8-OxoG-Dependent Regulation of Global Protein Responses Leads to Mutagenesis and Stress Survival in Bacillus subtilis.

The guanine oxidized (GO) system of Bacillus subtilis, composed of the YtkD (MutT), MutM and MutY proteins, counteracts the cytotoxic and genotoxic effects of the oxidized nucleobase 8-OxoG. Here, we report that in growing B. subtilis cells, the genetic inactivation of GO system potentiated mutagenesis (HPM), and subsequent hyperresistance, contributes to the damaging effects of hydrogen peroxide (H2O2) (HPHR). The mechanism(s) that connect the accumulation of the mutagenic lesion 8-OxoG with the ability of B. subtilis to evolve and survive the noxious effects of oxidative stress were dissected. Genetic and biochemical evidence indicated that the synthesis of KatA was exacerbated, in a PerR-independent manner, and the transcriptional coupling repair factor, Mfd, contributed to HPHR and HPM of the ΔGO strain. Moreover, these phenotypes are associated with wider pleiotropic effects, as revealed by a global proteome analysis. The inactivation of the GO system results in the upregulated production of KatA, and it reprograms the synthesis of the proteins involved in distinct types of cellular stress; this has a direct impact on (i) cysteine catabolism, (ii) the synthesis of iron-sulfur clusters, (iii) the reorganization of cell wall architecture, (iv) the activation of AhpC/AhpF-independent organic peroxide resistance, and (v) increased resistance to transcription-acting antibiotics. Therefore, to contend with the cytotoxic and genotoxic effects derived from the accumulation of 8-OxoG, B. subtilis activates the synthesis of proteins belonging to transcriptional regulons that respond to a wide, diverse range of cell stressors.

Pseudomonas environmental strain produces a DegQ-derived and PDZ domain containing peptide with protease activity.

In the search of new enzymatic activities with a possible industrial application, we focused on those microorganisms and their molecular mechanisms that allow them to succeed in the environment, particularly in the proteolytic activity and its central role in the microorganisms' successful permanence. The use of highly active serine proteases for industrial applications is a modern need, especially for the formulation of detergents, protein processing, and hair removal from animal skins. This report provides the isolation and identification of a highly proteolytic fragment derived from DegQ produced by a Pseudomonas fluorescens environmental strain isolated from a frog carcass. Zymograms demonstrate that a 10 kDa protein mainly generates the total proteolytic activity of this strain, which is enhanced by the detergent SDS. Mass spectroscopy analysis revealed that the protein derived a couple of peptides, the ones showing the highest coverage belonging to DegQ. Interestingly, this small protein fragment contains a PDZ domain but no obvious residues indicating that it is a protease. Protein model analysis shows that this fragment corresponds to the main PDZ domain from DegQ, and its unique sequence and structure render a proteolytic peptide. The results presented here indicate that a novel DegQ fragment is sufficient for obtaining high protease activity highlighting that the analysis of environmental microorganisms can render new strains or enzymes with helpful biotechnological characteristics.

Theoretical study of ArcB and its dimerization, interaction with anaerobic metabolites, and activation of ArcA.

The complex metabolism of Escherichia coli has been extensively studied, including its response to oxygen availability. The ArcA/B two-component system (TCS) is the key regulator for the transition between these two environmental conditions and has been thoroughly characterized using genetic and biochemical approaches. Still, to date, limited structural data is available. The breakthrough provided by AlphaFold2 in 2021 has brought a reliable tool to the scientific community for assessing the structural features of complex proteins. In this report, we analyzed the structural aspects of the ArcA/B TCS using AlphaFold2 models. The models are consistent with the experimentally determined structures of ArcB kinase. The predicted structure of the dimeric form of ArcB is consistent with the extensive genetic and biochemical data available regarding mechanistic signal perception and regulation. The predicted interaction of the dimeric form of ArcB with its cognate response regulator (ArcA) is also consistent with both the forward and reverse phosphotransfer mechanisms. The ArcB model was used to detect putative binding cavities to anaerobic metabolites, encouraging testing of these predictions experimentally. Finally, the highly accurate models of other ArcB homologs suggest that different experimental approaches are needed to determine signal perception in kinases lacking the PAS domain. Overall, ArcB is a kinase with features that need further testing, especially in determining its crystal structure under different conditions.

Two-Component System Sensor Kinases from Asgardian Archaea May Be Witnesses to Eukaryotic Cell Evolution.

The signal transduction paradigm in bacteria involves two-component systems (TCSs). Asgardarchaeota are archaea that may have originated the current eukaryotic lifeforms. Most research on these archaea has focused on eukaryotic-like features, such as genes involved in phagocytosis, cytoskeleton structure, and vesicle trafficking. However, little attention has been given to specific prokaryotic features. Here, the sequence and predicted structural features of TCS sensor kinases analyzed from two metagenome assemblies and a genomic assembly from cultured Asgardian archaea are presented. The homology of the sensor kinases suggests the grouping of Lokiarchaeum closer to bacterial homologs. In contrast, one group from a Lokiarchaeum and a meta-genome assembly from Candidatus Heimdallarchaeum suggest the presence of a set of kinases separated from the typical bacterial TCS sensor kinases. AtoS and ArcB homologs were found in meta-genome assemblies along with defined domains for other well-characterized sensor kinases, suggesting the close link between these organisms and bacteria that may have resulted in the metabolic link to the establishment of symbiosis. Several kinases are predicted to be cytoplasmic; some contain several PAS domains. The data shown here suggest that TCS kinases in Asgardian bacteria are witnesses to the transition from bacteria to eukaryotic organisms.

Attenuation of In Vitro and In Vivo Virulence Is Associated with Repression of Gene Expression of AIG1 Gene in Entamoeba histolytica.

Entamoeba histolytica virulence results from complex host-parasite interactions implicating multiple amoebic components (e.g., Gal/GalNAc lectin, cysteine proteinases, and amoebapores) and host factors (microbiota and immune response). UG10 is a strain derived from E. histolytica virulent HM-1:IMSS strain that has lost its virulence in vitro and in vivo as determined by a decrease of hemolytic, cytopathic, and cytotoxic activities, increased susceptibility to human complement, and its inability to form liver abscesses in hamsters. We compared the transcriptome of nonvirulent UG10 and its parental HM-1:IMSS strain. No differences in gene expression of the classical virulence factors were observed. Genes downregulated in the UG10 trophozoites encode for proteins that belong to small GTPases, such as Rab and AIG1. Several protein-coding genes, including iron-sulfur flavoproteins and heat shock protein 70, were also upregulated in UG10. Overexpression of the EhAIG1 gene (EHI_180390) in nonvirulent UG10 trophozoites resulted in augmented virulence in vitro and in vivo. Cocultivation of HM-1:IMSS with E. coli O55 bacteria cells reduced virulence in vitro, and the EhAIG1 gene expression was downregulated. In contrast, virulence was increased in the monoxenic strain UG10, and the EhAIG1 gene expression was upregulated. Therefore, the EhAIG1 gene (EHI_180390) represents a novel virulence determinant in E. histolytica.

Catalases in the pathogenesis of Sporothrix schenckii research.

Pathogenic fungal infection success depends on the ability to escape the immune response. Most strategies for fungal infection control are focused on the inhibition of virulence factors and increasing the effectiveness of antifungal drugs. Nevertheless, little attention has been focused on their physiological resistance to the host immune system. Hints may be found in pathogenic fungi that also inhabit the soil. In nature, the saprophyte lifestyle of fungi is also associated with predators that can induce oxidative stress upon cell damage. The natural sources of nutrients for fungi are linked to cellulose degradation, which in turn generates reactive oxygen species (ROS). Overall, the antioxidant arsenal needed to thrive both in free-living and pathogenic lifestyles in fungi is fundamental for success. In this review, we present recent findings regarding catalases and oxidative stress in fungi and how these can be in close relationship with pathogenesis. Additionally, special focus is placed on catalases of Sporothrix schenckii as a pathogenic model with a dual lifestyle. It is assumed that catalase expression is activated upon exposure to H2O2, but there are reports where this is not always the case. Additionally, it may be relevant to consider the role of catalases in S. schenckii survival in the saprophytic lifestyle and why their study can assess their involvement in the survival and therefore, in the virulence phenotype of different species of Sporothrix and when each of the three catalases are required. Also, studying antioxidant mechanisms in other isolates of pathogenic and free-living fungi may be linked to the virulence phenotype and be potential therapeutic and diagnostic targets. Thus, the rationale for this review to place focus on fungal catalases and their role in pathogenesis in addition to counteracting the effect of immune system reactive oxygen species. Fungi that thrive in soil and have mammal hosts could shed light on the importance of these enzymes in the two types of lifestyles. We look forward to encouraging more research in a myriad of areas on catalase biology with a focus on basic and applied objectives and placing these enzymes as virulence determinants.

Escherichia coli transcription factors of unknown function: sequence features and possible evolutionary relationships.

Organisms need mechanisms to perceive the environment and respond accordingly to environmental changes or the presence of hazards. Transcription factors (TFs) are required for cells to respond to the environment by controlling the expression of genes needed. Escherichia coli has been the model bacterium for many decades, and still, there are features embedded in its genome that remain unstudied. To date, 58 TFs remain poorly characterized, although their binding sites have been experimentally determined. This study showed that these TFs have sequence variation at the third codon position G+C content but maintain the same Codon Adaptation Index (CAI) trend as annotated functional transcription factors. Most of these transcription factors are in areas of the genome where abundant repetitive and mobile elements are present. Sequence divergence points to groups with distinctive sequence signatures but maintaining the same type of DNA binding domain. Finally, the analysis of the promoter sequences of the 58 TFs showed A+T rich regions that agree with the features of horizontally transferred genes. The findings reported here pave the way for future research of these TFs that may uncover their role as spare factors in case of lose-of-function mutations in core TFs and trace back their evolutionary history.

Chromogenic Escherichia coli reporter strain for screening DNA damaging agents.

The presence of pollutants in soil and water has given rise to diverse analytical and biological approaches to detect and measure contaminants in the environment. Using bacterial cells as reporter strains represents an advantage for detecting pollutants present in soil or water samples. Here, an Escherichia coli reporter strain expressing a chromoprotein capable of interacting with soil or water samples and responding to DNA damaging compounds is validated. The reporter strain generates a qualitative signal and is based on the expression of the coral chromoprotein AmilCP under the control of the recA promoter. This strain can be used simply by applying soil or water samples directly and rendering activation upon DNA damage. This reporter strain responds to agents that damage DNA (with an apparent detection limit of 1 µg of mitomycin C) without observable response to membrane integrity damage, protein folding or oxidative stress generating agents, in the latter case, DNA damage was observed. The developed reporter strain reported here is effective for the detection of DNA damaging agents present in soils samples. In a proof-of-concept analysis using soil containing chromium, showing activation at 15.56 mg/L of Cr(VI) present in soil and leached samples and is consistent with Cr(III) toxicity at high concentrations (130 µg). Our findings suggest that chromogenic reporter strains can be applied for simple screening, thus reducing the number of samples requiring analytical techniques.

Comparison of Cell Wall Polysaccharide Composition and Structure Between Strains of Sporothrix schenckii and Sporothrix brasiliensis.

Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are the main causative agents of sporotrichosis, a human subcutaneous mycosis. Differences in virulence patterns are associated with each species but remain largely uncharacterized. The S. schenckii and S. brasiliensis cell wall composition and virulence are influenced by the culturing media, with little or no influence on S. globosa. By keeping constant the culturing media, we compared the cell wall composition of three S. schenckii and two S. brasiliensis strains, previously described as presenting different virulence levels on a murine model of infection. The cell wall composition of the five Sporothrix spp. strains correlated with the biochemical composition of the cell wall previously reported for the species. However, the rhamnose-to-ß-glucan ratio exhibits differences among strains, with an increase in cell wall rhamnose-to-ß-glucan ratio as their virulence increased. This relationship can be expressed mathematically, which could be an important tool for the determination of virulence in Sporothrix spp. Also, structural differences in rhamnomannan were found, with longer side chains present in strains with lower virulence reported for both species here studied, adding insight to the importance of this polysaccharide in the pathogenic process of these fungi.

TIP4.0: Industrial Internet of Things Platform for Predictive Maintenance.

Industry 4.0, allied with the growth and democratization of Artificial Intelligence (AI) and the advent of IoT, is paving the way for the complete digitization and automation of industrial processes. Maintenance is one of these processes, where the introduction of a predictive approach, as opposed to the traditional techniques, is expected to considerably improve the industry maintenance strategies with gains such as reduced downtime, improved equipment effectiveness, lower maintenance costs, increased return on assets, risk mitigation, and, ultimately, profitable growth. With predictive maintenance, dedicated sensors monitor the critical points of assets. The sensor data then feed into machine learning algorithms that can infer the asset health status and inform operators and decision-makers. With this in mind, in this paper, we present TIP4.0, a platform for predictive maintenance based on a modular software solution for edge computing gateways. TIP4.0 is built around Yocto, which makes it readily available and compliant with Commercial Off-the-Shelf (COTS) or proprietary hardware. TIP4.0 was conceived with an industry mindset with communication interfaces that allow it to serve sensor networks in the shop floor and modular software architecture that allows it to be easily adjusted to new deployment scenarios. To showcase its potential, the TIP4.0 platform was validated over COTS hardware, and we considered a public data-set for the simulation of predictive maintenance scenarios. We used a Convolution Neural Network (CNN) architecture, which provided competitive performance over the state-of-the-art approaches, while being approximately four-times and two-times faster than the uncompressed model inference on the Central Processing Unit (CPU) and Graphical Processing Unit, respectively. These results highlight the capabilities of distributed large-scale edge computing over industrial scenarios.

Refinement of the Griess method for measuring nitrite in biological samples.

Nitric oxide (NO) is a reactive gas that participates in many physiological as well as pathogenic processes in higher eukaryotic organisms. Inflammatory responses elicit higher levels of this molecule. Nevertheless, there are many technical challenges to accurately measure the amount of NO produced. Previously, a method using whole-cell extracts from Escherichia coli was able to generate the conversion of nitrate into nitrite to measure the amount of nitrate or indirectly the NO present in a sample using the Griess reaction. Here we present an improvement to this method, by using E. coli whole-cell extracts lacking one of the two nitrite reductases, rendered a more precise measurement when coupled with the Griess reaction than our previous report. Alternatively, osmotic stress showed to downregulate the expression of both nitrate reductases, which can be an alternative for indirect nitrate and NO reduction. The results presented here show an easy method for nitrate and NO reduction to nitrite and avoid the reconversion to nitrate, also as an alternative for other analytical methods that are based on cadmium, purified nitrate reductase enzyme, or salicylic methods to reduce NO. This method can be widely used for measuring NO production in living organisms, soil, and other relevant microbiological samples.

Insect Immune Evasion by Dauer and Nondauer Entomopathogenic Nematodes.

The immune response of animals, including insects, is overcome by some parasites. For example, dauer larvae (DL) of the obligate entomopathogenic nematodes (EPNs) Heterorhabditis and Steinernema can invade insects, evade their defenses, and cause death. Although DL were long assumed to be the only infective stage of nematodes, recent reports suggest that L2-L3 larvae of facultative EPNs are also capable of killing insects. There are no studies, to our knowledge, about the role of nonimmunological barriers (the exoskeleton and its openings) in avoiding infection by DL and L2-L3 larvae, or whether these larval stages evade the host immune system in the same way. The objective of this study was to examine these questions by infecting Galleria mellonella with the facultative parasitic nematode Rhabditis regina. DL or L2-L3 larvae were either deposited on or near the moths or injected into their hemocoel. Once nematodes reached the hemocoel, the following host immune response parameters were quantified: prophenoloxidase, phenoloxidase, lytic activity, and the number of granular hemocytes. DL showed a greater ability to penetrate the exoskeleton than L2-L3 larvae. Once inside, however, both went unnoticed by the immune system and killed the insect. A higher number of granular hemocytes was activated by L2-L3 larvae than DL. We show for the first time that L2-L3 larvae can penetrate and evade the insect immune system. Further research is needed to compare facultative and specialized EPNs to determine which is more likely, with both DL and L2-L3 larvae, to evade insect defense barriers and produce death. The results will contribute to understanding the evolution of virulence in entomopathogenic nematodes.

Possible role played by the SINE2 element in gene regulation, as demonstrated by differential processing and polyadenylation in avirulent strains of E. histolytica.

Entamoeba histolytica represents a useful model in parasitic organisms due to its complex genomic organization and survival mechanisms. To counteract pathogenic organisms, it is necessary to characterize their molecular biology to design new strategies to combat them. In this report, we investigated a less-known genetic element, short interspersed nuclear element 2 (SINE2), that is present in this ameba and is highly transcribed and polyadenylated. In this study, we show that in two different nonvirulent strains of E. histolytica, SINE2 is differentially processed into two transcript fragments, that is, a full-length 560-nt fragment and a shorter 393-nt fragment bearing an approximately 18-nt polyadenylation tail. Sequence analysis of the SINE2 transcript showed that a Musashi-like protein may bind to it. Also, two putative Musashi-like sequences were identified on the transcript. Semiquantitative expression analysis of the two Musashi-like proteins identified in the E. histolytica genome (XP_648918 and XP_649094) showed that XP_64094 is overexpressed in the nonvirulent strains tested. The information available in the literature and the results presented in this report indicate that SINE2 may affect other genes, as observed with the epigenetic silencing of the G3 strain, by an antisense mechanism or via RNA-protein interactions that may ultimately be involved in the phenotype of nonvirulent strains of E. histolytica.

Chitosan-BaTiO3 nanostructured piezopolymer for tissue engineering.

Herein we report the synthesis of a piezopolymer composed of chitosan (CS)/hydroxylated BaTiO3 (OH-BTO) nanoparticles with enhanced biocompatibility, non-toxicity, and piezoelectric behavior that can be advantageously used in biomedical applications. Our CS/OH-BTO nanocomposites exhibit piezoelectric coefficient (d33 = 11.29 pC/N) between those of dry skin (0.05-0.19 pC/N) and bone (4-11 pC/N), demonstrating biocompatibility in contact with human fibroblasts (HF) cells after 24 h. SEM, XRD, FTIR and Raman measurements were performed to assess the mechanism of interaction between CS matrix and OH-BTO NPs and their correlation with the biological responses. Cytotoxicity assays with HF cells reveal that hydroxylation of BTO NPs does not affect the cell viability of CS/OH-BTO films with NPs concentration from 1 to 30 wt.%. In contrast, non-hydroxylated BTO NPs showed significant cell damage, which could be traced to uncontrollable NPs agglomeration. This behavior suggests that CS/OH-BTO nanocomposites can act as active material that promotes cell growth and can be used for biomedical purposes.

Loss of Kex2 Affects the Candida albicans Cell Wall and Interaction with Innate Immune Cells.

The secretory pathway in Candida albicans involves the protein translocation into the lumen of the endoplasmic reticulum and transport to the Golgi complex, where proteins undergo posttranslational modifications, including glycosylation and proteolysis. The Golgi-resident Kex2 protease is involved in such processing and disruption of its encoding gene affected virulence and dimorphism. These previous studies were performed using cells without URA3 or with URA3 ectopically placed into the KEX2 locus. Since these conditions are known to affect the cellular fitness and the host-fungus interaction, here we generated a kex2Δ null mutant strain with URA3 placed into the neutral locus RPS1. The characterization of this strain showed defects in the cell wall composition, with a reduction in the N-linked mannan content, and the increment in the levels of O-linked mannans, chitin, and ß-glucans. The defects in the mannan content are likely linked to changes in Golgi-resident enzymes, as the α-1,2-mannosyltransferase and α-1,6-mannosyltransferase activities were incremented and reduced, respectively. The mutant cells also showed reduced ability to stimulate cytokine production and phagocytosis by human mononuclear cells and macrophages, respectively. Collectively, these data showed that loss of Kex2 affected the cell wall composition, the protein glycosylation pathways, and interaction with innate immune cells.

aeBlue Chromoprotein Color is Temperature Dependent.

BACKGROUND: Marine sessile organisms display a color palette that is the result of the expression of fluorescent and non-fluorescent proteins. Fluorescent proteins have uncovered transcriptional regulation, subcellular localization of proteins, and the fate of cells during development. Chromoproteins have received less attention until recent years as bioreporters. Here, we studied the properties of aeBlue, a a 25.91 kDa protein from the anemone Actinia equina. OBJECTIVE: To assess the properties of aeBlue chromoprotein under different physicochemical conditions. METHODS: In this article, during the purification of aeBlue we uncovered that it suffered a color shift when frozen. We studied the color shift by different temperature incubation and physicochemical conditions and light spectroscopy. To assess the possible structural changes in the protein, circular dichroism analysis, size exclusion chromatography and native PAGE was performed. RESULTS: We uncover that aeBlue chromoprotein, when expressed from a synthetic construct in Escherichia coli, showed a temperature dependent color shift. Protein purified at 4 °C by metal affinity chromatography exhibited a pinkish color and shifts back at higher temperatures to its intense blue color. Circular dichroism analysis revealed that the structure in the pink form of the protein has reduced secondary structure at 4 °C, but at 35 °C and higher, the structure shifts to a native conformation and Far UV- vis CD spectra revealed the shift in an aromatic residue of the chromophore. Also, the chromophore retains its properties in a wide range of conditions (pH, denaturants, reducing and oxidants agents). Quaternary structure is also maintained as a tetrameric conformation as shown by native gel and size exclusion chromatography. CONCLUSION: Our results suggest that the chromophore position in aeBlue is shifted from its native position rendering the pink color and the process to return it to its native blue conformation is temperature dependent.

An in vitro characterisation of the Trichomonas vaginalis TATA box-binding proteins (TBPs).

The protozoan parasite Trichomonas vaginalis is a common human pathogen from one of the earliest-diverging eukaryotic lineages. At the transcriptional level, the highly conserved Inr element of RNA pol II-transcribed genes surrounds the transcription start site and is recognised by IBP39, a protein exclusive of T. vaginalis. Typical TATA boxes have not been identified in this organism but, in contrast, BLAST analyses of the T. vaginalis genome identified two genes encoding putative TATA-binding proteins (herein referred to as TvTBP1 and TvTBP2). The goal of this work was to characterise these two proteins at the molecular level. Our results show that both TvTBPs theoretically adopt the saddle-shaped structure distinctive to TBPs and both Tvtbp genes are expressed in T. vaginalis. TvTBP1 did not complement a Saccharomyces cerevisiae mutant lacking TBP; however, TvTBP1 and TvTBP2 proteins bound T. vaginalis DNA promoter sequences in EMSA assays. We propose that TvTBP1 may be part of the preinitiation transcription complex in T. vaginalis since TvTBP1 recombinant protein was able to bind IBP39 in vitro. This work represents the first approach towards the characterisation of general transcription factors in this early divergent organism.

Plasma membrane damage repair is mediated by an acid sphingomyelinase in Entamoeba histolytica.

Entamoeba histolytica is a pathogen that during its infective process confronts the host defenses, which damages the amoebic plasma membrane (PM), resulting in the loss of viability. However, it is unknown whether amoebic trophozoites are able to repair their PM when it is damaged. Acid sphingomyelinases (aSMases) have been reported in mammalian cells to promote endocytosis and removal of PM lesions. In this work, six predicted amoebic genes encoding for aSMases were found to be transcribed in the HM1:IMSS strain, finding that the EhaSM6 gene is the most transcribed in basal growth conditions and rendered a functional protein. The secreted aSMase activity detected was stimulated by Mg+2 and inhibited by Co+2. Trophozoites that overexpress the EhaSM6 gene (HM1-SM6HA) exhibit an increase of 2-fold in the secreted aSMase activity. This transfectant trophozoites exposed to pore-forming molecules (SLO, Magainin, ß-Defensin 2 and human complement) exhibited an increase from 6 to 25-fold in the secreted aSMase activity which correlated with higher amoebic viability in a Ca+2 dependent process. However, other agents that affect the PM such as hydrogen peroxide also induced an increase of secreted aSMase, but to a lesser extent. The aSMase6 enzyme is N- and C-terminal processed. Confocal and transmission electron microscopy showed that trophozoites treated with SLO presented a migration of lysosomes containing the aSMase towards the PM, inducing the formation of membrane patches and endosomes in the control strain. These cellular structures were increased in the overexpressing strain, indicating the involvement of the aSMase6 in the PM injury repair. The pore-forming molecules induced an increase in the expression of EhaSM1, 2, 5 and 6 genes, meanwhile, hydrogen peroxide induced an increase in all of them. In all the conditions evaluated, the EhaSM6 gene exhibited the highest levels of induction. Overall, these novel findings show that the aSMase6 enzyme from E. histolytica promotes the repair of the PM damaged with pore-forming molecules to prevent losing cell integrity. This novel system could act when encountered with the lytic defense systems of the host.

Preparation of Peppermint Oil-Based Nanodevices Loaded with Paclitaxel: Cytotoxic and Apoptosis Studies in HeLa Cells.

In this work, several normal, oil-in-water (o/w) microemulsions (MEs) were prepared using peppermint essential oil, jojoba oil, trans-anethole, and vitamin E as oil phases to test their capacity to load paclitaxel (PTX). Initially, pseudo-ternary partial phase diagrams were constructed in order to find the normal microemulsion region using d-α-tocopherol polyethylene glycol 1000 succinate (TPGS-1000) as surfactant and isobutanol (iso-BuOH) as co-surfactant. Selected ME formulations were loaded with PTX reaching concentrations of 0.6 mg mL-1 for the peppermint oil and trans-anethole MEs, while for the vitamin E and jojoba oil MEs, the maximum concentration was 0.3 mg mL-1. The PTX-loaded MEs were stable according to the results of heating-cooling cycles and mechanical force (centrifugation) test. Particularly, drug release profile for the PTX-loaded peppermint oil ME (MEPP) showed that ∼ 90% of drug was released in the first 48 h. Also, MEPP formulation showed 70% and 90% viability reduction on human cervical cancer (HeLa) cells after 24 and 48 h of exposure, respectively. In addition, HeLa cell apoptosis was confirmed by measuring caspase activity and DNA fragmentation. Results showed that the MEPP sample presented a major pro-apoptotic capability by comparing with the unloaded PTX ME sample.