Reengineering of an Artificial Protein Cage for Efficient Packaging of Active Enzymes.

Protein cages that readily encapsulate active enzymes of interest present useful nanotools for delivery and catalysis, wherein those with programmable disassembly characteristics serve as particularly attractive platforms. Here, a general guest packaging system based on an artificial protein cage, TRAP-cage, the disassembly of which can be induced by the addition of reducing agents, is established. In this system, TRAP-cage with SpyCatcher moieties in the lumen is prepared using genetic modification of the protein building block and assembled into a cage structure with either monovalent gold ions or molecular crosslinkers. The resulting protein cage can efficiently capture guest proteins equipped with a SpyTag by simply mixing them in an aqueous solution. This post-assembly loading system, which circumvents the exposure of guests to thiol-reactive crosslinkers, enables the packaging of enzymes possessing a catalytic cysteine or a metal cofactor while retaining their catalytic activity.

Evolutionary formation of melatonin and vitamin D in early life forms: insects take centre stage.

Melatonin, a product of tryptophan metabolism via serotonin, is a molecule with an indole backbone that is widely produced by bacteria, unicellular eukaryotic organisms, plants, fungi and all animal taxa. Aside from its role in the regulation of circadian rhythms, it has diverse biological actions including regulation of cytoprotective responses and other functions crucial for survival across different species. The latter properties are also shared by its metabolites including kynuric products generated by reactive oxygen species or phototransfomation induced by ultraviolet radiation. Vitamins D and related photoproducts originate from phototransformation of ∆5,7 sterols, of which 7-dehydrocholesterol and ergosterol are examples. Their ∆5,7 bonds in the B ring absorb solar ultraviolet radiation [290-315 nm, ultraviolet B (UVB) radiation] resulting in B ring opening to produce previtamin D, also referred to as a secosteroid. Once formed, previtamin D can either undergo thermal-induced isomerization to vitamin D or absorb UVB radiation to be transformed into photoproducts including lumisterol and tachysterol. Vitamin D, as well as the previtamin D photoproducts lumisterol and tachysterol, are hydroxylated by cyochrome P450 (CYP) enzymes to produce biologically active hydroxyderivatives. The best known of these is 1,25-dihydroxyvitamin D (1,25(OH)2D) for which the major function in vertebrates is regulation of calcium and phosphorus metabolism. Herein we review data on melatonin production and metabolism and discuss their functions in insects. We discuss production of previtamin D and vitamin D, and their photoproducts in fungi, plants and insects, as well as mechanisms for their enzymatic activation and suggest possible biological functions for them in these groups of organisms. For the detection of these secosteroids and their precursors and photoderivatives, as well as melatonin metabolites, we focus on honey produced by bees and on body extracts of Drosophila melanogaster. Common biological functions for melatonin derivatives and secosteroids such as cytoprotective and photoprotective actions in insects are discussed. We provide hypotheses for the photoproduction of other secosteroids and of kynuric metabolites of melatonin, based on the known photobiology of ∆5,7 sterols and of the indole ring, respectively. We also offer possible mechanisms of actions for these unique molecules and summarise differences and similarities of melatoninergic and secosteroidogenic pathways in diverse organisms including insects.

Autophagy as a new player in the regulation of clock neurons physiology of Drosophila melanogaster.

Axonal terminals of the small ventral lateral neurons (sLNvs), the circadian clock neurons of Drosophila, show daily changes in their arborization complexity, with many branches in the morning and their shrinkage during the night. This complex phenomenon is precisely regulated by several mechanisms. In the present study we describe that one of them is autophagy, a self-degradative process, also involved in changes of cell membrane size and shape. Our results showed that autophagosome formation and processing in PDF-expressing neurons (both sLNv and lLNv) are rhythmic and they have different patterns in the cell bodies and terminals. These rhythmic changes in the autophagy activity seem to be important for neuronal plasticity. We found that autophagosome cargos are different during the day and night, and more proteins involved in membrane remodeling are present in autophagosomes in the morning. In addition, we described for the first time that Atg8-positive vesicles are also present outside the sLNv terminals, which suggests that secretory autophagy might be involved in regulating the clock signaling network. Our data indicate that rhythmic autophagy in clock neurons affect the pacemaker function, through remodeling of terminal membrane and secretion of specific proteins from sLNvs.

Dysregulated iron homeostasis in dystrophin-deficient cardiomyocytes: correction by gene editing and pharmacological treatment.

AIMS: Duchenne muscular dystrophy (DMD)-associated cardiomyopathy is a serious life-threatening complication, the mechanisms of which have not been fully established, and therefore no effective treatment is currently available. The purpose of the study was to identify new molecular signatures of the cardiomyopathy development in DMD. METHODS AND RESULTS: For modelling of DMD-associated cardiomyopathy, we prepared three pairs of isogenic control and dystrophin-deficient human induced pluripotent stem cell (hiPSC) lines. Two isogenic hiPSC lines were obtained by CRISPR/Cas9-mediated deletion of DMD exon 50 in unaffected cells generated from healthy donor and then differentiated into cardiomyocytes (hiPSC-CM). The latter were subjected to global transcriptomic and proteomic analyses followed by more in-depth investigation of selected pathway and pharmacological modulation of observed defects. Proteomic analysis indicated a decrease in the level of mitoNEET protein in dystrophin-deficient hiPSC-CM, suggesting alteration in iron metabolism. Further experiments demonstrated increased labile iron pool both in the cytoplasm and mitochondria, a decrease in ferroportin level and an increase in both ferritin and transferrin receptor in DMD hiPSC-CM. Importantly, CRISPR/Cas9-mediated correction of the mutation in the patient-derived hiPSC reversed the observed changes in iron metabolism and restored normal iron levels in cardiomyocytes. Moreover, treatment of DMD hiPSC-CM with deferoxamine (DFO, iron chelator) or pioglitazone (mitoNEET stabilizing compound) decreased the level of reactive oxygen species in DMD hiPSC-CM. CONCLUSION: To our knowledge, this study demonstrated for the first time impaired iron metabolism in human DMD cardiomyocytes, and potential reversal of this effect by correction of DMD mutation or pharmacological treatment. This implies that iron overload-regulating compounds may serve as novel therapeutic agents in DMD-associated cardiomyopathy.

Stress Conditions Affect the Immunomodulatory Potential of Candida albicans Extracellular Vesicles and Their Impact on Cytokine Release by THP-1 Human Macrophages.

Human immune cells possess the ability to react complexly and effectively after contact with microbial virulence factors, including those transported in cell-derived structures of nanometer sizes termed extracellular vesicles (EVs). EVs are produced by organisms of all kingdoms, including fungi pathogenic to humans. In this work, the immunomodulatory properties of EVs produced under oxidative stress conditions or at host concentrations of CO2 by the fungal pathogen Candida albicans were investigated. The interaction of EVs with human pro-monocytes of the U-937 cell line was established, and the most notable effect was attributed to oxidative stress-related EVs. The immunomodulatory potential of tested EVs against human THP-1 macrophages was verified using cytotoxicity assay, ROS-production assay, and the measurement of cytokine production. All fungal EVs tested did not show a significant cytotoxic effect on THP-1 cells, although a slight pro-oxidative impact was indicated for EVs released by C. albicans cells grown under oxidative stress. Furthermore, for all tested types of EVs, the pro-inflammatory properties related to increased IL-8 and TNF-α production and decreased IL-10 secretion were demonstrated, with the most significant effect observed for EVs released under oxidative stress conditions.

Candida albicans Biofilm-Derived Extracellular Vesicles Are Involved in the Tolerance to Caspofungin, Biofilm Detachment, and Fungal Proteolytic Activity.

It has been repeatedly reported that the cells of organisms in all kingdoms of life produce nanometer-sized lipid membrane-enveloped extracellular vesicles (EVs), transporting and protecting various substances of cellular origin. While the composition of EVs produced by human pathogenic fungi has been studied in recent decades, another important challenge is the analysis of their functionality. Thus far, fungal EVs have been shown to play significant roles in intercellular communication, biofilm production, and modulation of host immune cell responses. In this study, we verified the involvement of biofilm-derived EVs produced by two different strains of Candida albicans-C. albicans SC5314 and 3147 (ATCC 10231)-in various aspects of biofilm function by examining its thickness, stability, metabolic activity, and cell viability in the presence of EVs and the antifungal drug caspofungin. Furthermore, the proteolytic activity against the kininogen-derived antimicrobial peptide NAT26 was confirmed by HPLC analysis for C. albicans EVs that are known to carry, among others, particular members of the secreted aspartic proteinases (Saps) family. In conclusion, EVs derived from C. albicans biofilms were shown to be involved in biofilm tolerance to caspofungin, biofilm detachment, and fungal proteolytic activity.

Changes in heme oxygenase level during development affect the adult life of Drosophila melanogaster.

Heme oxygenase (HO) has been shown to control various cellular processes in both mammals and Drosophila melanogaster. Here, we investigated how changes in HO levels in neurons and glial cells during development affect adult flies, by using the TARGET Drosophila system to manipulate the expression of the ho gene. The obtained data showed differences in adult survival, maximum lifespan, climbing, locomotor activity, and sleep, which depended on the level of HO (after ho up-regulation or downregulation), the timing of expression (chronic or at specific developmental stages), cell types (neurons or glia), sex (males or females), and age of flies. In addition to ho, the effects of changing the mRNA level of the Drosophila CNC factor gene (NRF2 homolog in mammals and master regulator of HO), were also examined to compare with those observed after changing ho expression. We showed that HO levels in neurons and glia must be maintained at an appropriate physiological level during development to ensure the well-being of adults. We also found that the downregulation of ho in either neurons or glia in the brain is compensated by ho expressed in the retina.

Melanogenesis Is Directly Affected by Metabolites of Melatonin in Human Melanoma Cells.

Melatonin (N-acetyl-5-methoxytryptamine, MEL), its kynurenic (N1-acetyl-N2-formyl-5-methoxykynurenine, AFMK) and indolic derivatives (6-hydroxymelatonin, 6(OH)MEL and 5-methoxytryptamine, 5-MT) are endogenously produced in human epidermis. Melatonin, produced by the pineal gland, brain and peripheral organs, displays a diversity of physiological functions including anti-inflammatory, immunomodulatory, and anti-tumor capacities. Herein, we assessed their regulatory effect on melanogenesis using amelanotic (A375, Sk-Mel-28) and highly pigmented (MNT-1, melanotic) human melanoma cell lines. We discovered that subjected compounds decrease the downstream pathway of melanin synthesis by causing a significant drop of cyclic adenosine monophosphate (cAMP) level, the microphthalmia-associated transcription factor (MITF) and resultant collapse of tyrosinase (TYR) activity, and melanin content comparatively to N-phenylthiourea (PTU, a positive control). We observed a reduction in pigment in melanosomes visualized by the transmission electron microscopy. Finally, we assessed the role of G-protein-coupled seven-transmembrane-domain receptors. Obtained results revealed that nonselective MT1 and MT2 receptor antagonist (luzindole) or selective MT2 receptor antagonist (4-P-PDOT) did not affect dysregulation of the melanin pathway indicating a receptor-independent mechanism. Our findings, together with the current state of the art, provide a convenient experimental model to study the complex relationship between metabolites of melatonin and the control of pigmentation serving as a future and rationale strategy for targeted therapies of melanoma-affected patients.

Investigating the potential effects of α-synuclein aggregation on susceptibility to chronic stress in a mouse Parkinson's disease model.

BACKGROUND: Parkinson's disease (PD) is a motor disorder characterized by the degeneration of dopaminergic neurons, putatively due to the accumulation of α-synuclein (α-syn) in Lewy bodies (LBs) in Substantia Nigra. PD is also associated with the formation of LBs in brain areas responsible for emotional and cognitive regulation such as the amygdala and prefrontal cortex, and concurrent depression prevalence in PD patients. The exact link between dopaminergic cell loss, α-syn aggregation, depression, and stress, a major depression risk factor, is unclear. Therefore, we aimed to explore the interplay between sensitivity to chronic stress and α-syn aggregation. METHODS: Bilateral injections of α-syn preformed fibrils (PFFs) into the striatum of C57Bl/6 J mice were used to induce α-syn aggregation. Three months after injections, animals were exposed to chronic social defeat stress. RESULTS: α-syn aggregation did not affect stress susceptibility but independently caused increased locomotor activity in the open field test, reduced anxiety in the light-dark box test, and increased active time in the tail suspension test. Ex vivo analysis revealed modest dopaminergic neuron loss in the substantia nigra and reduced dopaminergic innervation in the dorsal striatum in PFFs injected groups. α-Syn aggregates were prominent in the amygdala, prefrontal cortex, and substantia nigra, with minimal α-syn aggregation in the raphe nuclei and locus coeruleus. CONCLUSIONS: Progressive bilateral α-syn aggregation might lead to compensatory activity increase and alterations in emotionally regulated behavior, without affecting stress susceptibility. Understanding how α-syn aggregation and degeneration in specific brain structures contribute to depression and anxiety in PD patients requires further investigation.

Effects of adenosine receptor overexpression and silencing in neurons and glial cells on lifespan, fitness, and sleep of Drosophila melanogaster.

A single adenosine receptor gene (dAdoR) has been detected in Drosophila melanogaster. However, its function in different cell types of the nervous system is mostly unknown. Therefore, we overexpressed or silenced the dAdoR gene in eye photoreceptors, all neurons, or glial cells and examined the fitness of flies, the amount and daily pattern of sleep, and the influence of dAdoR silencing on Bruchpilot (BRP) presynaptic protein. Furthermore, we examined the dAdoR and brp gene expression in young and old flies. We found that a higher level of dAdoR in the retina photoreceptors, all neurons, and glial cells negatively influenced the survival rate and lifespan of male and female Drosophila in a cell-dependent manner and to a different extent depending on the age of the flies. In old flies, expression of both dAdoR and brp was higher than in young ones. An excess of dAdoR in neurons improved climbing in older individuals. It also influenced sleep by lengthening nighttime sleep and siesta. In turn, silencing of dAdoR decreased the lifespan of flies, although it increased the survival rate of young flies. It hindered the climbing of older males and females, but did not change sleep. Silencing also affected the daily pattern of BRP abundance, especially when dAdoR expression was decreased in glial cells. The obtained results indicate the role of adenosine and dAdoR in the regulation of fitness in flies that is based on communication between neurons and glial cells, and the effect of glial cells on synapses.

Isolation and Characteristics of Extracellular Vesicles Produced by Probiotics: Yeast Saccharomyces boulardii CNCM I-745 and Bacterium Streptococcus salivarius K12.

Numerous probiotic microorganisms have repeatedly been shown to produce nanometer-sized structures named extracellular vesicles (EVs). Recently, it has been suggested that similarly to whole microbial cells, EVs produced by probiotics may also demonstrate health benefits to the host, while their application does not involve the risk of infection caused by live microorganisms. In this work, we isolated EVs from two probiotic species originating from different taxonomic domains - yeast Saccharomyces boulardii CNCM I-745 and bacterium Streptococcus salivarius K12. The diameters of S. boulardii EVs were about 142 nm and for S. salivarius EVs about 123 nm. For S. boulardii EVs, 1641 proteins and for S. salivarius EVs, 466 proteins were identified with a liquid chromatography-coupled tandem mass spectrometry and then functionally classified. In both microbial species, metabolic proteins significantly contributed to the cargo of EVs comprising 25% and 26% of all identified vesicular proteins for fungi and bacteria, respectively. Moreover, enzymes associated with cell wall rearrangement, including enzymatically active glucanases, were also identified in EVs. Furthermore, probiotic EVs were shown to influence host cells and stimulate the production of IL-1ß and IL-8 by the human monocytic cell line THP-1, and, at the same time, did not cause any remarkable reduction in the survival rate of Galleria mellonella larvae in this invertebrate model commonly used to evaluate microbial EV toxicity. These observations suggest that the EVs produced by the investigated probiotic microorganisms may be promising structures for future use in pro-health applications.

The dual role of heme oxygenase in regulating apoptosis in the nervous system of Drosophila melanogaster.

Accumulating evidence from mammalian studies suggests the dual-faced character of heme oxygenase (HO) in oxidative stress-dependent neurodegeneration. The present study aimed to investigate both neuroprotective and neurotoxic effects of heme oxygenase after the ho gene chronic overexpression or silencing in neurons of Drosophila melanogaster. Our results showed early deaths and behavioral defects after pan-neuronal ho overexpression, while survival and climbing in a strain with pan-neuronal ho silencing were similar over time with its parental controls. We also found that HO can be pro-apoptotic or anti-apoptotic under different conditions. In young (7-day-old) flies, both the cell death activator gene (hid) expression and the initiator caspase Dronc activity increased in heads of flies when ho expression was changed. In addition, various expression levels of ho produced cell-specific degeneration. Dopaminergic (DA) neurons and retina photoreceptors are particularly vulnerable to changes in ho expression. In older (30-day-old) flies, we did not detect any further increase in hid expression or enhanced degeneration, however, we still observed high activity of the initiator caspase. In addition, we used curcumin to further show the involvement of neuronal HO in the regulation of apoptosis. Under normal conditions, curcumin induced both the expression of ho and hid, which was reversed after exposure to high-temperature stress and when supplemented in flies with ho silencing. These results indicate that neuronal HO regulates apoptosis and this process depends on ho expression level, age of flies, and cell type.

Light exposure during development affects physiology of adults in Drosophila melanogaster.

Light is one of most important factors synchronizing organisms to day/night cycles in the environment. In Drosophila it is received through compound eyes, Hofbauer-Buchner eyelet, ocelli, using phospholipase C-dependent phototransduction and by deep brain photoreceptors, like Cryptochrome. Even a single light pulse during early life induces larval-time memory, which synchronizes the circadian clock and maintains daily rhythms in adult flies. In this study we investigated several processes in adult flies after maintaining their embryos, larvae and pupae in constant darkness (DD) until eclosion. We found that the lack of external light during development affects sleep time, by reduction of night sleep, and in effect shift to the daytime. However, disruption of internal CRY- dependent photoreception annuls this effect. We also observed changes in the expression of genes encoding neurotransmitters and their receptors between flies kept in different light regime. In addition, the lack of light during development results in decreasing size of mushroom bodies, involved in sleep regulation. Taking together, our results show that presence of light during early life plays a key role in brain development and affects adult behavior.

The Role of Glia Clocks in the Regulation of Sleep in Drosophila melanogaster.

In Drosophila melanogaster, the pacemaker located in the brain plays the main role in maintaining circadian rhythms; however, peripheral oscillators including glial cells, are also crucial components of the circadian network. In the present study, we investigated an impact of oscillators located in astrocyte-like glia, the chiasm giant glia of the optic lobe, epithelial and subperineurial glia on sleep of Drosophila males. We described that oscillators located in astrocyte-like glia and chiasm giant glia are necessary to maintain daily changes in clock neurons arborizations, while those located in epithelial glia regulate amplitude of these changes. Finally, we showed that communication between glia and neurons through tripartite synapses formed by epithelial glia and, in effect, neurotransmission regulation plays important role in wake-promoting during the day.SIGNIFICANCE STATEMENTCircadian clock or pacemaker regulates many aspects of animals' physiology and behavior. The pacemaker is located in the brain and is composed of neurons. However, there are also additional oscillators, called peripheral clocks, which synchronize the main clock. Despite the critical role of glia in the clock machinery, little is known which type of glia houses peripheral oscillators and how they affect neuronal clocks. This study using Drosophila shows that oscillators in specific glia types maintain awakeness during the day by regulating the daily plasticity of clock neurons.

Insight Into the Properties and Immunoregulatory Effect of Extracellular Vesicles Produced by Candida glabrata, Candida parapsilosis, and Candida tropicalis Biofilms.

Currently, non-albicans Candida species, including C. tropicalis, C. glabrata, and C. parapsilosis, are becoming an increasing epidemiological threat, predominantly due to the distinct collection of virulence mechanisms, as well as emerging resistance to antifungal drugs typically used in the treatment of candidiasis. They can produce biofilms that release extracellular vesicles (EVs), which are nanometric spherical structures surrounded by a lipid bilayer, transporting diversified biologically active cargo, that may be involved in intercellular communication, biofilm matrix production, and interaction with the host. In this work, we characterize the size and protein composition of these structures for three species of non-albicans Candida fungi forming biofilm, indicating considerable heterogeneity of the investigated population of fungal EVs. Examination of the influence of EVs on cytokine production by the human monocytic cell line THP-1 differentiated into macrophage-like cells revealed that the tested vesicles have a stimulating effect on the secretion of tumor necrosis factor α and interleukin 8, while they reduce the production of interleukin 10. This may indicate the proinflammatory nature of the effect of EVs produced by these species on the host immune cells. Moreover, it has been indicated that vesicles may be involved in C. tropicalis biofilm resistance to fluconazole and caspofungin. This reveals the important role of EVs not only in the physiology of C. tropicalis, C. glabrata, and C. parapsilosis fungi but also in the pathogenesis of infections associated with the production of fungal biofilm.

Antimicrobial Properties of a Peptide Derived from the Male Fertility Factor kl2 Protein of Drosophila melanogaster.

Antimicrobial peptides (AMPs) are important components of innate immunity. Here, we report the antimicrobial properties of a peptide derived from the Male fertility factor kl2 (MFF-kl2) protein of Drosophila melanogaster, which was identified as a functional analog of the mammalian antibacterial chemerin-p4 peptide. The antimicrobial activity of multifunctional chemerin is mainly associated with a domain localized in the middle of the chemerin sequence, Val66-Pro85 peptide (chemerin-p4). Using bioinformatic tools, we found homologs of the chemerin-p4 peptide in the proteome of D. melanogaster. One of them is MFF-p1, which is a part of the MFF kl2 protein, encoded by the gene male fertility factor kl2 (kl-2) located on the long arm of the Y chromosome. The second detected peptide (Z-p1) is a part of the Zizimin protein belonging to DOCK family, which is involved in cellular signaling processes. After testing the antimicrobial properties of both peptides, we found that only MFF-p1 possesses these properties. Here, we demonstrate its antimicrobial potential both in vitro and in vivo after infecting D. melanogaster with bacteria. MFF-p1 strongly inhibits the viable counts of E. coli and B. subtilis after 2 h of treatment and disrupts bacterial cells. The expression of kl-2 is regulated by exposure to bacteria and by the circadian clock.

Glia-Neurons Cross-Talk Regulated Through Autophagy.

Autophagy is a self-degradative process which plays a role in removing misfolded or aggregated proteins, clearing damaged organelles, but also in changes of cell membrane size and shape. The aim of this phenomenon is to deliver cytoplasmic cargo to the lysosome through the intermediary of a double membrane-bound vesicle (autophagosome), that fuses with a lysosome to form autolysosome, where cargo is degraded by proteases. Products of degradation are transported back to the cytoplasm, where they can be re-used. In the present study we showed that autophagy is important for proper functioning of the glia and that it is involved in the regulation of circadian structural changes in processes of the pacemaker neurons. This effect is mainly observed in astrocyte-like glia, which play a role of peripheral circadian oscillators in the Drosophila brain.

Chemically induced protein cage assembly with programmable opening and cargo release.

Engineered protein cages are promising tools that can be customized for applications in medicine and nanotechnology. A major challenge is developing a straightforward strategy for endowing cages with bespoke, inducible disassembly. Such cages would allow release of encapsulated cargoes at desired timing and location. Here, we achieve such programmable disassembly using protein cages, in which the subunits are held together by different molecular cross-linkers. This modular system enables cage disassembly to be controlled in a condition-dependent manner. Structural details of the resulting cages were determined using cryo­electron microscopy, which allowed observation of bridging cross-linkers at intended positions. Triggered disassembly was demonstrated by high-speed atomic force microscopy and subsequent cargo release using an encapsulated Förster resonance energy transfer pair whose signal depends on the quaternary structure of the cage.

Cecropin D-derived synthetic peptides in the fight against Candida albicans cell filamentation and biofilm formation.

The recent progressive increase in the incidence of invasive fungal infections, especially in immunocompromised patients, makes the search for new therapies crucial in the face of the growing drug resistance of prevalent nosocomial yeast strains. The latest research focuses on the active compounds of natural origin, inhibiting fungal growth, and preventing the formation of fungal biofilms. Antimicrobial peptides are currently the subject of numerous studies concerning effective antifungal therapy. In the present study, the antifungal properties of two synthetic peptides (ΔM3, ΔM4) derived from an insect antimicrobial peptide - cecropin D - were investigated. The fungicidal activity of both compounds was demonstrated against the yeast forms of Candida albicans, Candida tropicalis, and Candida parapsilosis, reaching a MFC99.9 in the micromolar range, while Candida glabrata showed greater resistance to these peptides. The scanning electron microscopy revealed a destabilization of the yeast cell walls upon treatment with both peptides; however, their effectiveness was strongly modified by the presence of salt or plasma in the yeast environment. The transition of C. albicans cells from yeast to filamentous form, as well as the formation of biofilms, was effectively reduced by ΔM4. Mature biofilm viability was inhibited by a higher concentration of this peptide and was accompanied by increased ROS production, activation of the GPX3 and SOD5 genes, and finally, increased membrane permeability. Furthermore, both peptides showed a synergistic effect with caspofungin in inhibiting the metabolic activity of C. albicans cells, and an additive effect was also observed for the mixtures of peptides with amphotericin B. The results indicate the possible potential of the tested peptides in the prevention and treatment of candidiasis.

Regulation of Heme Oxygenase and Its Cross-Talks with Apoptosis and Autophagy under Different Conditions in Drosophila.

Heme oxygenase (HO) is one of the cytoprotective enzymes that can mitigate the effects of oxidative stress. Here, we found that the ho mRNA level oscillates in the brain of Drosophila melanogaster with two minima at the beginning of the day and night. This rhythm was partly masked by light as its pattern changed in constant darkness (DD). It followed a similar trend in the clock mutant per01 under light/dark regime (LD12:12); however, differences between time points were not statistically significant. In older flies (20 days old), the rhythm was vanished; however, 15 days of curcumin feeding restored this rhythm with an elevated ho mRNA level at all time points studied. In addition, flies exposed to paraquat had higher ho expression in the brain, but only at a specific time of the day which can be a protective response of the brain against stress. These findings suggest that the expression of ho in the fly's brain is regulated by the circadian clock, light, age, exposure to stress, and the presence of exogenous antioxidants. We also found that HO cross-talks with apoptosis and autophagy under different conditions. Induction of neuronal ho was accompanied by increased transcription of apoptosis and autophagy-related genes. However, this trend changed after exposure to curcumin and paraquat. Our results suggest that HO is involved in the control of apoptotic and autophagic key processes protecting the brain against oxidative damage.