Yeast-Based Screen to Identify Natural Compounds with a Potential Therapeutic Effect in Hailey-Hailey Disease

The term orthodisease defines human disorders in which the pathogenic gene has orthologs in model organism genomes. Yeasts have been instrumental for gaining insights into the molecular basis of many human disorders, particularly those resulting from impaired cellular metabolism. We and others have used yeasts as a model system to study the molecular basis of Hailey-Hailey disease (HHD), a human blistering skin disorder caused by haploinsufficiency of the gene ATP2C1 the orthologous of the yeast gene PMR1. We observed that K. lactis cells defective for PMR1 gene share several biological similarities with HHD derived keratinocytes. Based on the conservation of ATP2C1/PMR1 function from yeast to human, here we used a yeast-based assay to screen for molecules able to influence the pleiotropy associated with PMR1 deletion. We identified six compounds, Kaempferol, Indirubin, Lappaconite, Cyclocytidine, Azomycin and Nalidixic Acid that induced different major shape phenotypes in K. lactis. These include mitochondrial and the cell-wall morphology-related phenotypes. Interestingly, a secondary assay in mammalian cells confirmed activity for Kaempferol. Indeed, this compound was also active on human keratinocytes depleted of ATP2C1 function by siRNA-treatment used as an in-vitro model of HHD. We found that Kaempferol was a potent NRF2 regulator, strongly inducing its expression and its downstream target NQO1. In addition, Kaempferol could decrease oxidative stress of ATP2C1 defective keratinocytes, characterized by reduced NRF2-expression. Our results indicated that the activation of these pathways might provide protection to the HHD-skin cells. As oxidative stress plays pivotal roles in promoting the skin lesions of Hailey-Hailey, the NRF2 pathway could be a viable therapeutic target for HHD

Caenorhabditis elegans and probiotics interactions from a prolongevity perspective

Probiotics exert beneficial effects on host health through different mechanisms of action, such as production of antimicrobial substances, competition with pathogens, enhancement of host mucosal barrier integrity and immunomodulation. In the context of ageing, which is characterized by several physiological alterations leading to a low grade inflammatory status called inflammageing, evidences suggest a potential prolongevity role of probiotics. Unraveling the mechanisms underlying anti-ageing effects requires the use of simple model systems. To this respect, the nematode Caenorhabditis elegans represents a suitable model organism for the study of both host-microbe interactions and for ageing studies, because of conserved signaling pathways and host defense mechanisms involved in the regulation of its lifespan. Therefore, this review analyses the impact of probiotics on C. elegans age-related parameters, with particular emphasis on oxidative stress, immunity, inflammation and protection from pathogen infections. The picture emerging from our analysis highlights that several probiotic strains are able to exert anti-ageing effects in nematodes by acting on common molecular pathways, such as insulin/insulin-like growth factor-1 (IIS) and p38 mitogen-activated protein kinase (p38 MAPK). In this perspective, C. elegans appears to be advantageous for shedding light on key mechanisms involved in host prolongevity in response to probiotics supplementation

Lanthanum biosorption by different Saccharomyces cerevisiae strains

Biosorption can be a promising technology in rare earth metal separation and recovery due to the low costs of waste biomasses (used as biosorbents) and the high selectivity exploiting specific interaction between metals and biological active sites. In this work, Saccharomyces cerevisiae biomass was used to recover lanthanum. Biosorption properties of two S. cerevisiae strains, wild type and rim20. mutant, have been tested. Potentiometric titrations were carried out for rim20. mutant strain and compared with wild type. Nature of the main active sites and their concentration were determined by implementing mechanistic models. Carboxylic, amino and phosphoric sites are the main groups present. Higher concentration of negatively charged sites was found in rim20. (0.0024 mol/g) than in wild type (0.0022 mol/g). The rate of lanthanum biosorption process is very fast requiring only 10-20 minutes to reach equilibrium condition for both strains. Then biosorption equilibrium tests were done for both biomasses by testing two equilibrium pH (4.0 and 6.0). Maximum uptake capacities (qmax) were: 70 mg/g and 40 mg/g at pH 4.0 for rim20. and wild type, respectively, and 67 mg/g and 80 mg/g at pH 6.0 for wild type and rim20., respectively. These data evidenced that: rim20. mutant had a higher maximum biosorption capacity with respect to wild type counterpart, and that pH had a relevant effect on lanthanum removal. S. cerevisiae yeast denoted good lanthanum biosorption properties and, between tested strains, rim20. was found to be the most promising for such aim

Characterization of the transcription factor encoding gene, KlADR1: metabolic role in Kluyveromyces lactis and expression in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Adr1 is a zinc-finger transcription factor involved in the transcriptional activation of ADH2. Deletion of KlADR1, its putative ortholog in Kluyveromyces lactis, led to reduced growth in glycerol, oleate and yeast extract-peptone medium suggesting, as in S. cerevisiae, its requirement for glycerol, fatty acid and nitrogen utilization. Moreover, growth comparison on yeast extract and peptone plates showed in K. lactis a KlAdr1-dependent growth trait not present in S. cerevisiae, indicating different metabolic roles of the two factors in their environmental niches. KlADR1 is required for growth under respiratory and fermentative conditions like KlADH, alcohol dehydrogenase genes necessary for metabolic adaptation during the growth transition. Using in-gel native alcohol dehydrogenase assay, we showed that this factor affected the Adh pattern by altering the balance between these activities. Since the activity most affected by KlAdr1 is KlAdh3, a deletion analysis of the KlADH3 promoter allowed the isolation of a DNA fragment through which KlAdr1 modulated its expression. The expression of the KlADR1-GFP gene allowed the intracellular localization of the factor in K. lactis and S. cerevisiae, suggesting in the two yeasts a common mechanism of KlAdr1 translocation under fermentative and respiratory conditions. Finally, the chimeric Kl/ScADR1 gene encoding the zinc- finger domains of KlAdr1 fused to the transactivating domains of the S. cerevisiae factor activated in Scadr1D the transcription of ADH2 in a ScAdr1-dependent fashion

Impact of a complex food microbiota on energy metabolism in the model organism Caenorhabditis elegans

The nematode Caenorhabditis elegans is widely used as a model system for research on aging, development, and host-pathogen interactions. Little is currently known about the mechanisms underlying the effects exerted by foodborne microbes. We took advantage of C. elegans to evaluate the impact of foodborne microbiota on well characterized physiological features of the worms. Foodborne lactic acid bacteria (LAB) consortium was used to feed nematodes and its composition was evaluated by 16S rDNA analysis and strain typing before and after colonization of the nematode gut. Lactobacillus delbrueckii, L. fermentum, and Leuconostoc lactis were identified as the main species and shown to display different worm gut colonization capacities. LAB supplementation appeared to decrease nematode lifespan compared to the animals fed with the conventional Escherichia coli nutrient source or a probiotic bacterial strain. Reduced brood size was also observed in microbiota-fed nematodes. Moreover, massive accumulation of lipid droplets was revealed by BODIPY staining. Altered expression of nhr-49, pept-1, and tub-1 genes, associated with obesity phenotypes, was demonstrated by RT-qPCR. Since several pathways are evolutionarily conserved in C. elegans, our results highlight the nematode as a valuable model system to investigate the effects of a complex microbial consortium on host energy metabolism

Extension of Chronological Lifespan by Hexokinase Mutation in Kluyveromyces lactis Involves Increased Level of the Mitochondrial Chaperonin Hsp60

Oxidative damage, mitochondrial dysfunction, genomic instability, and telomere shortening represent all molecular processes proposed as causal factors in aging. Lifespan can be increased by metabolism through an influence on such processes. Glucose reduction extends chronological lifespan (CLS) of Saccharomyces cerevisiae through metabolic adaptation to respiration. To answer the question if the reduced CLS could be ascribed to glucose per se or to glucose repression of respiratory enzymes, we used the Kluyveromyces lactis yeast, where glucose repression does not affect the respiratory function. We identified the unique hexokinase, encoded by RAG5 gene, as an important player in influencing yeast lifespan by modulating mitochondrial functionality and the level of the mitochondrial chaperonin Hsp60. In this context, this hexokinase might have a regulatory role in the influence of CLS, shedding new light on the complex regulation played by hexokinases

Surface disinfections: present and future

The propagation of antibiotic resistance increases the chances of major infections for patients during hospitalization and the spread of health related diseases. Therefore finding new and effective solutions to prevent the proliferation of pathogenic microorganisms is critical, in order to protect hospital environment, such as the surfaces of biomedical devices. Modern nanotechnology has proven to be an effective countermeasure to tackle the threat of infections. On this note, recent scientific breakthroughs have demonstrated that antimicrobial nanomaterials are effective in preventing pathogens from developing resistance. Despite the ability to destroy a great deal of bacteria and control the outbreak of infections, nanomaterials present many other advantages. Moreover, it is unlikely for nanomaterials to develop resistance due to their multiple and simultaneous bactericidal mechanisms. In recent years, science has explored more complex antimicrobial coatings and nanomaterials based on graphene have shown great potential in antibacterial treatment. The purpose of this article is to deepen the discussion on the threat of infections related to surface disinfection and to assess the state of the art and potential solutions, with specific focus on disinfection procedures using nanomaterials

Combination of metabolomic and proteomic analysis revealed different features among Lactobacillus delbrueckii subspecies bulgaricus and lactis strains while in vivo testing in the model organism Caenorhabditis elegans highlighted probiotic properties

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus, lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans, with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis. Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates

Thermal adaptability of Kluyveromyces marxianus in recombinant protein production

Background: Kluyveromyces marxianus combines the ease of genetic manipulation and fermentation with the ability to efficiently secrete high molecular weight proteins, performing eukaryotic post-translational modifications. It is able to grow efficiently in a wide range of temperatures. The secretion performances were analyzed in the host K. marxianus L3 in the range between 5\ub0C and 40\ub0C by means of 3 different reporter proteins, since temperature appears a key parameter for production and secretion of recombinant proteins.Results: The recombinant strains were able to grow up to 40\ub0C and, along the tested temperature interval (5-40\ub0C), the specific growth rates (\u3bc) were generally lower as compared to those of the untransformed strain. Biomass yields were slightly affected by temperature, with the highest values reached at 15\ub0C and 30\ub0C. The secretion of the endogenous \u3b2-fructofuranosidase, used as an internal control, was efficient in the range of the tested temperature, as evaluated by assaying the enzyme activity in the culture supernatants. The endogenous \u3b2-fructofuranosidase production was temperature dependent, with the highest yield at 30\ub0C. The heterologous proteins HSA, GAA and Sod1p were all successfully produced and secreted between 5\ub0C and 40\ub0C, albeit each one presented a different optimal production temperature (15, 40, 5-30\ub0C for HSA, GAA and Sod1p, respectively).Conclusions: K. marxianus L3 has been identified as a promising and flexible cell factory. In a sole host, the optimization of growth temperatures for the efficient secretion of each individual protein can be carried out over a wide range of temperatures

Enamel remineralization and repair results of Biomimetic Hydroxyapatite toothpaste on deciduous teeth: an effective option to fluoride toothpaste

Background: Dental caries is a recognized worldwide public health problem. Despite being one of the most efective strategies against dental caries, the excessive use of fuorine may result in a potential risk of developing dental fuorosis especially in children under age of six. The purpose of this work is to analyze a fuorine-free toothpaste containing Biomimetic Hydroxyapatite to assess enamel re-mineralizing and repairing properties. Results: The study was performed in vitro and in vivo, comparing the hydroxyapatite toothpaste with two others toothpaste containing diferent fuorine concentrations. The coating efect of the micro-structured Hydroxyapatite nanoparticles reintegrates the enamel with a biomimetic flm reproducing the structure and the morphology of the biologic Hydroxyapatite of the enamel. As demonstrated, the coating is due to the deposit of a new layer of apatite, which presents fewer particles than the natural enamel, not based on the chemical—physical changes occurring in fuorinated toothpastes. Moreover, it shows resistance to brushing as a consequence of chemical bonds between the synthetic and natural crystals of the enamel. Conclusions: The use of Biomimetic Hydroxyapatite toothpastes has proven to be a valuable prevention measure against dental caries in primary dentition since it prevents the risk of fuorosis