptor Mchr1 mRNA hybridization or MCHR1-ir cells. This overlap was most prominent along the ventral and lateral border of the rostral part of the LS (LSr). Most MCHR1-labelled LS neurons lay adjacent to passing MCH-ir fibres, but some MCH-ir varicosities directly contacted the soma or cilium of MCHR1-labelled LS neurons. We thus performed whole-cell patch-clamp recordings from MCHR1-rich LSr regions to determine if and how LS cells respond to MCH. Bath application of MCH to acute brain slices activated a bicuculline-sensitive chloride current that directly hyperpolarized LS cells. This MCH-mediated hyperpolarization was blocked by calphostin C, which suggested that the inhibitory actions of MCH were mediated by protein kinase C-dependent activation of GABAA receptors. Taken together, these findings define potential hotspots within the LS that may elucidate the contributions of MCH to stress- or anxiety-related feeding behaviours. KEY POINTS: Melanin-concentrating hormone (MCH) neurons have dense nerve terminals within the lateral septum (LS), a key region underlying stress- and anxiety-like behaviours that are emerging roles of the MCH system, but the function of MCH in the LS is not known. We found spatial overlap between MCH-immunoreactive fibres, Mchr1 mRNA, and MCHR1 protein expression along the lateral border of the LS. Within MCHR1-rich regions, MCH directly inhibited LS cells by increasing chloride conductance via GABAA receptor activation in a protein kinase C-dependent manner. Electrophysiological MCH effects in brain slices have been elusive, and few studies have described the mechanisms of MCH action. Our findings demonstrated, to our knowledge, the first description of MCHR1 Gq-coupling in brain slices, which was previously predicted in cell or primary culture models only. Together, these findings defined hotspots and mechanistic underpinnings for MCH effects such as in feeding and anxiety-related behaviours.

Melanin-concentrating hormone receptor 1 is discarded by exosomes after internalization.

Melanin-concentrating hormone (MCH) receptor 1 (MCHR1), a G protein-coupled receptor, is poised for interaction with its ligands on the plasma membrane. Analyses of MCHR1 knockout mice suggest that this receptor could be a therapeutic target for the treatment of appetite disorders, glucose metabolism, psychiatric disorders, and inflammation. Binding of MCH to MCHR1 initiates calcium signaling, which is subsequently attenuated through receptor internalization. However, the ultimate destiny of the receptor post-internalization remains unexplored. In this study, we report the extracellular secretion of MCHR1 via exosomes. The recruitment of MCHR1 to exosomes occurs subsequent to its internalization, which is induced by stimulation with the ligand MCH. Although a highly glycosylated form of MCHR1, potentially representing a mature form, is selectively recruited to exosomes, the MCHR1 transferred into other cells does not exhibit functionality. The truncation of MCHR1 at the C-terminus not only impairs its response to MCH but also hinders its recruitment to exosomes. These findings imply that functional MCHR1 could be secreted extracellularly via exosomes, a process that may represent a mechanism for the termination of intracellular MCHR1 signaling.

Dual-Polymer Functionalized Melanin-AgNPs Nanocomposite with Hydroxyapatite Binding Ability to Penetrate and Retain in Biofilm Sequentially Treating Periodontitis.

Periodontitis is the leading cause of adult tooth missing. Thorny bacterial biofilm and high reactive oxygen species (ROS) levels in tissue are key elements for the periodontitis process. It is meaningful to develop an advanced therapeutic system with sequential antibacterial/ antioxidant ability to meet the overall goals of periodontitis therapy. Herein, a dual-polymer functionalized melanin-AgNPs (P/D-MNP-Ag) with biofilm penetration, hydroxyapatite binding, and sequentially treatment ability are fabricated. Polymer enriched with 2-(Dimethylamino)ethyl methacrylate (D), can be protonated in an acid environment with enhanced positive charge, promoting penetration in biofilm. The other polymer is rich in phosphate group (P) and can chelate Ca2+, promoting the polymer to adhere to the hydroxyapatite surface. Melanin has good ROS scavenging and photothermal abilities, after in situ reduction Ag, melanin-AgNPs composite has sequentially transitioned between antibacterial and antioxidative ability due to heat and acid accelerated Ag+ release. The released Ag+ and heat have synergistic antibacterial effects for bacterial killing. With Ag+ consumption, the antioxidant ability of MNP recovers to scavenge ROS in the inflammatory area. When applied in the periodontitis model, P/D-MNP-Ag has good therapeutical effects to ablate biofilm, relieve inflammation state, and reduce alveolar bone loss. P/D-MNP-Ag with sequential treatment ability provides a reference for developing advanced oral biofilm eradication systems.

Image-Guided Photothermal and Immune Therapy of Tumors via Melanin-Producing Genetically Engineered Bacteria.

Photothermal therapy (PTT) is a new treatment modality for tumors. However, the efficient delivery of photothermal agents into tumors remains difficult, especially in hypoxic tumor regions. In this study, an approach to deliver melanin, a natural photothermal agent, into tumors using genetically engineered bacteria for image-guided photothermal and immune therapy is developed. An Escherichia coli MG1655 is transformed with a recombinant plasmid harboring a tyrosinase gene to produce melanin nanoparticles. Melanin-producing genetically engineered bacteria (MG1655-M) are systemically administered to 4T1 tumor-bearing mice. The tumor-targeting properties of MG1655-M in the hypoxic environment integrate the properties of hypoxia targeting, photoacoustic imaging, and photothermal therapeutic agents in an "all-in-one" manner. This eliminates the need for post-modification to achieve image-guided hypoxia-targeted cancer photothermal therapy. Tumor growth is significantly suppressed by irradiating the tumor with an 808 nm laser. Furthermore, strong antitumor immunity is triggered by PTT, thereby producing long-term immune memory effects that effectively inhibit tumor metastasis and recurrence. This work proposes a new photothermal and immune therapy guided by an "all-in-one" melanin-producing genetically engineered bacteria, which can offer broad potential applications in cancer treatment.

Role of Pmk1, Mpk1, or Hog1 in the mitogen-activated protein kinase pathway of Aspergillus cristatus.

Aspergillus cristatus is a probiotic fungus known for its safety and abundant secondary metabolites, making it a promising candidate for various applications. However, limited progress has been made in researching A. cristatus due to challenges in genetic manipulation. The mitogen-activated protein kinase (MAPK) signaling pathway is involved in numerous physiological processes, but its specific role in A. cristatus remains unclear. In this study, we successfully developed an efficient polyethylene glycol (PEG)-mediated protoplast transformation method for A. cristatus, enabling us to investigate the function of Pmk1, Mpk1, and Hog1 in the MAPK signaling pathway. Our findings revealed that Pmk1, Mpk1, and Hog1 are crucial for sexual reproduction, melanin synthesis, and response to external stress in A. cristatus. Notably, the deletion of Pmk1, Mpk1, or Hog1 resulted in the loss of sexual reproduction capability in A. cristatus. Overall, this research on MAPK will contribute to the continued understanding of the reproductive strategy and melanin synthesis mechanism of A. cristatus.

The genomic architecture of continuous plumage colour variation in the European barn owl (Tyto alba).

The maintenance of colour variation in wild populations has long fascinated evolutionary biologists, although most studies have focused on discrete traits exhibiting rather simple inheritance patterns and genetic architectures. However, the study of continuous colour traits and their potentially oligo- or polygenic genetic bases remains rare in wild populations. We studied the genetics of the continuously varying white-to-rufous plumage coloration of the European barn owl (Tyto alba) using a genome-wide association approach on the whole-genome data of 75 individuals. We confirmed a mutation at the melanocortin-1-receptor gene (MC1R) is involved in the coloration and identified two new regions, located in super-scaffolds 9 and 42. The combination of the three regions explains most of the colour variation (80.37%, 95% credible interval 58.45-100%). One discovered region, located in the sex chromosome, differs between the most extreme colorations in owls sharing a specific MC1R genotype. This region may play a role in the colour sex dimorphism of this species, possibly in interaction with the autosomal MC1R. We thus provide insights into the genetic architecture of continuous colour variation, pointing to an oligogenic basis with potential epistatic effects among loci that should aid future studies understanding how continuous colour variation is maintained in nature.

Transcriptional convergence after repeated duplication of an amino acid transporter gene leads to the independent emergence of the black husk/pericarp trait in barley and rice.

The repeated emergence of the same trait (convergent evolution) in distinct species is an interesting phenomenon and manifests visibly the power of natural selection. The underlying genetic mechanisms have important implications to understand how the genome evolves under environmental challenges. In cereal crops, both rice and barley can develop black-coloured husk/pericarp due to melanin accumulation. However, it is unclear if this trait shares a common origin. Here, we fine-mapped the barley HvBlp gene controlling the black husk/pericarp trait and confirmed its function by gene silencing. The result was further supported by a yellow husk/pericarp mutant with deletion of the HvBlp gene, derived from gamma ray radiation of the wild-type W1. HvBlp encodes a putative tyrosine transporter homologous to the black husk gene OsBh4 in rice. Surprisingly, synteny and phylogenetic analyses showed that HvBlp and OsBh4 belonged to different lineages resulted from dispersed and tandem duplications, respectively, suggesting that the black husk/pericarp trait has emerged independently. The dispersed duplication (dated at 21.23 MYA) yielding HvBlp occurred exclusively in the common ancestor of Triticeae. HvBlp and OsBh4 displayed converged transcription in husk/pericarp tissues, contributing to the black husk/pericarp trait. Further transcriptome and metabolome data identified critical candidate genes and metabolites related to melanin production in barley. Taken together, our study described a compelling case of convergent evolution resulted from transcriptional convergence after repeated gene duplication, providing valuable genetic insights into phenotypic evolution. The identification of the black husk/pericarp genes in barley also has great potential in breeding for stress-resilient varieties with higher nutritional values.

Deletion of the polyketide synthase-encoding gene pks1 prevents melanization in the extremophilic fungus Cryomyces antarcticus.

Cryomyces antarcticus, a melanized cryptoendolithic fungus endemic to Antarctica, can tolerate environmental conditions as severe as those in space. Particularly, its ability to withstand ionizing radiation has been attributed to the presence of thick and highly melanized cell walls, which-according to a previous investigation-may contain both 1,8-dihydroxynaphthalene (DHN) and L-3,4 dihydroxyphenylalanine (L-DOPA) melanin. The genes putatively involved in the synthesis of DHN melanin were identified in the genome of C. antarcticus. Most important is capks1 encoding a non-reducing polyketide synthase (PKS) and being the ortholog of the functionally characterized kppks1 from the rock-inhabiting fungus Knufia petricola. The co-expression of CaPKS1 or KpPKS1 with a 4'-phosphopantetheinyl transferase in Saccharomyces cerevisiae resulted in the formation of a yellowish pigment, suggesting that CaPKS1 is the enzyme providing the precursor for DHN melanin. To dissect the composition and function of the melanin layer in the outer cell wall of C. antarcticus, non-melanized mutants were generated by CRISPR/Cas9-mediated genome editing. Notwithstanding its slow growth (up to months), three independent non-melanized Δcapks1 mutants were obtained. The mutants exhibited growth similar to the wild type and a light pinkish pigmentation, which is presumably due to carotenoids. Interestingly, visible light had an adverse effect on growth of both melanized wild-type and non-melanized Δcapks1 strains. Further evidence that light can pass the melanized cell walls derives from a mutant expressing a H2B-GFP fusion protein, which can be detected by fluorescence microscopy. In conclusion, the study reports on the first genetic manipulation of C. antarcticus, resulting in non-melanized mutants and demonstrating that the melanin is rather of the DHN type. These mutants will allow to elucidate the relevance of melanization for surviving extreme conditions found in the natural habitat as well as in space.

Chronic Stress-Induced Elevation of Melanin-Concentrating Hormone in the Locus Coeruleus Inhibits Norepinephrine Production and Associated With Depression-Like Behaviors in Rats.

BACKGROUND: Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that projects throughout the central nervous system, including the noradrenergic locus coeruleus (LC). Our previous study suggested that MCH/MCH receptor 1 (MCHR1) in the LC may be involved in the regulation of depression. The present study investigated whether the role of MCH/MCHR1 in the LC in depression-like behaviors is associated with the regulation of norepinephrine. METHOD: Chronic unpredictable stress (CUS) and an acute intra-LC microinjection of MCH induced depression-like behaviors in rats. The MCHR1 antagonist SNAP-94847 was also microinjected in the LC in rats that were suffering CUS or treated with MCH. The sucrose preference, forced swim, and locomotor tests were used for behavioral evaluation. Immunofluorescence staining, enzyme-linked immunosorbent assay, western blot, and high-performance liquid chromatography with electrochemical detection were used to explore the mechanism of MCH/MCHR1 in the regulation of depression-like behaviors. RESULTS: CUS induced an abnormal elevation of MCH levels and downregulated MCHR1 in the LC, which was highly correlated with the formation of depression-like behaviors. SNAP-94847 exerted antidepressant effects in CUS-exposed rats by normalizing tyrosine hydroxylase, dopamine ß hydroxylase, and norepinephrine in the LC. An acute microinjection of MCH induced depression-like behaviors through its action on MCHR1. MCHR1 antagonism in the LC significantly reversed the MCH-induced downregulation of norepinephrine production by normalizing MCHR1-medicated cAMP-PKA signaling. CONCLUSIONS: Our study confirmed that the MCH/MCHR1 system in the LC may be involved in depression-like behaviors by downregulating norepinephrine production. These results improve our understanding of the pathogenesis of depression that is related to the MCH/MCHR1 system in the LC.

Fast freezing inhibits melanin synthesis of melanocytes by modulating the Wnt/ß-catenin signalling pathway.

Skin hyperpigmentation is mainly caused by excessive synthesis of melanin; however, there is still no safe and effective therapy for its removal. Here, we found that the dermal freezer was able to improve UVB-induced hyperpigmentation of guinea pigs without causing obvious epidermal damage. We also mimic freezing stimulation at the cellular level by rapid freezing and observed that freezing treatments <2.5 min could not decrease cell viability or induce cell apoptosis in B16F10 and Melan-A cells. Critically, melanin content and tyrosinase activity in two cells were greatly reduced after freezing treatments. The dramatic decrease in tyrosinase activity was associated with the downregulation of MITF, TYR, TRP-1 and TRP-2 protein expression in response to freezing treatments for two cells. Furthermore, our results first demonstrated that freezing treatments significantly reduced the levels of p-GSK3ß and ß-catenin and the nuclear accumulation of ß-catenin in B16F10 and Melan-A cells. Together, these data suggest that fast freezing treatments can inhibit melanogenesis-related gene expression in melanocytes by regulating the Wnt/ß-catenin signalling pathway. The inhibition of melanin production eventually contributed to the improvement in skin hyperpigmentation induced by UVB. Therefore, fast freezing treatments may be a new alternative of skin whitening in the clinic in the future.

The in vitro and in vivo depigmentation activity of coenzyme Q0, a major quinone derivative from Antrodia camphorata, through autophagy induction in human melanocytes and keratinocytes.

BACKGROUND: Coenzyme Q0 (CoQ0), a novel quinone derivative of Antrodia camphorata, has been utilized as a therapeutic agent (including antioxidant, anti-inflammatory, antiangiogenic, antiatherosclerotic, and anticancer agents); however, its depigmenting efficiency has yet to be studied. METHODS: We resolved the depigmenting efficiency of CoQ0 through autophagy induction in melanoma (B16F10) and melanin-feeding keratinocyte (HaCaT) cells and in vivo Zebrafish model. Then, MPLC/HPLC analysis, MTT assay, Western blotting, immunofluorescence staining, LC3 transfection, melanin formation, GFP-LC3 puncta, AVO formation, tyrosinase activity, and TEM were used. RESULTS: CoQ0-induced autophagy in B16F10 cells was shown by enhanced LC3-II accumulation, ATG7 expression, autophagosome GFP-LC3 puncta, and AVOs formation, and ATG4B downregulation, and Beclin-1/Bcl-2 dysregulation. In α-MSH-stimulated B16F10 cells, CoQ0 induced antimelanogenesis by suppressing CREB-MITF pathway, tyrosinase expression/activity, and melanin formation via autophagy. TEM data disclosed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in α-MSH-stimulated B16F10 cells. CoQ0-inhibited melanogenesis in α-MSH-stimulated B16F10 cells was reversed by pretreatment with the autophagy inhibitor 3-MA or silencing of LC3. Additionally, CoQ0-induced autophagy in HaCaT cells was revealed by enhanced LC3-II accumulation, autophagosome GFP-LC3 puncta and AVO formation, ATG4B downregulation, ATG5/ATG7 expression, and Beclin-1/Bcl-2 dysregulation. In melanin-feeding HaCaT cells, CoQ0 induced melanin degradation by suppressing melanosome gp100 and melanin formation via autophagy. TEM confirmed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in melanin-feeding HaCaT cells. Treatment with 3-MA reversed CoQ0-mediated melanin degradation in melanin-feeding HaCaT cells. In vivo study showed that CoQ0 suppressed endogenous body pigmentation by antimelanogenesis and melanin degradation through autophagy induction in a zebrafish model. CONCLUSIONS: Our results showed that CoQ0 exerted antimelanogenesis and melanin degradation by inducing autophagy. CoQ0 could be used in skin-whitening formulations as a topical cosmetic application.

Investigation of the influence of pH and temperature on melanization and survival under oxidative stress in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic pathogenic fungus that produces melanin during infection, an important virulence factor in Cryptococcal infections that enhances the ability of the fungus to resist immune defense. This fungus can synthesize melanin from a variety of substrates, including L-DOPA (L-3,4-dihydroxyphenylalanine). Since melanin protects the fungus from various stress factors such as oxidative, nitrosative, extreme heat and cold stress; we investigated the effects of environmental conditions on melanin production and survival. In this study, we investigated the effects of different pH values (5.6, 7.0 and 8.5) and temperatures (30 °C and 37 °C) on melanization and cell survival using a microtiter plate-based melanin production assay and an oxidative stress assay, respectively. In addition, the efficacy of compounds known to inhibit laccase involved in melanin synthesis, i.e., tunicamycin, ß-mercaptoethanol, dithiothreitol, sodium azide and caspofungin on melanization was evaluated and their sensitivity to temperature and pH changes was measured. The results showed that melanin content correlated with pH and temperature changes and that pH 8.5 and 30 °C, were best for melanin production. Besides that, melanin production protects the fungal cells from oxidative stress induced by hydrogen peroxide. Thus, changes in pH and temperature drastically alter melanin production in C. neoformans and it correlates with the fungal survival. Due to the limited antifungal repertoire and the development of resistance in cryptococcal infections, the investigation of environmental conditions in the regulation of melanization and survival of C. neoformans could be useful for future research and clinical phasing.

Novel melanin-derived stationary phase for immobilized metal ion affinity chromatography in recombinant His-tagged protein purification.

The matrix of the stationary phase is a crucial element in affinity chromatography for protein purification. Various materials, including polymer or magnetic materials, have been employed as the matrix in the purification of His-tagged protein. Here, for the first time, we utilized a combination of melanin and alginate, both natural polymer materials, to synthesize Ni-melanin/alginate (Ni-M/A) beads for His-tagged protein purification. We investigated the binding of His-tagged Mpro on the Ni-M/A beads, referred to as Ni-M/A-Mpro, and assessed the elution efficiency of Mpro from the beads. Our examination involved FTIR, EDS, XRD, SDS-PAGE, and Western blotting methods. FTIR spectra revealed notable changes in the stretching patterns and intensities of hydroxyl, amine, carbonyl, imine and amide chemical groups, when Mpro protein was present in the Ni-M/A sample. XRD spectra demonstrated the occurrence of two Nickel peaks at 35-40 deg and 40-45 deg in Ni-M/A, but only one nickel peak at 35-40 deg in Ni-M/A-Mpro, indicating the binding of Mpro on the Nickel ions. EDS analysis reported a decrease in the concentration of Nickel on the surface of Ni-M/A from 16% to 7% when Mpro protein was loaded into the stationary phase. Importantly, our data indicated that the purity of the His-tagged protein Mpro after purification reached 97% after just one-step purification using the Ni-M/A stationary phase. Moreover, the binding capacity of Ni-M/A for Mpro was approximately 5.2 mg/g with recovery efficiency of 40%. Our results suggested Ni-M/A as a highly potential solid phase for affinity chromatography in the purification of His-tagged protein.

Regulation of the MCHergic Neural Circuit to Dorsal Raphe Nucleus on Emotion-Related Behaviors and Intestinal Dysfunction in Mice Model of Irritable Bowel Syndrome with Diarrhea.

INTRODUCTION: Irritable bowel syndrome with diarrhea (IBS-D) is frequently accompanied by depression and anxiety, resulting in a reduced quality of life and increased medical expenditures. Although psychological factors are known to play an important role in the genesis and development of IBS-D, an understanding of the central neural control of intestinal dysfunction remains elusive. Melanin-concentrating hormone (MCH) is a gut-brain peptide involved in regulating feeding, sleep-wake rhythms, and emotional states. METHODS: This study investigated the regulation of the MCHergic neural circuit from the lateral hypothalamic area (LHA) to the dorsal raphe nucleus (DRN) on anxiety- and depression-like behaviors, intestinal motility, and visceral hypersensitivity in a mice model of IBS-D. The models of IBS-D were prepared by inducing chronic unpredictable mild stress. RESULTS: Chemogenetic activation of the MCH neurons in the LHA could excite serotonin (5-HT) neurons in the DRN and induce anxiety- and depression-like behaviors and IBS-D-like symptoms, which could be recovered by microinjection of the MCH receptor antagonist SNAP94847 into the DRN. The mice model of IBS-D showed a reduction of 5-HT and brain-derived neurotrophic factor (BDNF) expression in the DRN, while an elevation of 5-HT and BDNF was observed in the colon through immunofluorescent staining, ELISA, and Western blot analysis. SNAP94847 treatment in the DRN alleviated anxiety- and depression-like behaviors, improved intestinal motility, and alleviated visceral hypersensitivity responses by normalizing the 5-HT and BDNF expression in the DRN and colon. CONCLUSION: This study suggests that the activation of MCH neurons in the LHA may induce IBS-D symptoms via the DRN and that the MCH receptor antagonist could potentially have therapeutic effects.