The Emerging Role of Human Gut Bacteria Extracellular Vesicles in Mental Disorders and Developing New Pharmaceuticals.

In recent years, further evidence has emerged regarding the involvement of extracellular vesicles in various human physiopathological conditions such as Alzheimer's disease, Parkinson's disease, irritable bowel syndrome, and mental disorders. The biogenesis and cargo of such vesicles may reveal their impact on human health nd disease and set the underpinnings for the development of novel chemical compounds and pharmaceuticals. In this review, we examine the link between bacteria-derived exosomes in the gastrointestinal tract and mental disorders, such as depression and anxiety disorders. Crucially, we focus on whether changes in the gut environment affect the human mental state or the other way around. Furthermore, the possibility of handling bacteria-derived exosomes as vectors of chemicals to treat such conditions is examined.

Comparative Analysis of Prokaryotic Extracellular Vesicle Proteins and Their Targeting Signals.

Prokaryotic extracellular vesicles (EVs) are vesicles that bud from the cell membrane and are secreted by bacteria and archaea. EV cargo in Gram-negative bacteria includes mostly periplasmic and outer membrane proteins. EVs are clinically important as their cargo can include toxins associated with bacterial virulence and toxicity; additionally, they have been proposed as efficient vaccine agents and as the ancestors of the eukaryotic endomembrane system. However, the mechanistic details behind EV cargo selection and release are still poorly understood. In this study, we have performed bioinformatics analysis of published data on EV proteomes from 38 species of bacteria and 4 archaea. Focusing on clusters of orthologous genes (COGs) and using the EggNOG mapper function, we have identified cargo proteins that are commonly found in EVs across species. We discuss the putative role of these prominent proteins in EV biogenesis and function. We also analyzed the published EV proteomes for conserved signal sequences and discuss the potential role of these signal sequences for EV cargo selection.

Evolution of two-component quorum sensing systems.

Quorum sensing (QS) is a cell-to-cell communication system that enables bacteria to coordinate their gene expression depending on their population density, via the detection of small molecules called autoinducers. In this way bacteria can act collectively to initiate processes like bioluminescence, virulence and biofilm formation. Autoinducers are detected by receptors, some of which are part of two-component signal transduction systems (TCS), which comprise of a (usually membrane-bound) sensor histidine kinase (HK) and a cognate response regulator (RR). Different QS systems are used by different bacterial taxa, and their relative evolutionary relationships have not been extensively studied. To address this, we used the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to identify all the QS HKs and RRs that are part of TCSs and examined their conservation across microbial taxa. We compared the combinations of the highly conserved domains in the different families of receptors and response regulators using the Simple Modular Architecture Research Tool (SMART) and KEGG databases, and we also carried out phylogenetic analyses for each family, and all families together. The distribution of the different QS systems across taxa, indicates flexibility in HK-RR pairing and highlights the need for further study of the most abundant systems. For both the QS receptors and the response regulators, our results indicate close evolutionary relationships between certain families, highlighting a common evolutionary history which can inform future applications, such as the design of novel inhibitors for pathogenic QS systems.

Conservation biology of threatened Mediterranean chasmophytes: The case of Asperula naufraga endemic to Zakynthos island (Ionian islands, Greece).

Asperula naufraga is a rare and threatened obligate chasmophyte, endemic to Zakynthos island (Ionian islands, Greece). In this study, we provide a combined approach (including monitoring of demographic and reproductive parameters and study of genetic diversity) to assess the current conservation status of the species and to estimate its future extinction risk. The five subpopulations of A. naufraga were monitored for five years (2014-2018). Population size markedly fluctuated between 68-130 mature individuals during the monitoring period. The extent of occurrence (EOO) was estimated at 28.7 km2 and the area of occupancy (AOO) was 8 km2. Stage-structure recordings were similar for all subpopulations, characterized by high proportions of adult and senescent individuals, following a common pattern, which has been observed in other cliff-dwelling plants. Preliminary genetic analysis with SSRs markers revealed low heterozygosity within subpopulations and significant departure from H-W equilibrium, which combined with small population size suggest increased threat of genetic diversity loss. Our results indicate that the species should be placed in the Critically Endangered (CR) IUCN threat category, while according to Population Viability Analysis results its extinction risk increases to 47.8% in the next 50 years. The small population size combined with large fluctuations in its size, low recruitment and low genetic diversity, indicate the need of undertaking effective in situ and ex situ conservation measures.

Structural models for the design of novel antiviral agents against Greek Goat Encephalitis.

The Greek Goat Encephalitis virus (GGE) belongs to the Flaviviridae family of the genus Flavivirus. The GGE virus constitutes an important pathogen of livestock that infects the goat's central nervous system. The viral enzymes of GGE, helicase and RNA-dependent RNA polymerase (RdRP), are ideal targets for inhibitor design, since those enzymes are crucial for the virus' survival, proliferation and transmission. In an effort to understand the molecular structure underlying the functions of those viral enzymes, the three dimensional structures of GGE NS3 helicase and NS5 RdRP have been modelled. The models were constructed in silico using conventional homology modelling techniques and the known 3D crystal structures of solved proteins from closely related species as templates. The established structural models of the GGE NS3 helicase and NS5 RdRP have been evaluated for their viability using a repertoire of in silico tools. The goal of this study is to present the 3D conformations of the GGE viral enzymes as reliable structural models that could provide the platform for the design of novel anti-GGE agents.

Evolution of the F0F1 ATP synthase complex in light of the patchy distribution of different bioenergetic pathways across prokaryotes.

Bacteria and archaea are characterized by an amazing metabolic diversity, which allows them to persist in diverse and often extreme habitats. Apart from oxygenic photosynthesis and oxidative phosphorylation, well-studied processes from chloroplasts and mitochondria of plants and animals, prokaryotes utilize various chemo- or lithotrophic modes, such as anoxygenic photosynthesis, iron oxidation and reduction, sulfate reduction, and methanogenesis. Most bioenergetic pathways have a similar general structure, with an electron transport chain composed of protein complexes acting as electron donors and acceptors, as well as a central cytochrome complex, mobile electron carriers, and an ATP synthase. While each pathway has been studied in considerable detail in isolation, not much is known about their relative evolutionary relationships. Wanting to address how this metabolic diversity evolved, we mapped the distribution of nine bioenergetic modes on a phylogenetic tree based on 16S rRNA sequences from 272 species representing the full diversity of prokaryotic lineages. This highlights the patchy distribution of many pathways across different lineages, and suggests either up to 26 independent origins or 17 horizontal gene transfer events. Next, we used comparative genomics and phylogenetic analysis of all subunits of the F0F1 ATP synthase, common to most bacterial lineages regardless of their bioenergetic mode. Our results indicate an ancient origin of this protein complex, and no clustering based on bioenergetic mode, which suggests that no special modifications are needed for the ATP synthase to work with different electron transport chains. Moreover, examination of the ATP synthase genetic locus indicates various gene rearrangements in the different bacterial lineages, ancient duplications of atpI and of the beta subunit of the F0 subcomplex, as well as more recent stochastic lineage-specific and species-specific duplications of all subunits. We discuss the implications of the overall pattern of conservation and flexibility of the F0F1 ATP synthase genetic locus.

Evolution of the plasma and tissue kallikreins, and their alternative splicing isoforms.

Kallikreins are secreted serine proteases with important roles in human physiology. Human plasma kallikrein, encoded by the KLKB1 gene on locus 4q34-35, functions in the blood coagulation pathway, and in regulating blood pressure. The human tissue kallikrein and kallikrein-related peptidases (KLKs) have diverse expression patterns and physiological roles, including cancer-related processes such as cell growth regulation, angiogenesis, invasion, and metastasis. Prostate-specific antigen (PSA), the product of the KLK3 gene, is the most widely used biomarker in clinical practice today. A total of 15 KLKs are encoded by the largest contiguous cluster of protease genes in the human genome (19q13.3-13.4), which makes them ideal for evolutionary analysis of gene duplication events. Previous studies on the evolution of KLKs have traced mammalian homologs as well as a probable early origin of the family in aves, amphibia and reptilia. The aim of this study was to address the evolutionary and functional relationships between tissue KLKs and plasma kallikrein, and to examine the evolution of alternative splicing isoforms. Sequences of plasma and tissue kallikreins and their alternative transcripts were collected from the NCBI and Ensembl databases, and comprehensive phylogenetic analysis was performed by Bayesian as well as maximum likelihood methods. Plasma and tissue kallikreins exhibit high sequence similarity in the trypsin domain (>50%). Phylogenetic analysis indicates an early divergence of KLKB1, which groups closely with plasminogen, chymotrypsin, and complement factor D (CFD), in a monophyletic group distinct from trypsin and the tissue KLKs. Reconstruction of the earliest events leading to the diversification of the tissue KLKs is not well resolved, indicating rapid expansion in mammals. Alternative transcripts of each KLK gene show species-specific divergence, while examination of sequence conservation indicates that many annotated human KLK isoforms are missing the catalytic triad that is crucial for protease activity.

Gene fusion analysis in the battle against the African endemic sleeping sickness.

The protozoan Trypanosoma brucei causes African Trypanosomiasis or sleeping sickness in humans, which can be lethal if untreated. Most available pharmacological treatments for the disease have severe side-effects. The purpose of this analysis was to detect novel protein-protein interactions (PPIs), vital for the parasite, which could lead to the development of drugs against this disease to block the specific interactions. In this work, the Domain Fusion Analysis (Rosetta Stone method) was used to identify novel PPIs, by comparing T. brucei to 19 organisms covering all major lineages of the tree of life. Overall, 49 possible protein-protein interactions were detected, and classified based on (a) statistical significance (BLAST e-value, domain length etc.), (b) their involvement in crucial metabolic pathways, and (c) their evolutionary history, particularly focusing on whether a protein pair is split in T. brucei and fused in the human host. We also evaluated fusion events including hypothetical proteins, and suggest a possible molecular function or involvement in a certain biological process. This work has produced valuable results which could be further studied through structural biology or other experimental approaches so as to validate the protein-protein interactions proposed here. The evolutionary analysis of the proteins involved showed that, gene fusion or gene fission events can happen in all organisms, while some protein domains are more prone to fusion and fission events and present complex evolutionary patterns.

A holistic evolutionary and structural study of flaviviridae provides insights into the function and inhibition of HCV helicase.

Viral RNA helicases are involved in duplex unwinding during the RNA replication of the virus. It is suggested that these helicases represent very promising antiviral targets. Viruses of the flaviviridae family are the causative agents of many common and devastating diseases, including hepatitis, yellow fever and dengue fever. As there is currently no available anti-Flaviviridae therapy, there is urgent need for the development of efficient anti-viral pharmaceutical strategies. Herein, we report the complete phylogenetic analysis across flaviviridae alongside a more in-depth evolutionary study that revealed a series of conserved and invariant amino acids that are predicted to be key to the function of the helicase. Structural molecular modelling analysis revealed the strategic significance of these residues based on their relative positioning on the 3D structures of the helicase enzymes, which may be used as pharmacological targets. We previously reported a novel series of highly potent HCV helicase inhibitors, and we now re-assess their antiviral potential using the 3D structural model of the invariant helicase residues. It was found that the most active compound of the series, compound C4, exhibited an IC50 in the submicromolar range, whereas its stereoisomer (compound C12) was completely inactive. Useful insights were obtained from molecular modelling and conformational search studies via molecular dynamics simulations. C12 tends to bend and lock in an almost "U" shape conformation, failing to establish vital interactions with the active site of HCV. On the contrary, C4 spends most of its conformational time in a straight, more rigid formation that allows it to successfully block the passage of the oligonucleotide in the ssRNA channel of the HCV helicase. This study paves the way and provides the necessary framework for the in-depth analysis required to enable the future design of new and potent anti-viral agents.