Heavy metal whole-cell biosensors using eukaryotic microorganisms: An updated critical review

This review analyzes the advantages and disadvantages of using eukaryotic microorganisms to design whole-cell biosensors (WCBs) for monitoring environmental heavy metal pollution in soil or aquatic habitats. Basic considerations for designing a eukaryotic WCB are also shown. A comparative analysis of the promoter genes used to design WCBs is carried out, and the sensitivity and reproducibility of the main reporter genes used is also reviewed. Three main eukaryotic taxonomic groups are considered: yeasts, microalgae, and ciliated protozoa. Models that have been widely analyzed as potential WCBs are the Saccharomyces cerevisiae model among yeasts, the Tetrahymena thermophila model for ciliates and Chlamydomonas model for microalgae. The advantages and disadvantages of each microbial group are discussed, and a ranking of sensitivity to the same type of metal pollutant from reported eukaryotic WCBs is also shown. General conclusions and possible future developments of eukaryotic WCBs are reported

Environmental Biosensors: A Microbiological View

In this mini-review, the potential of using microorganisms to design biosensors for detecting environmental pollutants is analyzed and discussed. A distinction is made between a classical biosensor (CB) and a whole-cell biosensor (WCB), emphasizing their structural components and the possibility of using whole microorganisms as their bioreceptor elements. The advantages and disadvantages of using prokaryotic microorganisms as opposed to eukaryotic microorganisms are described. Likewise, the advantages of using protozoa ciliates) over other eukaryotic microorganisms are also shown. We analyze the current bibliography on biosensors built on microorganisms as bioreceptors of pollutant molecules, such as inorganic (metal (loid)s) or organic (xenobiotics). New trends, such as the prokaryotic riboswitches, microbial two-component systems where the pollutant can be simultaneously detected and bioremediated, along with advances in synthetic biology, are shown as promising tools in the design of environmental biosensors

Hints for Metal-Preference Protein Sequence Determinants: Different Metal Binding Features of the Five Tetrahymena thermophila Metallothioneins

The metal binding preference of metallothioneins (MTs) groups them in two extreme subsets, the Zn/Cd- and the Cu-thioneins. Ciliates harbor the largest MT gene/protein family reported so far, in-cluding 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tet-rahymena thermophila, three MTs (MTT1, MTT3 and MTT5) were considered Cd-thioneins and two (MTT2 and MTT4) Cu-thioneins, according to gene expression inducibility and phylogenetic analysis. In this study, the metal-binding abilities of the five MTT proteins were characterized, to obtain information about the folding and stability of their cognate- and non-cognate metal complexes, and to characterize the T. thermophila MT system at protein level. Hence, the five MTTs were recombinantly synthesized as Zn2+-, Cd2+- or Cu+-complexes, which were analyzed by electrospray mass spectrometry (ESI-MS), circular dichroism (CD), and UV-vis spectrophotometry. Among the Cd-thioneins, MTT1 and MTT5 were optimal for Cd2+ coordination, yielding unique Cd17- and Cd8- complexes, respectively. When binding Zn2+, they rendered a mixture of Zn-species. Only MTT5 was capable to coordinate Cu+, although yielding heteronuclear Zn-, Cu-species or highly unstable Cu-homometallic species. MTT3 exhibited poor binding abilities both for Cd2+ and for Cu+, and although not optimally, it yielded the best result when coordinating Zn2+. The two Cu-thioneins, MTT2 and MTT4 isoforms formed homometallic Cu-complexes (major Cu20-MTT) upon synthesis in Cu-supplemented hosts. Contrarily, they were unable to fold into stable Cd-complexes, while Zn-MTT species were only recovered for MTT4 (major Zn10-MTT4). Thus, the metal binding preferences of the five T. thermophila MTs correlate well with their previous classification as Cd- and Cu-thioneins, and globally, they can be classified from Zn/Cd- to Cu-thioneins according to the gradation: MTT1>MTT5>MTT3>MTT4>MTT2. The main mechanisms underlying the evolution and specialization of the MTT metal binding preferences may have been in-ternal tandem duplications, presence of doublet and triplet Cys patterns in Zn/Cd-thioneins, and op-timization of site specific amino acid determinants (Lys for Zn/Cd- and Asn for Cu-coordination)

Autophagy and lipid droplets are a defense mechanism against toxic copper oxide nanotubes in the eukaryotic microbial model Tetrahymena thermophila

The widespread use of inorganic nanomaterials of anthropogenic origin has significantly increased in the last decade, being now considered as emerging pollutants. This makes it necessary to carry out studies to further understand their toxicity and interactions with cells. In the present work we analyzed the toxicity of CuO nanotubes (CuONT) in the ciliate Tetrahymena thermophila, a eukaryotic unicellular model with animal biology. CuONT exposure rapidly induced ROS generation in the cell leading to oxidative stress and upregulation of genes encoding antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase), metal-chelating metallothioneins and cytochrome P450 monooxygenases. Comet assays and overexpression of genes involved in DNA repair confirmed oxidative DNA damage in CuONT-treated cells. Remarkably, both electron and fluorescent microscopy revealed numerous lipid droplets and autophagosomes containing CuONT aggregates and damaged mitochondria, indicating activation of macroautophagy, which was further confirmed by a dramatic upregulation of ATG (AuTophaGy related) genes. Treatment with autophagy inhibitors significantly increased CuONT toxicity, evidencing the protective role of autophagy towards CuONTinduced damage. Moreover, increased formation of lipid droplets appears as an additional mechanism of CuONT detoxification. Based on these results, we present a hypothetical scenario summarizing how T. thermophila responds to CuONT toxicity. This study corroborates the use of this ciliate as an excellent eukaryotic microbial model for analyzing the cellular response to stress caused by toxic metal nanoparticles

Arsenate and arsenite differential toxicity in Tetrahymena thermophila

A comparative analysis of toxicities of both arsenic forms (arsenite and arsenate) in the model eukaryotic microorganism Tetrahymena thermophila (ciliate protozoa) has shown the presence of various detoxification mechanisms and cellular effects comparable to those of animal cells under arsenic stress. In the wild type strain SB1969 arsenate is almost 2.5 times more toxic than arsenite. According to the concentration addition model used in binary metallic mixtures their toxicities show an additive effect. Using fluorescent assays and flow cytometry, it has been detected that As(V) generates elevated levels of ROS/RNS compared to As(III). Both produce the same levels of superoxide anion, but As(V) also causes greater increases in hydrogen peroxide and peroxynitrite. The mitochondrial membrane potential is affected by both As(V) and As(III), and electron microscopy has also revealed that mitochondria are the main target of both arsenic ionic forms. Fusion/fission and swelling mitochondrial and mitophagy, together with macroautophagy, vacuolization and mucocyst extruction are mainly associated to As(V) toxicity, while As(III) induces an extensive lipid metabolism dysfunction (adipotropic effect). Quantitative RT-PCR analysis of some genes encoding antioxidant proteins or enzymes has shown that glutathione and thioredoxin metabolisms are involved in the response to arsenic stress. Likewise, the function of metallothioneins seems to be crucial in arsenic detoxification processes, after using both metallothionein knockout and knockdown strains and cells overexpressing metallothionein genes from this ciliate. The analysis of the differential toxicity of As(III) and As(V) shown in this study provides cytological and molecular tools to be used as biomarkers for each of the two arsenic ionic forms

Effectiveness of Thrombectomy in Stroke According to Baseline Prognostic Factors: Inverse Probability of Treatment Weighting Analysis of a Population-Based Registry

Background and Purpose In real-world practice, the benefit of mechanical thrombectomy (MT) is uncertain in stroke patients with very favorable or poor prognostic profiles at baseline. We studied the effectiveness of MT versus medical treatment stratifying by different baseline prognostic factors. Methods Retrospective analysis of 2,588 patients with an ischemic stroke due to large vessel occlusion nested in the population-based registry of stroke code activations in Catalonia from January 2017 to June 2019. The effect of MT on good functional outcome (modified Rankin Score 85 years, National Institutes of Health Stroke Scale [NIHSS] >25, time from onset >6 hours, Alberta Stroke Program Early CT Score 3), good (if NIHSS <6 or distal occlusion, in the absence of poor prognostic factors), or reference (not meeting other groups' criteria). Results Patients receiving MT (n=1,996, 77%) were younger, had less pre-stroke disability, and received systemic thrombolysis less frequently. These differences were balanced after the IPTW stratified by prognosis. MT was associated with good functional outcome in the reference (odds ratio [OR], 2.9; 95% confidence interval [CI], 2.0 to 4.4), and especially in the poor baseline prognostic stratum (OR, 3.9; 95% CI, 2.6 to 5.9), but not in the good prognostic stratum. MT was associated with survival only in the poor prognostic stratum (OR, 2.6; 95% CI, 2.0 to 3.3). Conclusions Despite their worse overall outcomes, the impact of thrombectomy over medical management was more substantial in patients with poorer baseline prognostic factors than patients with good prognostic factors

Integrated genomic characterization of oesophageal carcinoma

Oesophageal cancers are prominent worldwide; however, there are few targeted therapies and survival rates for these cancers remain dismal. Here we performed a comprehensive molecular analysis of 164 carcinomas of the oesophagus derived from Western and Eastern populations. Beyond known histopathological and epidemiologic distinctions, molecular features differentiated oesophageal squamous cell carcinomas from oesophageal adenocarcinomas. Oesophageal squamous cell carcinomas resembled squamous carcinomas of other organs more than they did oesophageal adenocarcinomas. Our analyses identified three molecular subclasses of oesophageal squamous cell carcinomas, but none showed evidence for an aetiological role of human papillomavirus. Squamous cell carcinomas showed frequent genomic amplifications of CCND1 and SOX2 and/or TP63, whereas ERBB2, VEGFA and GATA4 and GATA6 were more commonly amplified in adenocarcinomas. Oesophageal adenocarcinomas strongly resembled the chromosomally unstable variant of gastric adenocarcinoma, suggesting that these cancers could be considered a single disease entity. However, some molecular features, including DNA hypermethylation, occurred disproportionally in oesophageal adenocarcinomas. These data provide a framework to facilitate more rational categorization of these tumours and a foundation for new therapies