High microbiome and metabolome diversification in coexisting sponges with different bio-ecological traits.

Marine Porifera host diverse microbial communities, which influence host metabolism and fitness. However, functional relationships between sponge microbiomes and metabolic signatures are poorly understood. We integrate microbiome characterization, metabolomics and microbial predicted functions of four coexisting Mediterranean sponges -Petrosia ficiformis, Chondrosia reniformis, Crambe crambe and Chondrilla nucula. Microscopy observations reveal anatomical differences in microbial densities. Microbiomes exhibit strong species-specific trends. C. crambe shares many rare amplicon sequence variants (ASV) with the surrounding seawater. This suggests important inputs of microbial diversity acquired by selective horizontal acquisition. Phylum Cyanobacteria is mainly represented in C. nucula and C. crambe. According to putative functions, the microbiome of P. ficiformis and C. reniformis are functionally heterotrophic, while C. crambe and C. nucula are autotrophic. The four species display distinct metabolic profiles at single compound level. However, at molecular class level they share a "core metabolome". Concurrently, we find global microbiome-metabolome association when considering all four sponge species. Within each species still, sets of microbe/metabolites are identified driving multi-omics congruence. Our findings suggest that diverse microbial players and metabolic profiles may promote niche diversification, but also, analogous phenotypic patterns of "symbiont evolutionary convergence" in sponge assemblages where holobionts co-exist in the same area.

Chemical and Pharmacological Prospection of the Ascidian Cystodytes dellechiajei.

Marine invertebrates are a traditional source of natural products with relevant biological properties. Tunicates are soft-bodied, solitary or colonial, sessile organisms that provide compounds unique in their structure and activity. The aim of this work was to investigate the chemical composition of the ascidian Cystodytes dellechiajei, selected on the basis of a positive result in biological screening for ligands of relevant receptors of the innate immune system, including TLR2, TLR4, dectin-1b, and TREM2. Bioassay-guided screening of this tunicate extract yielded two known pyridoacridine alkaloids, shermilamine B (1) and N-deacetylshermilamine B (2), and a family of methyl-branched cerebrosides (3). Compounds 2 and 3 showed selective binding to TREM2 in a dose-dependent manner. N-deacetylshermilamine B (2), together with its acetylated analogue, shermilamine B (1), was also strongly cytotoxic against multiple myeloma cell lines. TREM2 is involved in immunomodulatory processes and neurodegenerative diseases. N-deacetylshermilamine B (2) is the first example of a polycyclic alkaloid to show an affinity for this receptor.

BODIPY-Based Analogue of the TREM2-Binding Molecular Adjuvant Sulfavant A, a Chemical Tool for Imaging and Tracking Biological Systems.

Recently, we described synthetic sulfolipids named Sulfavants as a novel class of molecular adjuvants based on the sulfoquinovosyl-diacylglycerol skeleton. The members of this family, Sulfavant A (1), Sulfavant R (2), and Sulfavant S (3), showed important effects on triggering receptor expressed on myeloid cells 2 (TREM2)-induced differentiation and maturation of human dendritic cells (hDC), through a novel cell mechanism underlying the regulation of the immune response. As these molecules are involved in biological TREM2-mediated processes crucial for cell survival, here, we report the synthesis and application of a fluorescent analogue of Sulfavant A bearing the 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene moiety (Me4-BODIPY). The fluorescent derivative, named PB-SULF A (4), preserving the biological activity of Sulfavants, opens the way to chemical biology and cell biology experiments to better understand the interactions with cellular and in vivo organ targets and to improve our comprehension of complex molecular mechanisms underlying the not fully understood ligand-induced TREM2 activity.

Potential of Lipid Biosynthesis under Heterotrophy in the Marine Diatom Cyclotella cryptica.

Despite the theoretical high productivity, microalgae-based oil production is not economically sustainable due to the high cost of photoautotrophic cultures. Heterotrophic growth is a suitable economic alternative to overcoming light dependence and climatic/geographic fluctuations. Here we report data about growth performance, biomass production, and lipid composition of the marine diatom Cyclotella cryptica, chosen as a model strain for biodiesel production in heterothrophy. A repeated-batch process of heterotrophic cultivation has also been investigated to assess the robustness and phenotypic stability. The process consisting of six constant cycle repetitions was carried out for 42 days and led to an average dry biomass production of 1.5 ± 0.1 g L-1 of which 20% lipids composed of 60% triglycerides, 20% phospholipids. and 20% glycolipids. The major fatty acids were C16:0 (∼26%), C16:1 ω-7 (∼57%), and C20:5 ω-3 (∼12%), with a significant reduction in the unsaturated fatty acids in comparison to other microalgae grown in heterotrophy. Fatty acids were differently distributed among the glycerolipid classes, and the lipid composition was used to compare the potential properties of C. cryptica oil with traditional vegetable biofuels.

Marine natural product lepadin A as a novel inducer of immunogenic cell death via CD91-dependent pathway.

Immunogenic Cell Death (ICD) represents a mechanism of enhancing T cell-driven response against tumor cells. The process is enabled by release of damage-associated molecular patterns (DAMPs) and cytokines by dying cells. Based on molecular studies and clinical marker assessment, ICD can be a new target for cancer chemotherapy hitherto restricted to a few conventional anticancer drugs. In view of the development of small molecules in targeted cancer therapy, we reported the preliminary evidence on the role of the natural product lepadin A (1) as a novel ICD inducer. Here we describe the ICD mechanism of lepadin A (1) by proving the translocation of the protein calreticulin (CRT) to the plasma membrane of human A2058 melanoma cells. CRT exposure is an ICD marker in clinical studies and was associated with the activation of the intrinsic apoptotic pathway in A2058 cells with lepadin A (1). After the treatment, the tumour cells acquired the ability to activate dendritic cells (DCs) with cytokine release and costimulatory molecule expression that is consistent with a phenotypic profile committed to priming T lymphocytes via a CD91-dependent mechanism. The effect of lepadin A (1) was dose-dependent and comparable to the response of the chemotherapy drug doxorubicin (2), a well-established ICD inducer.

The immunoregulatory effect of the TREM2-agonist Sulfavant A in human allogeneic mixed lymphocyte reaction.

Introduction: Sulfavant A (SULF A) is a synthetic derivative of naturally occurring sulfolipids. The molecule triggers TREM2-related maturation of dendritic cells (DCs) and has shown promising adjuvant activity in a cancer vaccine model. Methods: the immunomodulatory activity of SULF A is tested in an allogeneic mixed lymphocyte reaction (MLR) assay based on monocyte-derived dendritic cells and naïve T lymphocytes from human donors. Flow cytometry multiparametric analyses and ELISA assays were performed to characterize the immune populations, T cell proliferation, and to quantify key cytokines. Results: Supplementation of 10 µg/mL SULF A to the co-cultures induced DCs to expose the costimulatory molecules ICOSL and OX40L and to reduce release of the pro-inflammatory cytokine IL-12. After 7 days of SULF A treatment, T lymphocytes proliferated more and showed increased IL-4 synthesis along with downregulation of Th1 signals such as IFNγ, T-bet and CXCR3. Consistent with these findings, naïve T cells polarized toward a regulatory phenotype with up-regulation of FOXP3 expression and IL-10 synthesis. Flow cytometry analysis also supported the priming of a CD127-/CD4+/CD25+ subpopulation positive for ICOS, the inhibitory molecule CTLA-4, and the activation marker CD69. Discussion: These results prove that SULF A can modulate DC-T cell synapse and stimulate lymphocyte proliferation and activation. In the hyperresponsive and uncontrolled context of the allogeneic MLR, the effect is associated to differentiation of regulatory T cell subsets and dampening of inflammatory signals.

Discovery of lead natural products for developing pan-SARS-CoV-2 therapeutics.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global public health crisis. The reduced efficacy of therapeutic monoclonal antibodies against emerging SARS-CoV-2 variants of concern (VOCs), such as omicron BA.5 subvariants, has underlined the need to explore a novel spectrum of antivirals that are effective against existing and evolving SARS-CoV-2 VOCs. To address the need for novel therapeutic options, we applied cell-based high-content screening to a library of natural products (NPs) obtained from plants, fungi, bacteria, and marine sponges, which represent a considerable diversity of chemical scaffolds. The antiviral effect of 373 NPs was evaluated using the mNeonGreen (mNG) reporter SARS-CoV-2 virus in a lung epithelial cell line (Calu-3). The screening identified 26 NPs with half-maximal effective concentrations (EC50) below 50 µM against mNG-SARS-CoV-2; 16 of these had EC50 values below 10 µM and three NPs (holyrine A, alotaketal C, and bafilomycin D) had EC50 values in the nanomolar range. We demonstrated the pan-SARS-CoV-2 activity of these three lead antivirals against SARS-CoV-2 highly transmissible Omicron subvariants (BA.5, BA.2 and BA.1) and highly pathogenic Delta VOCs in human Calu-3 lung cells. Notably, holyrine A, alotaketal C, and bafilomycin D, are potent nanomolar inhibitors of SARS-CoV-2 Omicron subvariants BA.5 and BA.2. The pan-SARS-CoV-2 activity of alotaketal C [protein kinase C (PKC) activator] and bafilomycin D (V-ATPase inhibitor) suggest that these two NPs are acting as host-directed antivirals (HDAs). Future research should explore whether PKC regulation impacts human susceptibility to and the severity of SARS-CoV-2 infection, and it should confirm the important role of human V-ATPase in the VOC lifecycle. Interestingly, we observed a synergistic action of bafilomycin D and N-0385 (a highly potent inhibitor of human TMPRSS2 protease) against Omicron subvariant BA.2 in human Calu-3 lung cells, which suggests that these two highly potent HDAs are targeting two different mechanisms of SARS-CoV-2 entry. Overall, our study provides insight into the potential of NPs with highly diverse chemical structures as valuable inspirational starting points for developing pan-SARS-CoV-2 therapeutics and for unravelling potential host factors and pathways regulating SARS-CoV-2 VOC infection including emerging omicron BA.5 subvariants.

Occurrence of Capnophilic Lactic Fermentation in the Hyperthermophilic Anaerobic Bacterium Thermotoga sp. Strain RQ7.

Capnophilic lactic fermentation (CLF) is an anaplerotic pathway exclusively identified in the anaerobic hyperthermophilic bacterium Thermotoga neapolitana, a member of the order Thermotogales. The CO2-activated pathway enables non-competitive synthesis of hydrogen and L-lactic acid at high yields, making it an economically attractive process for bioenergy production. In this work, we discovered and characterized CLF in Thermotoga sp. strain RQ7, a naturally competent strain, opening a new avenue for molecular investigation of the pathway. Evaluation of the fermentation products and expression analyses of key CLF-genes by RT-PCR revealed similar CLF-phenotypes between T. neapolitana and T. sp. strain RQ7, which were absent in the non-CLF-performing strain T. maritima. Key CLF enzymes, such as PFOR, HYD, LDH, RNF, and NFN, are up-regulated in the two CLF strains. Another important finding is the up-regulation of V-ATPase, which couples ATP hydrolysis to proton transport across the membranes, in the two CLF-performing strains. The fact that V-ATPase is absent in T. maritima suggested that this enzyme plays a key role in maintaining the necessary proton gradient to support high demand of reducing equivalents for simultaneous hydrogen and lactic acid synthesis in CLF.

ADViSELipidomics: a workflow for analyzing lipidomics data.

SUMMARY: ADViSELipidomics is a novel Shiny app for preprocessing, analyzing and visualizing lipidomics data. It handles the outputs from LipidSearch and LIQUID for lipid identification and quantification and the data from the Metabolomics Workbench. ADViSELipidomics extracts information by parsing lipid species (using LIPID MAPS classification) and, together with information available on the samples, performs several exploratory and statistical analyses. When the experiment includes internal lipid standards, ADViSELipidomics can normalize the data matrix, providing normalized concentration values per lipids and samples. Moreover, it identifies differentially abundant lipids in simple and complex experimental designs, dealing with batch effect correction. Finally, ADViSELipidomics has a user-friendly graphical user interface and supports an extensive series of interactive graphics. AVAILABILITY AND IMPLEMENTATION: ADViSELipidomics is freely available at https://github.com/ShinyFabio/ADViSELipidomics. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Organic extract of Geodia cydonium induces cell cycle block in human mesothelioma cells.

The serious side effects caused by chemotherapeutics and the development of cancer chemoresistance represent the most significant limitations in the treatment of cancer. Some alternative approaches have been developed in recent years, which are based on natural compounds, and have allowed important advances in cancer therapeutics. During the last 50 years, sponges have been considered a promising source of natural products from the marine environment, representing ~30% of all marine natural products. Among sponges, the Mediterranean species Geodia cydonium represents a potential source of these type of products with considerable biotechnological interest as pharmaceutical agents. The present study demonstrated the antiproliferative effect of an organic G. cydonium extract (GEOCYDO) against three human mesothelioma cell lines, MSTO-211H (MSTO), NCI-H2452 (NCI) and Ist-Mes2 (Mes2), which differ in their sensitivity (MSTO and NCI) and resistance (Mes2) to standard combined treatment with cisplatin and piroxicam. To this aim, the activity of the extract was evaluated by analyzing its effects on cell viability, cancer properties and cell cycle progression by means of colony formation assay, cell cycle analysis and protein expression analysis. The results revealed, in mesothelioma, this extract was able to reduce self-renewal, cell migration and it could induce cell cycle arrest in G0/G1 stage, thus blocking cell proliferation. In conclusion, to the best of our knowledge, the present results indicated for the first time that GEOCYDO can contain active compounds able to affect cell proliferation in mesothelioma, suggesting that it could be considered as a potential novel drug source for cancer treatment.

Antitumor Potential of Immunomodulatory Natural Products.

Cancer is one of the leading causes of death globally. Anticancer drugs aim to block tumor growth by killing cancerous cells in order to prevent tumor progression and metastasis. Efficient anticancer drugs should also minimize general toxicity towards organs and healthy cells. Tumor growth can also be successfully restrained by targeting and modulating immune response. Cancer immunotherapy is assuming a growing relevance in the fight against cancer and has recently aroused much interest for its wider safety and the capability to complement conventional chemotherapeutic approaches. Natural products are a traditional source of molecules with relevant potential in the pharmacological field. The huge structural diversity of metabolites with low molecular weight (small molecules) from terrestrial and marine organisms has provided lead compounds for the discovery of many modern anticancer drugs. Many natural products combine chemo-protective and immunomodulant activity, thus offering the potential to be used alone or in association with conventional cancer therapy. In this review, we report the natural products known to possess antitumor properties by interaction with immune system, as well as discuss the possible immunomodulatory mechanisms of these molecules.

Sulfavant A as the first synthetic TREM2 ligand discloses a homeostatic response of dendritic cells after receptor engagement.

OBJECTIVE: The immune response arises from a fine balance of mechanisms that provide for surveillance, tolerance, and elimination of dangers. Sulfavant A (SULF A) is a sulfolipid with a promising adjuvant activity. Here we studied the mechanism of action of SULF A and addressed the identification of its molecular target in human dendritic cells (hDCs). METHODS: Adjuvant effect and immunological response to SULF A were assessed on DCs derived from human donors. In addition to testing various reporter cells, target identification and downstream signalling was supported by a reverse pharmacology approach based on antibody blocking and gene silencing, crosstalk with TLR pathways, use of human allogeneic mixed lymphocyte reaction. RESULTS: SULF A binds to the Triggering Receptor Expressed on Myeloid cells-2 (TREM2) and initiates an unconventional maturation of hDCs leading to enhanced migration activity and up-regulation of MHC and co-stimulatory molecules without release of conventional cytokines. This response involves the SYK-NFAT axis and is compromised by blockade or gene silencing of TREM2. Activation by SULF A preserved the DC functions to excite the allogeneic T cell response, and increased interleukin-10 release after lipopolysaccharide stimulation. CONCLUSION: SULF A is the first synthetic small molecule that binds to TREM2. The receptor engagement drives differentiation of an unprecedented DC phenotype (homeDCs) that contributes to immune homeostasis without compromising lymphocyte activation and immunogenic response. This mechanism fully supports the adjuvant and immunoregulatory activity of SULF A. We also propose that the biological properties of SULF A can be of interest in various physiopathological mechanisms and therapies involving TREM2.

Fractionation Protocol of Marine Metabolites.

Marine organisms are well known for their capability to produce chemically unique and biological relevant small molecules. However, marine extracts are complex mixtures of compounds with a huge presence of salts. Thus, biological screening and chemical analysis of marine extracts pose specific technical constraints and require adequate sample preparation. We have developed an automated solid-phase extraction (SPE)-based method to desalt marine extracts and recover metabolites, adapt to be integrated in platform of high-throughput screening. The procedure uses a poly(styrene-divynylbenzene)-based support and a stepwise organic solvent elution herein described.

Identification of the Marine Alkaloid Lepadin A as Potential Inducer of Immunogenic Cell Death.

Natural products and their synthetic analogs and derivatives are a traditional source of bioactive molecules with potential development as drug candidates. In this context, Marine Natural Products (MNPs) represent a rich reservoir of diverse molecular skeletons with potential pharmacological activity that, so far, has been mostly explored in cancer and infectious diseases. Starting from the development of a novel bioassay-guided screening platform for immunomodulatory compounds from an in-house MNPs library, we report the identification of the alkaloid lepadin A as a new model compound for immune-based anticancer activity with characteristics that suggest a possible mechanism as Immunogenic Cell Death inducer. The work describes the molecular-based bioprospecting in the Gulf of Naples together with the bioassay-guided fractionation, the chemical characterization of the alkaloid, and the biological activity in mouse dendritic cells (D1).

Untargeted and Targeted LC-MS/MS Based Metabolomics Study on In Vitro Culture of Phaeoacremonium Species.

Grapevine (Vitis vinifera L.) can be affected by many different biotic agents, including tracheomycotic fungi such as Phaeomoniella chlamydospora and Phaeoacremonium minimum, which are the main causal agent of Esca and Petri diseases. Both fungi produce phytotoxic naphthalenone polyketides, namely scytalone and isosclerone, that are related to symptom development. The main objective of this study was to investigate the secondary metabolites produced by three Phaeoacremonium species and to assess their phytotoxicity by in vitro bioassay. To this aim, untargeted and targeted LC-MS/MS-based metabolomics were performed. High resolution mass spectrometer UHPLC-Orbitrap was used for the untargeted profiling and dereplication of secondary metabolites. A sensitive multi reaction monitoring (MRM) method for the absolute quantification of scytalone and isosclerone was developed on a UPLC-QTrap. Different isolates of P. italicum, P. alvesii and P. rubrigenum were grown in vitro and the culture filtrates and organic extracts were assayed for phytotoxicity. The toxic effects varied within and among fungal isolates. Isosclerone and scytalone were dereplicated by matching retention times and HRMS and MS/MS data with pure standards. The amount of scytalone and isosclerone differed within and among fungal species. To our best knowledge, this is the first study that applies an approach of LC-MS/MS-based metabolomics to investigate differences in the metabolic composition of organic extracts of Phaeoacremonium species culture filtrates.

Direct evidence of the impact of aqueous self-assembly on biological behavior of amphiphilic molecules: The case study of molecular immunomodulators Sulfavants.

Sulfavant A and Sulfavant R, sulfoquinovoside-glycerol lipids under study as vaccine adjuvants, structurally differ only for the configuration of glyceridic carbon, R/S and R respectively. The in vitro activity of these substances follows a bell-shaped dose-response curve, but Sulfavant A gave the best response around 20 µM, while Sulfavant R at 10 nM. Characterization of aqueous self-assembly of these molecules by a multi-technique approach clarified the divergent and controversial biological outcome. Supramolecular structures were present at concentrations much lower than critical aggregation concentration for both products. The kind and size of these aggregates varied as a function of the concentration differently for Sulfavant A and Sulfavant R. At nanomolar range, Sulfavant A formed cohesive vesicles, while Sulfavant R arranged in spherical micellar particles whose reduced stability was probably responsible for an increase of monomer concentration in accordance with immunomodulatory profile. Instead, at micromolar concentrations transition from micellar to vesicular state of Sulfavant R occurred and thermodynamic stability of the aggregates, assessed by surface tensiometry, correlated with the bioactivity of Sulfavant A at 20 µM and the complete loss of efficacy of Sulfavant R. The study of Sulfavants provides clear evidence of how self-aggregation, often neglected, and the equilibria between monomers and aqueous supramolecular forms of lipophilic molecules deeply determine the overall bio-response.

Probing the Therapeutic Potential of Marine Phyla by SPE Extraction.

The marine environment is potentially a prolific source of small molecules with significant biological activities. In recent years, the development of new chromatographic phases and the progress in cell and molecular techniques have facilitated the search for marine natural products (MNPs) as novel pharmacophores and enhanced the success rate in the selection of new potential drug candidates. However, most of this exploration has so far been driven by anticancer research and has been limited to a reduced number of taxonomic groups. In this article, we report a test study on the screening potential of an in-house library of natural small molecules composed of 285 samples derived from 57 marine organisms that were chosen from among the major eukaryotic phyla so far represented in studies on bioactive MNPs. Both the extracts and SPE fractions of these organisms were simultaneously submitted to three different bioassays-two phenotypic and one enzymatic-for cytotoxic, antidiabetic, and antibacterial activity. On the whole, the screening of the MNP library selected 11 potential hits, but the distribution of the biological results showed that SPE fractionation increased the positive score regardless of the taxonomic group. In many cases, activity could be detected only in the enriched fractions after the elimination of the bulky effect due to salts. On a statistical basis, sponges and molluscs were confirmed to be the most significant source of cytotoxic and antimicrobial products, but other phyla were found to be effective with the other therapeutic targets.

Bioactivity Screening of Antarctic Sponges Reveals Anticancer Activity and Potential Cell Death via Ferroptosis by Mycalols.

Sponges are known to produce a series of compounds with bioactivities useful for human health. This study was conducted on four sponges collected in the framework of the XXXIV Italian National Antarctic Research Program (PNRA) in November-December 2018, i.e., Mycale (Oxymycale) acerata, Haliclona (Rhizoniera) dancoi, Hemimycale topsenti, and Hemigellius pilosus. Sponge extracts were fractioned and tested against hepatocellular carcinoma (HepG2), lung carcinoma (A549), and melanoma cells (A2058), in order to screen for antiproliferative or cytotoxic activity. Two different chemical classes of compounds, belonging to mycalols and suberitenones, were identified in the active fractions. Mycalols were the most active compounds, and their mechanism of action was also investigated at the gene and protein levels in HepG2 cells. Of the differentially expressed genes, ULK1 and GALNT5 were the most down-regulated genes, while MAPK8 was one of the most up-regulated genes. These genes were previously associated with ferroptosis, a programmed cell death triggered by iron-dependent lipid peroxidation, confirmed at the protein level by the down-regulation of GPX4, a key regulator of ferroptosis, and the up-regulation of NCOA4, involved in iron homeostasis. These data suggest, for the first time, that mycalols act by triggering ferroptosis in HepG2 cells.

Improvement of CO2 and Acetate Coupling into Lactic Acid by Genetic Manipulation of the Hyperthermophilic Bacterium Thermotoga neapolitana.

Capnophilic lactic fermentation (CLF) represents an attractive biotechnological process for biohydrogen production and synthesis of L-lactic acid from acetate and CO2. The present study focuses on a genetic manipulation approach of the Thermotoga neapolitana DSM33003 strain to enhance lactic acid synthesis by the heterologous expression of a thermostable acetyl-CoA synthetase that catalyses the irreversible acetate assimilation. Because of the scarcity of available genetic tools, each transformation step was optimized for T. neapolitana DSM33003 to cope with the specific needs of the host strain. Batch fermentations with and without an external source of acetate revealed a strongly increased lactate production (up to 2.5 g/L) for the recombinant strain compared to wild type. In the engineered bacterium, the assimilation of CO2 into lactic acid was increased 1.7 times but the hydrogen yield was impaired in comparison to the wild type strain. Analysis of fermentation yields revealed an impaired metabolism of hydrogen in the recombinant strain that should be addressed in future studies. These results offer an important prospective for the development of a sustainable approach that combines carbon capture, energy production from renewable source, and the synthesis of high value-added products, which will be addressed in future studies.