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.

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.

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.

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.

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.

Flotillin homologue is involved in the swimming behavior of Escherichia coli.

Cellular membrane is a key component for maintaining cell shape and integrity. The classical membrane structure and function by Singer and Nicolson groundbreaking model has depicted the membrane as a homogeneous fluid structure. This view has changed by the discovery of discrete domains containing different lipid compositions, called lipid rafts, which play a key role in signal transduction in eukaryotic cells. In the past few years, lipid raft-like structures have been found in bacteria also, constituted by cardiolipin and other modified lipids, perhaps involved in generating a specific site for protein clustering. Here, we report the analysis of a protein termed YqiK from Escherichia coli, a prohibitin homolog that has been implicated in stress sensing by the formation of membrane-associated microdomains. The E. coli yqiK-deficient mutant strain showed an enhanced swimming behavior and was resistant to ampicillin but its response to other stressing conditions was similar to that of the wild-type strain. The abnormal swimming behavior is reversed when the protein is expressed in trans from a plasmid. Also, we demonstrate that YqiK is not redundant with QmcA, another flotillin homolog found in E. coli. Our results, along with the data available in the literature, suggest that YqiK may be involved in the formation of discrete membrane-associated signaling complexes that regulate and agglomerate signaling proteins to generate cell response to chemotaxis.

Does juvenile hormone prompt oxidative stress in male damselflies?

In invertebrates, it has recently been reported that secondary sexual characteristics (SSCs) reflect the antioxidant defense of their bearers, but it is not known what physiological link maintains the honesty of those signals. Here, we used the damselfly Hetaerina americana to test whether juvenile hormone plays such a role. First, we analyzed whether oxidative damage is a real threat in natural damselfly populations by examining the accumulation of oxidized guanines as a function of age in males. Then, we injected paraquat (a pro-oxidant agent) and added the juvenile hormone analog methoprene (JHa) to the experimental group and the JHa vehicle (acetone) to the control group, to determine whether JHa increases the levels of pro-oxidants and antioxidants. We found that DNA oxidation increased with age, and that levels of hydrogen peroxide and superoxide dismutase, but not catalase or glutathione, were elevated in the JHa group compared with the control group. We propose that juvenile hormone is a mediator of the relationship between SSCs and antioxidant capacity and, based on the literature, we know that JHa suppresses the immune response. We therefore suggest that juvenile hormone is a molecular mediator of the general health of males, which is reflected in their SSCs.

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.

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.

Role of protein phosphomannosylation in the Candida tropicalis-macrophage interaction.

Candida tropicalis is an opportunistic fungal pathogen responsible for mucosal and systemic infections. The cell wall is the initial contact point between a fungal cell and the host immune system, and mannoproteins are important components that play key roles when interacting with host cells. In Candida albicans, mannans are modified by mannosyl-phosphate moieties, named phosphomannans, which can work as molecular scaffolds to synthesize ß1,2-mannooligosaccharides, and MNN4 is a positive regulator of the phosphomannosylation pathway. Here, we showed that C. tropicalis also displays phosphomannans on the cell surface, but the amount of this cell wall component varies depending on the fungal strain. We also identified a functional ortholog of CaMNN4 in C. tropicalis. Disruption of this gene caused depletion of phosphomannan content. The C. tropicalis mnn4Δ did not show defects in the ability to stimulate cytokine production by human mononuclear cells but displayed virulence attenuation in an insect model of candidiasis. When the mnn4Δ-macrophage interaction was analyzed, results showed that presence of cell wall phosphomannan was critical for C. tropicalis phagocytosis. Finally, our results strongly suggest a differential role for phosphomannans during phagocytosis of C. albicans and C. tropicalis.