Breaking the Mucin Barrier: A New Affinity Chromatography-Mass Spectrometry Approach to Unveil Potential Cell Markers and Pathways Altered in Pseudomyxoma Peritonei.

BACKGROUND: Pseudomyxoma peritonei (PMP) is a rare peritoneal mucinous carcinomatosis with largely unknown underlying molecular mechanisms. Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy is the only therapeutic option; however, despite its use, recurrence with a fatal outcome is common. The lack of molecular characterisation of PMP and other mucinous tumours is mainly due to the physicochemical properties of mucin. RESULTS: This manuscript describes the first protocol capable of breaking the mucin barrier and isolating proteins from mucinous tumours. Briefly, mucinous tumour samples were homogenised and subjected to liquid chromatography using two specific columns to reduce mainly glycoproteins, albumins and immunoglobulin G. The protein fractions were then subjected to mass spectrometry analysis and the proteomic profile obtained was analysed using various bioinformatic tools. Thus, we present here the first proteome analysed in PMP and identified a distinct mucin isoform profile in soft compared to hard mucin tumour tissues as well as key biological processes/pathways altered in mucinous tumours. Importantly, this protocol also allowed us to identify MUC13 as a potential tumour cell marker in PMP. CONCLUSIONS: In sum, our results demonstrate that this protein isolation protocol from mucin will have a high impact, allowing the oncology research community to more rapidly advance in the knowledge of PMP and other mucinous neoplasms, as well as develop new and effective therapeutic strategies.

Constructing a de novo transcriptome and a reference proteome for the bivalve Scrobicularia plana: Comparative analysis of different assembly strategies and proteomic analysis.

Scrobicularia plana is a coastal and estuarine bivalve widely used in ecotoxicological studies. However, the underlying molecular mechanisms for S. plana pollutant responses are hardly known due to the lack of molecular databases. Thus, in this study we present a holistic approach to assess a robust reference transcriptome and proteome of this clam. A mixture of control and metal-exposed individuals was used for mRNA isolation. Four sets of high quality filtered preprocessed reads were generated (two quality scores and two sequenced lengths) and assembled with Mira, Ray and Trinity algorithms. The sixty-four generated assemblies were refined, filtered and evaluated for their proteomic quality. Eight assemblies presented top Detonate scores but one was selected due to its compactness and biological representation, which was generated: (i) from the highest quality dataset (Q20L100), (ii) using Trinity algorithm with all k-mers (AtKa), (iii) removing redundancy by CD-HIT (RR80), and (iv) filtering out poor contigs (F), that was subsequently named Q20L100AtKaRR80F. S. plana proteomic analysis revealed 10,017 peptide groups that corresponded to 2066 proteins with a wide coverage of molecular functions and biological processes, confirming the strength of the database generated.

Meta-omic evaluation of bacterial microbial community structure and activity for the environmental assessment of soils: overcoming protein extraction pitfalls.

Microorganisms play unique, essential and integral roles in the biosphere. This work aims to assess the utility of soil's metaomics for environmental diagnosis. Doñana National Park (DNP) was selected as a natural lab since it contains a strictly protected core that is surrounded by numerous threats of pollution. Culture-independent high-throughput molecular tools were used to evaluate the alterations of the global structure and metabolic activities of the microbiome. 16S rRNA sequencing shows lower bacterial abundance and diversity in areas historically exposed to contamination that surround DNP. For metaproteomics, an innovative post-alkaline protein extraction protocol was developed. After NaOH treatment, successive washing with Tris-HCl buffer supplemented with glycerol was essential to eliminate interferences. Starting from soils with different physicochemical characteristics, the method renders proteins with a remarkable resolution on SDS-PAGE gels. The proteins extracted were analysed by using an in-house database constructed from the rRNA data. LC-MS/MS analysis identified 2182 non-redundant proteins with 135 showing significant differences in relative abundance in the soils around DNP. Relevant global biological processes were altered in response to the environmental changes, such as protective and antioxidant mechanisms, translation, folding and homeostasis of proteins, membrane transport and aerobic respiratory metabolism.

Overlapping responses between salt and oxidative stress in Debaryomyces hansenii.

Debaryomyces hansenii is a halotolerant yeast of importance in basic and applied research. Previous reports hinted about possible links between saline and oxidative stress responses in this yeast. The aim of this work was to study that hypothesis at different molecular levels, investigating after oxidative and saline stress: (i) transcription of seven genes related to oxidative and/or saline responses, (ii) activity of two main anti-oxidative enzymes, (iii) existence of common metabolic intermediates, and (iv) generation of damages to biomolecules as lipids and proteins. Our results showed how expression of genes related to oxidative stress was induced by exposure to NaCl and KCl, and, vice versa, transcription of some genes related to osmotic/salt stress responses was regulated by H2O2. Moreover, and contrary to S. cerevisiae, in D. hansenii HOG1 and MSN2 genes were modulated by stress at their transcriptional level. At the enzymatic level, saline stress also induced antioxidative enzymatic defenses as catalase and glutathione reductase. Furthermore, we demonstrated that both stresses are connected by the generation of intracellular ROS, and that hydrogen peroxide can affect the accumulation of in-cell sodium. On the other hand, no significant alterations in lipid oxidation or total glutathione content were observed upon exposure to both stresses tested. The results described in this work could help to understand the responses to both stressors, and to improve the biotechnological potential of D. hansenni.

Trk1, the sole potassium-specific transporter in Candida glabrata, contributes to the proper functioning of various cell processes.

Candida glabrata is a haploid yeast that is considered to be an emergent pathogen since it is the second most prevalent cause of candidiasis. Contrary to most yeasts, this species carries only one plasma membrane potassium transporter named CgTrk1. We show in this work that the activity of this transporter is regulated at the posttranslational level, and thus Trk1 contributes to potassium uptake under very different external cation concentrations. In addition to its function in potassium uptake, we report a diversity of physiological effects related to this transporter. CgTRK1 contributes to proper cell size, intracellular pH and membrane-potential homeostasis when expressed in Saccharomyces cerevisiae. Moreover, lithium influx experiments performed both in C. glabrata and S. cerevisiae indicate that the salt tolerance phenotype linked to CgTrk1 can be related to a high capacity to discriminate between potassium and lithium (or sodium) during the transport process. In summary, we show that CgTRK1 exerts a diversity of pleiotropic physiological roles and we propose that the corresponding protein may be an attractive pharmacological target for the development of new antifungal drugs.

The halotolerant Debaryomyces hansenii, the Cinderella of non-conventional yeasts.

Debaryomyces hansenii is a halotolerant yeast with a high biotechnological potential, particularly in the food industry. However, research in this yeast is limited by its molecular peculiarities. In this review we summarize the state of the art of research in this microorganisms, describing both pros and cons. We discuss (i) its halotolerance, (ii) the molecular factors involved in saline and osmotic stress, (iii) its high gene density and ambiguous CUG decoding, and (iv) its biotechnological and medical interests. We trust that all the bottlenecks in its study will soon be overcome and D. hansenii will become a fundamental organism for food biotechnological processes. Copyright © 2016 John Wiley & Sons, Ltd.

Are there any completely sterile organs or tissues in the human body? Is there any sacred place?

The human microbiome comprises an ample set of organisms that inhabit and interact within the human body, contributing both positively and negatively to our health. In recent years, several research groups have described the presence of microorganisms in organs or tissues traditionally considered as 'sterile' under healthy and pathological conditions. In this sense, microorganisms have been detected in several types of cancer, including those in 'sterile' organs. But how can the presence of microorganisms be detected? In most studies, 16S and internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing has led to the identification of prokaryotes and fungi. However, a major limitation of this technique is that it cannot distinguish between living and dead organisms. RNA-based methods have been proposed to overcome this limitation, as the shorter half-life of the RNA would identify only the transcriptionally active microorganisms, although perhaps not all the viable ones. In this sense, metaproteomic techniques or the search for molecular metabolic signatures could be interesting alternatives for the identification of living microorganisms. In summary, new technological advances are challenging the notion of 'sterile' organs in our body. However, to date, evidence for a structured living microbiome in most of these organs is scarce or non-existent. The implementation of new technological approaches will be necessary to fully understand the importance of the microbiome in these organs, which could pave the way for the development of a wide range of new therapeutic strategies.

Ions and nanoparticles of Ag and/or Cd metals in a model aquatic microcosm: Effects on the abundance, diversity and functionality of the sediment bacteriome.

Metals can be adsorbed on particulate matter, settle in sediments and cause alterations in aquatic environments. This study assesses the effect of Ag and/or Cd, both in ionic and nanoparticle (NP) forms, on the microbiome of sediments. For that purpose, aquatic controlled-microcosm experiments were exposed to an environmentally relevant and at tenfold higher doses of each form of the metals. Changes in the bacteriome were inferred by 16S rDNA sequencing. Ionic Ag caused a significant decrease of several bacterial families, whereas the effect was opposite when mixed with Cd, e.g., Desulfuromonadaceae family; in both cases, the bacteriome functionalities were greatly affected, particularly the nitrogen and sulfur metabolism. Compared to ionic forms, metallic NPs produced hardly any change in the abundance of microbial families, although the α-biodiversity of the bacteriome was reduced, and the functionality altered, when exposed to the NPs´ mixture. Our goal is to understand how metals, in different forms and combinations, released into the environment may endanger the health of aquatic ecosystems. This work may help to understand how aquatic metal pollution alters the structure and functionality of the microbiome and biogeochemical cycles, and how these changes can be addressed.

A concept for international societally relevant microbiology education and microbiology knowledge promulgation in society.

EXECUTIVE SUMMARY: Microbes are all pervasive in their distribution and influence on the functioning and well-being of humans, life in general and the planet. Microbially-based technologies contribute hugely to the supply of important goods and services we depend upon, such as the provision of food, medicines and clean water. They also offer mechanisms and strategies to mitigate and solve a wide range of problems and crises facing humanity at all levels, including those encapsulated in the sustainable development goals (SDGs) formulated by the United Nations. For example, microbial technologies can contribute in multiple ways to decarbonisation and hence confronting global warming, provide sanitation and clean water to the billions of people lacking them, improve soil fertility and hence food production and develop vaccines and other medicines to reduce and in some cases eliminate deadly infections. They are the foundation of biotechnology, an increasingly important and growing business sector and source of employment, and the centre of the bioeconomy, Green Deal, etc. But, because microbes are largely invisible, they are not familiar to most people, so opportunities they offer to effectively prevent and solve problems are often missed by decision-makers, with the negative consequences this entrains. To correct this lack of vital knowledge, the International Microbiology Literacy Initiative-the IMiLI-is recruiting from the global microbiology community and making freely available, teaching resources for a curriculum in societally relevant microbiology that can be used at all levels of learning. Its goal is the development of a society that is literate in relevant microbiology and, as a consequence, able to take full advantage of the potential of microbes and minimise the consequences of their negative activities. In addition to teaching about microbes, almost every lesson discusses the influence they have on sustainability and the SDGs and their ability to solve pressing problems of societal inequalities. The curriculum thus teaches about sustainability, societal needs and global citizenship. The lessons also reveal the impacts microbes and their activities have on our daily lives at the personal, family, community, national and global levels and their relevance for decisions at all levels. And, because effective, evidence-based decisions require not only relevant information but also critical and systems thinking, the resources also teach about these key generic aspects of deliberation. The IMiLI teaching resources are learner-centric, not academic microbiology-centric and deal with the microbiology of everyday issues. These span topics as diverse as owning and caring for a companion animal, the vast range of everyday foods that are produced via microbial processes, impressive geological formations created by microbes, childhood illnesses and how they are managed and how to reduce waste and pollution. They also leverage the exceptional excitement of exploration and discovery that typifies much progress in microbiology to capture the interest, inspire and motivate educators and learners alike. The IMiLI is establishing Regional Centres to translate the teaching resources into regional languages and adapt them to regional cultures, and to promote their use and assist educators employing them. Two of these are now operational. The Regional Centres constitute the interface between resource creators and educators-learners. As such, they will collect and analyse feedback from the end-users and transmit this to the resource creators so that teaching materials can be improved and refined, and new resources added in response to demand: educators and learners will thereby be directly involved in evolution of the teaching resources. The interactions between educators-learners and resource creators mediated by the Regional Centres will establish dynamic and synergistic relationships-a global societally relevant microbiology education ecosystem-in which creators also become learners, teaching resources are optimised and all players/stakeholders are empowered and their motivation increased. The IMiLI concept thus embraces the principle of teaching societally relevant microbiology embedded in the wider context of societal, biosphere and planetary needs, inequalities, the range of crises that confront us and the need for improved decisioning, which should ultimately lead to better citizenship and a humanity that is more sustainable and resilient. ABSTRACT: The biosphere of planet Earth is a microbial world: a vast reactor of countless microbially driven chemical transformations and energy transfers that push and pull many planetary geochemical processes, including the cycling of the elements of life, mitigate or amplify climate change (e.g., Nature Reviews Microbiology, 2019, 17, 569) and impact the well-being and activities of all organisms, including humans. Microbes are both our ancestors and creators of the planetary chemistry that allowed us to evolve (e.g., Life's engines: How microbes made earth habitable, 2023). To understand how the biosphere functions, how humans can influence its development and live more sustainably with the other organisms sharing it, we need to understand the microbes. In a recent editorial (Environmental Microbiology, 2019, 21, 1513), we advocated for improved microbiology literacy in society. Our concept of microbiology literacy is not based on knowledge of the academic subject of microbiology, with its multitude of component topics, plus the growing number of additional topics from other disciplines that become vitally important elements of current microbiology. Rather it is focused on microbial activities that impact us-individuals/communities/nations/the human world-and the biosphere and that are key to reaching informed decisions on a multitude of issues that regularly confront us, ranging from personal issues to crises of global importance. In other words, it is knowledge and understanding essential for adulthood and the transition to it, knowledge and understanding that must be acquired early in life in school. The 2019 Editorial marked the launch of the International Microbiology Literacy Initiative, the IMiLI. HERE, WE PRESENT: our concept of how microbiology literacy may be achieved and the rationale underpinning it; the type of teaching resources being created to realise the concept and the framing of microbial activities treated in these resources in the context of sustainability, societal needs and responsibilities and decision-making; and the key role of Regional Centres that will translate the teaching resources into local languages, adapt them according to local cultural needs, interface with regional educators and develop and serve as hubs of microbiology literacy education networks. The topics featuring in teaching resources are learner-centric and have been selected for their inherent relevance, interest and ability to excite and engage. Importantly, the resources coherently integrate and emphasise the overarching issues of sustainability, stewardship and critical thinking and the pervasive interdependencies of processes. More broadly, the concept emphasises how the multifarious applications of microbial activities can be leveraged to promote human/animal, plant, environmental and planetary health, improve social equity, alleviate humanitarian deficits and causes of conflicts among peoples and increase understanding between peoples (Microbial Biotechnology, 2023, 16(6), 1091-1111). Importantly, although the primary target of the freely available (CC BY-NC 4.0) IMiLI teaching resources is schoolchildren and their educators, they and the teaching philosophy are intended for all ages, abilities and cultural spectra of learners worldwide: in university education, lifelong learning, curiosity-driven, web-based knowledge acquisition and public outreach. The IMiLI teaching resources aim to promote development of a global microbiology education ecosystem that democratises microbiology knowledge.

Salt and oxidative stress tolerance in Debaryomyces hansenii and Debaryomyces fabryi.

We report the characterization of five strains belonging to the halotolerant highly related Debaryomyces hansenii/fabryi species. The analysis performed consisted in studying tolerance properties, membrane characteristics, and cation incell amounts. We have specifically investigated (1) tolerance to different chemicals, (2) tolerance to osmotic and salt stress, (3) tolerance and response to oxidative stress, (4) reactive oxygen species (ROS) content, (5) relative membrane potential, (6) cell volume, (7) K(+) and Na(+) ion content, and (8) membrane fluidity. Unexpectedly, no direct relationship was found between one particular strain, Na(+) content and its tolerance to NaCl or between its ROS content and its tolerance to H(2)O(2). Results show that, although in general, human origin D. fabryi strains were more resistant to oxidative stress and presented shorter doubling times and smaller cell volume than food isolated D. hansenii ones, strains belonging to the same species can be significantly different. Debaryomyces fabryi CBS1793 strain highlighted for its extremely tolerant behavior when exposed to the diverse stress factors studied.

Intraoperative oxygen tension and redox homeostasis in Pseudomyxoma peritonei: A short case series.

Introduction: Pseudomyxoma peritonei (PMP) is a rare malignant disease characterized by a massive multifocal accumulation of mucin within the peritoneal cavity. The current treatment option is based on complete cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy. However, the recurrence is frequent with subsequent progression and death. To date, most of the studies published in PMP are related to histological and genomic analyses. Thus, the need for further studies unveiling the underlying PMP molecular mechanisms is urgent. In this regard, hypoxia and oxidative stress have been extensively related to tumoral pathologies, although their contribution to PMP has not been elucidated. Methods: In this manuscript, we have evaluated, for the first time, the intratumoral real-time oxygen microtension (pO2mt) in the tumor (soft and hard mucin) and surrounding healthy tissue from five PMP patients during surgery. In addition, we measured hypoxia (Hypoxia Inducible Factor-1a; HIF-1α) and oxidative stress (catalase; CAT) markers in soft and hard mucin from the same five PMP patient samples and in five control samples. Results: The results showed low intratumoral oxygen levels, which were associated with increased HIF-1α protein levels, suggesting the presence of a hypoxic environment in these tumors. We also found a significant reduction in CAT activity levels in soft and hard mucin compared with healthy tissue samples. Discussion: In conclusion, our study provides the first evidence of low intratumoral oxygen levels in PMP patients associated with hypoxia and oxidative stress markers. However, further investigation is required to understand the potential role of oxidative stress in PMP in order to find new therapeutic strategies.

Antitumor effect of a small-molecule inhibitor of KRASG12D in xenograft models of mucinous appendicular neoplasms.

Pseudomyxoma peritonei (PMP) is a rare disease characterized by a massive accumulation of mucus in the peritoneal cavity. The only effective treatment is aggressive surgery, aimed at removing all visible tumors. However, a high percentage of patients relapse, with subsequent progression and death. Recently, there has been an increase in therapies that target mutated oncogenic proteins. In this sense, KRAS has been reported to be highly mutated in PMP, with KRASG12D being the most common subtype. Here, we tested the efficacy of a small-molecule KRASG12D inhibitor, MRTX1133, in a high-grade PMP xenograft mouse model carrying a KRASG12D mutation. The results obtained in this work showed a profound inhibition of tumor growth, which was associated with a reduction in cell proliferation, an increase in apoptosis, and a reduction in the MAPK and PI3K/AKT/mTOR signaling pathways. In conclusion, these results demonstrate the high potency and efficacy of MRTX1133 in KRASG12D-PMP tumors and provide a rationale for clinical trials.

Monitoring COVID-19 through SARS-CoV-2 quantification in wastewater: progress, challenges and prospects.

Wastewater-Based Epidemiology (WBE) is widely used to monitor the progression of the current SARS-CoV-2 pandemic at local levels. In this review, we address the different approaches to the steps needed for this surveillance: sampling wastewaters (WWs), concentrating the virus from the samples and quantifying them by qPCR, focusing on the main limitations of the methodologies used. Factors that can influence SARS-CoV-2 monitoring in WWs include: (i) physical parameters as temperature that can hamper the detection in warm seasons and tropical regions, (ii) sampling methodologies and timetables, being composite samples and Moore swabs the less variable and more sensitive approaches, (iii) virus concentration methodologies that need to be feasible and practicable in simpler laboratories and (iv) detection methodologies that should tend to use faster and cost-effective procedures. The efficiency of WW treatments and the use of WWs for SARS-CoV-2 variants detection are also addressed. Furthermore, we discuss the need for the development of common standardized protocols, although these must be versatile enough to comprise variations among target communities. WBE screening of risk populations will allow for the prediction of future outbreaks, thus alerting authorities to implement early action measurements.

Induction of Pseudomonas syringae pv. tomato DC3000 MexAB-OprM multidrug efflux pump by flavonoids is mediated by the repressor PmeR.

In this study, we have analyzed the expression of the Pseudomonas syringae pv. tomato DC3000 mexAB-oprM efflux pump operon and of the regulatory gene pmeR, and we have investigated the role of the PmeR protein on transcription from both promoters. We demonstrate that mexAB-oprM and pmeR are expressed in vivo at a relatively high and moderate basal level, respectively, which, in both cases, increases in the presence of different flavonoids and other compounds, such as butyl and methylparaben. We show that PmeR is the local repressor of the mexAB-oprM promoter and is able to regulate its own expression. The mechanism for this regulation includes binding to a pseudopalindromic operator site which overlaps both mexAB-oprM and pmeR promoters. We have also proven that flavonoids are able to interact with PmeR and induce a conformational change that interferes with the DNA binding ability of PmeR, thereby modulating mexAB-oprM and pmeR expression. Finally, we demonstrate by in vivo experiments that the PmeR/MexAB-OprM system contributes to the colonization of tomato plants. These results provide new insight into a transcriptional regulator and a transport system that play essential roles in the ability of P. syringae pv. tomato DC3000 to resist the action of flavonoids produced by the host.

High-throughput molecular analyses of microbiomes as a tool to monitor the wellbeing of aquatic environments.

Aquatic environments are the recipients of many sources of environmental stress that trigger both local and global changes. To evaluate the associated risks to organisms and ecosystems more sensitive and accurate strategies are required. The analysis of the microbiome is one of the most promising candidates for environmental diagnosis of aquatic systems. Culture-independent interconnected meta-omic approaches are being increasing used to fill the gaps that classical microbial approaches cannot resolve. Here, we provide a prospective view of the increasing application of these high-throughput molecular technologies to evaluate the structure and functional activity of microbial communities in response to changes and disturbances in the environment, mostly of anthropogenic origin. Some relevant topics are reviewed, such as: (i) the use of microorganisms for water quality assessment, highlighting the incidence of antimicrobial resistance as an increasingly serious threat to global public health; (ii) the crucial role of microorganisms and their complex relationships with the ongoing climate change, and other stress threats; (iii) the responses of the environmental microbiome to extreme pollution conditions, such as acid mine drainage or oil spills. Moreover, protists and viruses, due to their huge impacts on the structure of microbial communities, are emerging candidates for the assessment of aquatic environmental health.

The Potassium Transporter Hak1 in Candida Albicans, Regulation and Physiological Effects at Limiting Potassium and under Acidic Conditions.

The three families of yeast plasma membrane potassium influx transporters are represented in Candida albicans: Trk, Acu, and Hak proteins. Hak transporters work as K+-H+ symporters, and the genes coding for Hak proteins are transcriptionally activated under potassium limitation. This work shows that C. albicans mutant cells lacking CaHAK1 display a severe growth impairment at limiting potassium concentrations under acidic conditions. This is the consequence of a defective capacity to transport K+, as indicated by potassium absorption experiments and by the kinetics parameters of Rb+ (K+) transport. Moreover, hak1- cells are more sensitive to the toxic cation lithium. All these phenotypes became much less robust or even disappeared at alkaline growth conditions. Finally, transcriptional studies demonstrate that the hak1- mutant, in comparison with HAK1+ cells, activates the expression of the K+/Na+ ATPase coded by CaACU1 in the presence of Na+ or in the absence of K+.

Regulation and activity of CaTrk1, CaAcu1 and CaHak1, the three plasma membrane potassium transporters in Candida albicans.

Wild-type cells of Candida albicans, the most common human fungal pathogen, are able to grow at very low micromolar concentrations of potassium in the external milieu. One of the reasons behind that behaviour is the existence of three different types of K+ transporters in their plasma membrane: Trk1, Acu1 and Hak1. This work shows that the transporters are very differently regulated at the transcriptional level upon exposure to saline stress, pH alterations or K+ starvation. We propose that different transporters take the lead in the diverse environmental conditions, Trk1 being the "house-keeping" one, and Acu1/Hak1 dominating upon K+ limiting conditions. Heterologous expression of the genes coding for the three transporters in a Saccharomyces cerevisiae strain lacking its endogenous potassium transporters showed that all of them mediated cation transport but with very different efficiencies. Moreover, expression of the transporters in S. cerevisiae also affected other physiological characteristics such as sodium and lithium tolerance, membrane potential or intracellular pH, being, in general, CaTrk1 the most effective in keeping these parameters close to the usual wild-type physiological levels.

Bacteria, archae, fungi and viruses: it takes a community to eliminate waste.

Importance of the microbiota communities for improving the efficiency in Waste Water Treatment Plants.

Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons.

Coastal transitional waters are exposed to many anthropogenic threats. This study aims to assess the trace metals' pollution status of transitional waters by evaluating its biological effects in the clam Venerupis decussata. Among the studied sites along the Tunisian littoral, South Tunis and Boughrara were the most impacted, since clams from these two lagoons presented significant differences in: (i) trace metal contents, (ii) in-cell hydrogen peroxide, (iii) enzymatic and non-enzymatic defenses, (iv) damage to lipids and proteins, and (v) protein post-translational modifications. These changes related to evident histopathological traits. PCA showed a clear separation between the digestive gland and gills tissues and illustrated an impact gradient in Tunisian coastal lagoons. Water temperature was revealed as an added natural stressor that, when concurring with high pollution, may jeopardize an ecosystem's health and contribute to the accumulation of hazardous metals in organisms.