Goat milk extracellular vesicles: immuno-modulation effects on porcine monocyte-derived macrophages in vitro.

Introduction: Extracellular vesicles (EVs) are nanometric-membrane-bound sub-cellular structures, which can be recovered from milk. Milk EVs have drawn increasing interest due to their potential biomedical applications, therefore it is important to investigate their impact on key immune cells, such as macrophages. Methods: In this work, the immunomodulatory effects of goat milk EVs on untreated (moMФ) and classically activated (moM1) porcine monocyte-derived macrophages were investigated using flow cytometry, ELISA, and gene expression assays. Results: These particles were efficiently internalized by macrophages and high doses (60 mg protein weight) triggered the upregulation of MHC I and MHC II DR on moMФ, but not on moM1. In moMФ, exposure to low doses (0.6 mg) of mEVs enhanced the gene expression of IL10, EBI3, and IFNB, whereas high doses up-regulated several pro-inflammatory cytokines. These nanosized structures slightly modulated cytokine gene expression on moM1. Accordingly, the cytokine (protein) contents in culture supernatants of moMФ were mildly affected by exposure to low doses of mEVs, whereas high doses promoted the increased release of TNF, IL-8, IL-1a, IL-1b, IL-1Ra, IL-6, IL-10, and IL-12. The cytokines content in moM1 supernatants was not critically affected. Discussion: Overall, our data support a clinical application of these molecules: they polarized macrophages toward an M1-like phenotype, but this activation seemed to be controlled, to prevent potentially pathological over-reaction to stressors.

Corrigendum: Goat milk extracellular vesicles: immuno-modulation effects on porcine monocyte-derived macrophages in vitro.

[This corrects the article DOI: 10.3389/fimmu.2023.1209898.].

Genetic Characterization of a Novel Equus caballus Papillomavirus Isolated from a Thoroughbred Mare.

Papillomaviruses (PVs) are small, non-enveloped viruses, ubiquitous across the animal kingdom. PVs induce diverse forms of infection, such as cutaneous papillomas, genital papillomatosis, and carcinomas. During a survey on the fertility status of a mare, a novel Equus caballus PV (EcPV) has been identified using Next Generation Sequencing, and it was further confirmed with genome-walking PCR and Sanger sequencing. The complete circular genome 7607 bp long shares 67% average percentage of identity with EcPV9, EcPV2, EcPV1, and EcPV6, justifying a new classification as Equus caballus PV 10 (EcPV10). All EcPV genes are conserved in EcPV10, and phylogenetic analysis indicates that EcPV10 is closely related to EcPV9 and EcPV2, genus Dyoiota 1. A preliminary EcPV10 genoprevalence study, carried out on 216 horses using Real Time PCRs, suggested a low incidence of this isolate (3.7%) compared to EcPVs of the same genus such as EcPV2 and EcPV9 in the same horse population. We hypothesize a transmission mechanism different from the one observed in the closely related EcPV9 and EcPV2 that particularly infect Thoroughbreds. This horse breed is usually submitted to natural mating, thus indicating a possible sexual diffusion. No differences were detected for breeds in terms of susceptibility to EcPV10. Further studies are needed to investigate the molecular mechanisms behind the host and EcPV10 infection to explain the reduced viral spread.

Heterogeneity of Phenotypic and Functional Changes to Porcine Monocyte-Derived Macrophages Triggered by Diverse Polarizing Factors In Vitro.

Swine are attracting increasing attention as a biomedical model, due to many immunological similarities with humans. However, porcine macrophage polarization has not been extensively analyzed. Therefore, we investigated porcine monocyte-derived macrophages (moMΦ) triggered by either IFN-γ + LPS (classical activation) or by diverse "M2-related" polarizing factors: IL-4, IL-10, TGF-ß, and dexamethasone. IFN-γ and LPS polarized moMΦ toward a proinflammatory phenotype, although a significant IL-1Ra response was observed. Exposure to IL-4, IL-10, TGF-ß, and dexamethasone gave rise to four distinct phenotypes, all antithetic to IFN-γ and LPS. Some peculiarities were observed: IL-4 and IL-10 both enhanced expression of IL-18, and none of the "M2-related" stimuli induced IL-10 expression. Exposures to TGF-ß and dexamethasone were characterized by enhanced levels of TGF-ß2, whereas stimulation with dexamethasone, but not TGF-ß2, triggered CD163 upregulation and induction of CCL23. Macrophages stimulated with IL-10, TGF-ß, or dexamethasone presented decreased abilities to release proinflammatory cytokines in response to TLR2 or TLR3 ligands: IL-10 showed a powerful inhibitory activity for CXCL8 and TNF release, whereas TGF-ß provided a strong inhibitory signal for IL-6 production. While our results emphasized porcine macrophage plasticity broadly comparable to human and murine macrophages, they also highlighted some peculiarities in this species.

Microbiome enrichment from contaminated marine sediments unveils novel bacterial strains for petroleum hydrocarbon and heavy metal bioremediation.

Petroleum hydrocarbons and heavy metals are some of the most widespread contaminants affecting marine ecosystems, urgently needing effective and sustainable remediation solutions. Microbial-based bioremediation is gaining increasing interest as an effective, economically and environmentally sustainable strategy. Here, we hypothesized that the heavily polluted coastal area facing the Sarno River mouth, which discharges >3 tons of polycyclic aromatic hydrocarbons (PAHs) and ∼15 tons of heavy metals (HMs) into the sea annually, hosts unique microbiomes including marine bacteria useful for PAHs and HMs bioremediation. We thus enriched the microbiome of marine sediments, contextually selecting for HM-resistant bacteria. The enriched mixed bacterial culture was subjected to whole-DNA sequencing, metagenome-assembled-genomes (MAGs) annotation, and further sub-culturing to obtain the major bacterial species as pure strains. We obtained two novel isolates corresponding to the two most abundant MAGs (Alcanivorax xenomutans strain-SRM1 and Halomonas alkaliantarctica strain-SRM2), and tested their ability to degrade PAHs and remove HMs. Both strains exhibited high PAHs degradation (60-100%) and HMs removal (21-100%) yield, and we described in detail >60 genes in their MAGs to unveil the possible genetic basis for such abilities. Most promising yields (∼100%) were obtained towards naphthalene, pyrene and lead. We propose these novel bacterial strains and related genetic repertoire to be further exploited for effective bioremediation of marine environments contaminated with both PAHs and HMs.

Novel Insights on Pyoverdine: From Biosynthesis to Biotechnological Application.

Pyoverdines (PVDs) are a class of siderophores produced mostly by members of the genus Pseudomonas. Their primary function is to accumulate, mobilize, and transport iron necessary for cell metabolism. Moreover, PVDs also play a crucial role in microbes' survival by mediating biofilm formation and virulence. In this review, we reorganize the information produced in recent years regarding PVDs biosynthesis and pathogenic mechanisms, since PVDs are extremely valuable compounds. Additionally, we summarize the therapeutic applications deriving from the PVDs' use and focus on their role as therapeutic target themselves. We assess the current biotechnological applications of different sectors and evaluate the state-of-the-art technology relating to the use of synthetic biology tools for pathway engineering. Finally, we review the most recent methods and techniques capable of identifying such molecules in complex matrices for drug-discovery purposes.

Fungi Can Be More Effective than Bacteria for the Bioremediation of Marine Sediments Highly Contaminated with Heavy Metals.

The contamination of coastal marine sediments with heavy metals (HMs) is a widespread phenomenon that requires effective remediation actions. Bioremediation based on the use of bacteria is an economically and environmentally sustainable effective strategy for reducing HM contamination and/or toxicity in marine sediments. However, information on the efficiency of marine-derived fungi for HM decontamination of marine sediments is still largely lacking, despite evidence of the performance of terrestrial fungal strains on other contaminated matrixes (e.g., soils, freshwater sediments, industrial wastes). Here, we carried out for the first time an array of parallel laboratory experiments by using different combinations of chemical and microbial amendments (including acidophilic autotrophic and heterotrophic bacteria, as well as filamentous marine fungi) for the bioremediation of highly HM-contaminated sediments of the Portman Bay (NW Mediterranean Sea), an area largely affected by long-term historical discharges of mine tailings. Our results indicate that the bioleaching performance of metals from the sediment is based on the addition of fungi (Aspergillus niger and Trichoderma sp.), either alone or in combination with autotrophic bacteria, was higher when compared to other treatments. In particular, fungal addition allowed obtaining bioleaching yields for As eight times higher than those by chemical treatments and double compared with the addition of bacteria alone. Moreover, in our study, the fungal addition was the only treatment allowing effective bioleaching of otherwise not mobile fractions of Zn and Cd, thus overtaking bacterial treatments. We found that the lower the sediment pH reached by the experimental conditions, as in the case of fungal addition, the higher the solubilization yield of metals, suggesting that the specific metabolic features of A. niger and Trichoderma sp. enable lowering sediment pH and enhance HM bioleaching. Overall, our findings indicate that fungi can be more effective than acidophilic autotrophic and heterotrophic bacteria in HM bioleaching, and as such, their use can represent a promising and efficient strategy for the bioremediation of marine sediments highly contaminated with heavy metals.

Metagenome-assembled genome (MAG) of Oceancaulis alexandrii NP7 isolated from Mediterranean Sea polluted marine sediments and its bioremediation potential.

Oceanicaulis alexandrii strain NP7 is a marine bacterium which belongs to the Hyphomonadaceae family and was isolated from sediments highly contaminated with metals and polycyclic aromatic hydrocarbons released for decades by industrial activities in the Gulf of Naples (Mediterranean Sea). Here, we report the partial genome sequence and annotation of O. alexandrii strain NP7 that contains a chromosome of 2,954,327 bp and encodes for 2914 predicted coding sequences (CDSs) and 44 RNA-encoding genes. Although the presence of some CDSs for genes involved in hydrocarbon degradation processes (e.g., alkB) have already been described in the literature associated with the Oceanicaulis, this is the first time that more than 100 genes involved in metal detoxification processes and hydrocarbon degradation are reported belonging to this genus. The presence of a heterogeneous set of genes involved in stress response, hydrocarbon degradation, heavy metal resistance, and detoxification suggests a possible role for O. alexandrii NP7 in the bioremediation of these highly contaminated marine sediments.

Bacteria, Fungi and Microalgae for the Bioremediation of Marine Sediments Contaminated by Petroleum Hydrocarbons in the Omics Era.

Petroleum hydrocarbons (PHCs) are one of the most widespread and heterogeneous organic contaminants affecting marine ecosystems. The contamination of marine sediments or coastal areas by PHCs represents a major threat for the ecosystem and human health, calling for urgent, effective, and sustainable remediation solutions. Aside from some physical and chemical treatments that have been established over the years for marine sediment reclamation, bioremediation approaches based on the use of microorganisms are gaining increasing attention for their eco-compatibility, and lower costs. In this work, we review current knowledge concerning the bioremediation of PHCs in marine systems, presenting a synthesis of the most effective microbial taxa (i.e., bacteria, fungi, and microalgae) identified so far for hydrocarbon removal. We also discuss the challenges offered by innovative molecular approaches for the design of effective reclamation strategies based on these three microbial components of marine sediments contaminated by hydrocarbons.

Highly Contaminated Marine Sediments Can Host Rare Bacterial Taxa Potentially Useful for Bioremediation.

Coastal areas impacted by high anthropogenic pressures typically display sediment contamination by polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HMs). Microbial-based bioremediation represents a promising strategy for sediment reclamation, yet it frequently fails due to poor knowledge of the diversity and dynamics of the autochthonous microbial assemblages and to the inhibition of the target microbes in the contaminated matrix. In the present study, we used an integrated approach including a detailed environmental characterization, high-throughput sequencing and culturing to identify autochthonous bacteria with bioremediation potential in the sediments of Bagnoli-Coroglio (Gulf of Naples, Mediterranean Sea), a coastal area highly contaminated by PAHs, aliphatic hydrocarbons and HMs. The analysis of the benthic prokaryotic diversity showed that the distribution of the dominant taxon (Gammaproteobacteria) was mainly influenced by PAHs, As, and Cd concentrations. The other abundant taxa (including Alphaproteobacteria, Deltaproteobacteria, Bacteroidetes, Acidobacteria, Actinobacteria, NB1-j, Desulfobacterota, and Myxococcota) were mainly driven by sediment grain size and by Cu and Cr concentrations, while the rare taxa (i.e., each contributing <1%) by As and aliphatic hydrocarbons concentrations and by sediment redox potential. These results suggest a differential response of bacterial taxa to environmental features and chemical contamination and those different bacterial groups may be inhibited or promoted by different contaminants. This hypothesis was confirmed by culturing and isolating 80 bacterial strains using media highly enriched in PAHs, only nine of which were contextually resistant to high HM concentrations. Such resistant isolates represented novel Gammaproteobacteria strains affiliated to Vibrio, Pseudoalteromonas, and Agarivorans, which were only scarcely represented in their original assemblages. These findings suggest that rare but culturable bacterial strains resistant/tolerant to high levels of mixed contaminants can be promising candidates useful for the reclamation by bioaugmentation strategies of marine sediments that are highly contaminated with PAHs and HMs.

Genome Sequence of an Alkaliphilus Species Isolated from Historically Contaminated Sediments of the Gulf of Naples (Mediterranean Sea).

Here, we report the draft genome sequence of a metagenome-assembled genome (MAG) of a new Alkaliphilus bacterium, NP8, of the Clostridiaceae family. This bacterium was isolated from polluted sediment collected from an abandoned industrial site located in the Gulf of Naples (Mediterranean Sea) as part of a microbial consortium.

Degradation of Hydrocarbons and Heavy Metal Reduction by Marine Bacteria in Highly Contaminated Sediments.

Investigations on the ability of bacteria to enhance removal of hydrocarbons and reduce heavy metal toxicity in sediments are necessary to design more effective bioremediation strategies. In this study, five bacterial strains, Halomonas sp. SZN1, Alcanivorax sp. SZN2, Pseudoalteromonas sp. SZN3, Epibacterium sp. SZN4, and Virgibacillus sp. SZN7, were isolated from polluted sediments from an abandoned industrial site in the Gulf of Naples, Mediterranean Sea, and tested for their bioremediation efficiency on sediment samples collected from the same site. These bacteria were added as consortia or as individual cultures into polluted sediments to assess biodegradation efficiency of polycyclic aromatic hydrocarbons and heavy metal immobilisation capacity. Our results indicate that these bacteria were able to remove polycyclic aromatic hydrocarbons, with a removal rate up to ca. 80% for dibenzo-anthracene. In addition, these bacteria reduced arsenic, lead, and cadmium mobility by promoting their partitioning into less mobile and bioavailable fractions. Microbial consortia generally showed higher performance toward pollutants as compared with pure isolates, suggesting potential synergistic interactions able to enhance bioremediation capacity. Overall, our findings suggest that highly polluted sediments select for bacteria efficient at reducing the toxicity of hazardous compounds, paving the way for scaled-up bioremediation trials.

Microalgal Metallothioneins and Phytochelatins and Their Potential Use in Bioremediation.

The persistence of heavy metals (HMs) in the environment causes adverse effects to all living organisms; HMs accumulate along the food chain affecting different levels of biological organizations, from cells to tissues. HMs enter cells through transporter proteins and can bind to enzymes and nucleic acids interfering with their functioning. Strategies used by microalgae to minimize HM toxicity include the biosynthesis of metal-binding peptides that chelate metal cations inhibiting their activity. Metal-binding peptides include genetically encoded metallothioneins (MTs) and enzymatically produced phytochelatins (PCs). A number of techniques, including genetic engineering, focus on increasing the biosynthesis of MTs and PCs in microalgae. The present review reports the current knowledge on microalgal MTs and PCs and describes the state of art of their use for HM bioremediation and other putative biotechnological applications, also emphasizing on techniques aimed at increasing the cellular concentrations of MTs and PCs. In spite of the broad metabolic and chemical diversity of microalgae that are currently receiving increasing attention by biotechnological research, knowledge on MTs and PCs from these organisms is still limited to date.

Combined characterization of bovine polyhemoglobin microcapsules by UV-Vis absorption spectroscopy and cyclic voltammetry.

Polyhemoglobin produced from pure bovine hemoglobin by reaction with PEG bis(N-succynimidil succinate) as a cross-linking agent was encapsulated in gelatin and dehydrated by freeze-drying. Free carboxyhemoglobin and polyhemoglobin microcapsules were characterized by UV-Vis spectroscopy in the absorption range 450-650 nm and cyclic voltammetry in the voltage range from -0.8 to 0.6 mV to evaluate the ability to break the bond with carbon monoxide and to study the carrier's affinity for oxygen, respectively. SEM used to observe the shape of cross-linked gelatin-polyhemoglobin microparticles showed a regular distribution of globular shapes, with mean size of ~750 nm, which was ascribed to gelatin. Atomic absorption spectroscopy was also performed to detect iron presence in microparticles. Cyclic voltammetry using an Ag-AgCl electrode highlighted characteristic peaks at around -0.6 mV that were attributed to reversible oxygen bonding with iron in oxy-polyhemoglobin structure. These results suggest this technique as a powerful, direct and alternative method to evaluate the extent of hemoglobin oxygenation.

Preparation and characterization of a microencapsulated polyethylene glycol cross-linked polyhemoglobin.

Many complications are associated to the therapeutic use of blood, among which are not only transfusion adverse events but also other issues such as lack of donors and high costs for collecting, testing, preserving, and distributing blood packages. Therefore, a clinically viable "blood substitute" is considered the holy grail of traumatology and may greatly benefit medicine. One of the most successful approaches to date is conjugating hemoglobin with polyethylene glycol (PEG). This conjugation aims mainly at overcoming free cell hemoglobin toxicity, which makes its use as oxygen carrier in pure form unfeasible. To improve PEG-hemoglobin conjugates feasibility, we propose applying dual functional PEG cross-linking hemoglobin molecules encapsulated by a protein carrier. The new oxygen carrier showed mean values of the hydrodynamic diameter, dispersity, and zeta potential of 1370 nm, 0.029 and -36 mV, respectively, evidencing the successful synthesis of PEG bis(N-succinimidyl succinate) and polyhemoglobin as well as the structuring of protein carrier.