Identifying macroplastic pathobiomes and antibiotic resistance in a subtropical fish farm.

Macroplastics are ubiquitous in aquaculture ecosystems. However, to date the potential role of plastics as a support for bacterial biofilm that can include potential human pathogenic bacteria (PHPB) and antibiotic-resistant bacteria (ARB) has been largely overlooked. In this study, we used a combination of metabarcoding and standard antibiotic susceptibility testing to study the pathobiome and resistome of macroplastics, fish guts and the environment in a marine aquaculture farm in Mauritius. Aquaculture macroplastics were found to be higher in PHPB, dominated by the Vibrionaceae family (0.34 % of the total community), compared with environmental samples. Moreover, isolates from aquaculture plastics showed higher significant multiple antibiotic resistance (MAR) compared to non-plastic samples of seawater, sediment and fish guts. These results suggest that plastics act as a reservoir and fomite of PHPB and ARB in aquaculture, potentially threatening the health of farmed fish and human consumers.

Local acclimatisation-driven differential gene and protein expression patterns of Hsp70 in Acropora muricata: Implications for coral tolerance to bleaching.

Corals show spatial acclimatisation to local environment conditions. However, the various cellular mechanisms involved in local acclimatisation and variable bleaching patterns in corals remain to be thoroughly understood. In this study, the modulation of a protein implicated in cellular heat stress tolerance, the heat shock protein 70, was compared at both gene (hsp70) and protein (Hsp70) expression level in bleaching tolerant near-coast Acropora muricata colonies and bleaching susceptible reef colonies, in the lagoon of Belle Mare (Mauritius). The relative Hsp70 levels varied significantly between colonies from the two different locations, colonies having different health conditions and the year of collection. Before the bleaching event of 2016, near-coast colonies had higher basal levels of both Hsp70 gene and protein compared to reef colonies. During the bleaching event, the near-coast colonies did not bleach and had significantly higher relative levels of both Hsp70 gene and protein compared to bleached reef colonies. No significant genetic differentiation between the two studied coral populations was observed and all the colonies analysed were associated with Symbiodiniaceae of the genus Symbiodinium (Clade A) irrespective of location and sampling period. These findings provide further evidence of the involvement of Hsp70 in conferring bleaching tolerance to corals. Moreover, the consistent expression differences of Hsp70 gene and protein between the near-coast and reef coral populations in a natural setting indicate that the modulation of this Hsp is involved in local acclimatisation of corals to their environments.

Gene expression biomarkers of heat stress in scleractinian corals: Promises and limitations.

Gene expression biomarkers (GEBs) are emerging as powerful diagnostic tools for identifying and characterizing coral stress. Their capacity to detect sublethal stress prior to the onset of signs at the organismal level that might already indicate significant damage makes them more precise and proactive compared to traditional monitoring techniques. A high number of candidate GEBs, including certain heat shock protein genes, metabolic genes, oxidative stress genes, immune response genes, ion transport genes, and structural genes have been investigated, and some genes, including hsp16, Cacna1, MnSOD, SLC26, and Nf-kB, are already showing excellent potential as reliable indicators of thermal stress in corals. In this mini-review, we synthesize the current state of knowledge of scleractinian coral GEBs and highlight gaps in our understanding that identify directions for future work. We also address the underlying sources of variation that have sometimes led to contrasting results between studies, such as differences in experimental set-up and approach, intrinsic variation in the expression profiles of different experimental organisms (such as between different colonies or their algal symbionts), diel cycles, varying thermal history, and different expression thresholds. Despite advances in our understanding there is still no universally accepted biomarker of thermal stress, the molecular response of corals to heat stress is still unclear, and biomarker research in Symbiodinium still lags behind that of the host. These gaps should be addressed in future work.

Cytoglobin as a Biomarker in Cancer: Potential Perspective for Diagnosis and Management.

The search for biomarkers to detect the earliest glimpse of cancer has been one of the primary objectives of cancer research initiatives. These endeavours, in spite of constant clinical challenges, are now more focused as early cancer detection provides increased opportunities for different interventions and therapies, with higher potential for improving patient survival and quality of life. With the progress of the omics technologies, proteomics and metabolomics are currently being used for identification of biomarkers. In this line, cytoglobin (Cygb), a ubiquitously found protein, has been actively reviewed for its functional role. Cytoglobin is dynamically responsive to a number of insults, namely, fibrosis, oxidative stress, and hypoxia. Recently, it has been reported that Cygb is downregulated in a number of malignancies and that an induced overexpression reduces the proliferative characteristics of cancer cells. Thus, the upregulation of cytoglobin can be indicative of a tumour suppressor ability. Nevertheless, without a comprehensive outlook of the molecular and functional role of the globin, it will be most unlikely to consider cytoglobin as a biomarker for early detection of cancer or as a therapeutic option. This review provides an overview of the proposed role of cytoglobin and explores its potential functional role as a biomarker for cancer and other diseases.

Localisation of a family of complex-forming ß-barrels in the T. vaginalis hydrogenosomal membrane.

Crucial to organellogenesis was the development of membrane translocases responsible for delivering proteins to new cellular compartments. This investigation examines the Trichomonas vaginalis hydrogenosome, a mitochondrially derived organelle. We identify an expanded family of putative ß-barrel proteins (THOM A-I) comprising nine related sequences. Sub-cellular localisation by immunofluorescence and biochemical fractionation is consistent with THOMs being localised to the hydrogenosomal membrane. Native gel electrophoresis and chemical cross-linking support the ability of THOM proteins to be components of membrane-bound oligomeric protein complexes, consistent with a role in protein translocation.

The ATP-binding cassette proteins of the deep-branching protozoan parasite Trichomonas vaginalis.

The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters.

The Trichomonas vaginalis hydrogenosome proteome is highly reduced relative to mitochondria, yet complex compared with mitosomes.

The human pathogen Trichomonas vaginalis lacks conventional mitochondria and instead contains divergent mitochondrial-related organelles. These double-membrane bound organelles, called hydrogenosomes, produce molecular hydrogen. Phylogenetic and biochemical analyses of hydrogenosomes indicate a common origin with mitochondria; however identification of hydrogenosomal proteins and studies on its metabolism have been limited. Here we provide a detailed proteomic analysis of the T. vaginalis hydrogenosome. The proteome of purified hydrogenosomes consists of 569 proteins, a number substantially lower than the 1,000-1,500 proteins reported for fungal and animal mitochondrial proteomes, yet considerably higher than proteins assigned to mitosomes. Pathways common to and distinct from both mitochondria and mitosomes were revealed by the hydrogenosome proteome. Proteins known to function in amino acid and energy metabolism, Fe-S cluster assembly, flavin-mediated catalysis, oxygen stress response, membrane translocation, chaperonin functions, proteolytic processing and ATP hydrolysis account for ∼30% of the hydrogenosome proteome. Of the 569 proteins in the hydrogenosome proteome, many appear to be associated with the external surface of hydrogenosomes, including large numbers of GTPases and ribosomal proteins. Glycolytic proteins were also found to be associated with the hydrogenosome proteome, similar to that previously observed for mitochondrial proteomes. Approximately 18% of the hydrogenosomal proteome is composed of hypothetical proteins of unknown function, predictive of multiple activities and properties yet to be uncovered for these highly adapted organelles.

The 7th East Midlands Proteomics Workshop 12 November 2008, Nottingham, UK.

Over 200 scientists with a common interest in proteomic techniques and their application to fundamental biological and biomedical problems participated in the 7th East Midlands Proteomics Workshop. This annual one day meeting was held in Nottingham in November 2008 and is a joint venture of colleagues from three local Universities: The University of Nottingham, Nottingham Trent University, and Loughborough University.

Non-mitochondrial complex I proteins in a hydrogenosomal oxidoreductase complex.

Trichomonas vaginalis is a unicellular microaerophilic eukaryote that lacks mitochondria yet contains an alternative organelle, the hydrogenosome, involved in pyruvate metabolism. Pathways between the two organelles differ substantially: in hydrogenosomes, pyruvate oxidation is catalysed by pyruvate:ferredoxin oxidoreductase (PFOR), with electrons donated to an [Fe]-hydrogenase which produces hydrogen. ATP is generated exclusively by substrate-level phosphorylation in hydrogenosomes, as opposed to oxidative phosphorylation in mitochondria. PFOR and hydrogenase are found in eubacteria and amitochondriate eukaryotes, but not in typical mitochondria. Analyses of mitochondrial genomes indicate that mitochondria have a single endosymbiotic origin from an alpha-proteobacterial-type progenitor. The absence of a genome in trichomonad hydrogenosomes precludes such comparisons, leaving the endosymbiotic history of this organelle unclear. Although phylogenetic reconstructions of a few proteins indicate that trichomonad hydrogenosomes share a common origin with mitochondria, others do not. Here we describe a novel NADH dehydrogenase module of respiratory complex I that is coupled to the central hydrogenosomal fermentative pathway to form a hydrogenosomal oxidoreductase complex that seems to function independently of quinones. Phylogenetic analyses of hydrogenosomal complex I-like proteins Ndh51 and Ndh24 reveal that neither has a common origin with mitochondrial homologues. These studies argue against a vertical origin of trichomonad hydrogenosomes from the proto-mitochondrial endosymbiont.

Expression profiling of the Leishmania life cycle: cDNA arrays identify developmentally regulated genes present but not annotated in the genome.

As genomic sequencing of Leishmania nears completion, functional analyses that provide a global genetic perspective on biological processes are important. Despite polycistronic transcription, RNA transcript abundance can be measured using microarrays. To provide a resource to evaluate cDNA arrays, we undertook 5' expressed sequence tag analysis of 2183 full-length randomly selected cDNAs from Leishmania major promastigote (days 3, 7, 10 of culture in vitro), and lesion-derived amastigote libraries. PCR-amplified inserts from 1830 of these cDNA representing 1001 unique genes were spotted onto microarrays, and compared internally with PCR-amplified open reading frames (ORFs) from 904 genes representing 842 unique genes annotated in the L. major genome. Microarrays were screened with RNA from procyclic, metacyclic and amastigote populations of L. major. Redundant clones on the array gave highly reproducible results, providing confidence in identification of stage-specific gene expression. Four hundred and thirty unique (i.e. non-redundant) stage-specific genes were identified. A higher percentage of stage-specific gene expression was observed in amastigotes ( approximately 35%) compared to metacyclics ( approximately 12%) for both cDNAs and ORFs, but cDNAs provided a richer source of regulated genes than currently annotated ORFs from the Leishmania genome. In mapping cDNAs onto the Leishmania genome, we noted that approximately 42% aligned to regions not recognised as genes using current predictive annotation tools. These genes are highly represented in our stage-specific genes, and therefore represent important drug targets and vaccine candidates. Careful annotation of cDNAs onto the Leishmania genome will be important before producing the next generation of oligonucleotide arrays based on annotated genes of the genomic sequencing project.

Ancient invasions: from endosymbionts to organelles.

The acquisitions of mitochondria and plastids were important events in the evolution of the eukaryotic cell, supplying it with compartmentalized bioenergetic and biosynthetic factories. Ancient invasions by eubacteria through symbiosis more than a billion years ago initiated these processes. Advances in geochemistry, molecular phylogeny, and cell biology have offered insight into complex molecular events that drove the evolution of endosymbionts into contemporary organelles. In losing their autonomy, endosymbionts lost the bulk of their genomes, necessitating the evolution of elaborate mechanisms for organelle biogenesis and metabolite exchange. In the process, symbionts acquired many host-derived properties, lost much of their eubacterial identity, and were transformed into extraordinarily diverse organelles that reveal complex histories that we are only beginning to decipher.

Trichomonas vaginalis Hmp35, a putative pore-forming hydrogenosomal membrane protein, can form a complex in yeast mitochondria.

An abundant integral membrane protein, Hmp35, has been isolated from hydrogenosomes of Trichomonas vaginalis. This protein has no known homologue and exists as a stable 300-kDa complex, termed HMP35, in membranes of the hydrogenosome. By using blue native gel electrophoresis, we found the HMP35 complex to be stable in 2 m NaCl and up to 5 m urea. The endogenous Hmp35 protein was largely protease-resistant. The protein has a predominantly beta-sheet structure and predicted transmembrane domains that may form a pore. Interestingly, the protein has a high number of cysteine residues, some of which are arranged in motifs that resemble the RING finger, suggesting that they could be coordinating zinc or another divalent cation. Our data show that Hmp35 forms one intramolecular but no intermolecular disulfide bonds. We have isolated the HMP35 complex by expressing a His-tagged Hmp35 protein in vivo followed by purification with nickel-agarose beads. The purified 300-kDa complex consists of mostly Hmp35 with lesser amounts of 12-, 25-27-, and 32-kDa proteins. The stoichiometry of proteins in the complex indicates that Hmp35 exists as an oligomer. Hmp35 can be targeted heterologously into yeast mitochondria, despite the lack of homology with any yeast protein, demonstrating the compatibility of mitochondrial and hydrogenosomal protein translocation machineries.

The dynamic dimerization of the yeast ADP/ATP carrier in the inner mitochondrial membrane is affected by conserved cysteine residues.

The ADP/ATP carrier (AAC) that facilitates the translocation of ATP made in mitochondria is inserted at the inner mitochondrial membrane by the TIM10-TIM22 protein import system. Here we addressed the state of the AAC precursor during insertion (stage IV of import) and identified residues of the carrier important for dimerization. By a combination of (i) import of a mix of His-tagged and untagged versions of AAC either 35S-labeled or unlabeled, (ii) import of a tandem covalent dimer AAC into wild-type mitochondria, and (iii) import of monomeric AAC into mitochondria expressing only the tandem covalent dimer AAC, we found that the stage IV intermediate is a monomer, and this stage is probably the rate-limiting step of insertion in the membrane. Subsequent dimerization occurs extremely rapidly (within less than a minute). The incoming monomer dimerizes with monomeric endogenous AAC suggesting that the AAC dimer is very dynamic. Conserved Cys residues were found not to affect insertion significantly, but they are crucial for the dimerization process to obtain a functional carrier.