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The transport through the nuclear pore complex is used by cancer cells to evade tumor-suppressive mechanisms. Several tumor-suppressors have been shown to be excluded from the cell nucleus in cancer cells by the nuclear export receptor CRM1 and abnormal expression of CRM1 is oncogenic. Inhibition of CRM1 has long been postulated as potential approach for the treatment of cancer and to overcome therapy resistance. Furthermore, the nuclear export of viral components mediated by the CRM1 is crucial in various stages of the viral lifecycle and assembly of many viruses from diverse families, including coronavirus. However, the first nuclear export inhibitors failed or never entered into clinical trials. More recently CRM1 reemerged as a cancer target and a successful proof of concept was achieved with the clinical approval of Selinexor. The chemical complexity of natural products is a promising perspective for the discovery of new nuclear export inhibitors with a favorable toxicity profile. Several screening campaigns have been performed and several natural product-based nuclear export inhibitors have been identified. With this review we give an overview over the role of CRM1-mediated nuclear export in cancer and the effort made to identify and develop nuclear export inhibitors in particular from natural sources., This work was supported by Fundação para a Ciência e a Tecnologia (FCT) Research Center Grant UID/BIM/04773/2013 Centre for Biomedical Research 1334 and by the Spanish Ministry of Science, Innovation and Universities through Grant RTI2018-094629-B-I00 to WL. BF was supported by FCT-SFRH/BPD/100434/2014 and Marie Curie Individual Fellowship project TRIBBLES (#748585). This work was also supported by two LPCC-NRS/Terry Fox grants (2016/2017; 2017/2018).

Humanity faces great challenges, such as the rise of bacterial antibiotic resistance and cancer incidence. Thus, the discovery of novel therapeutics from underexplored environments, such as marine habitats, is fundamental. In this study, twelve strains from the phylum Firmicutes and thirty-four strains from the phylum Proteobacteria, isolated from marine sponges of the Erylus genus, collected in Portuguese waters, were tested for bioactivities and the secondary metabolites were characterised. Bioactivity screenings comprised antimicrobial, anti-fungal, anti-parasitic and anti-cancer assays. Selected bioactive extracts were further analysed for already described molecules through high performance liquid chromatography and mass spectrometry. Several bioactivities were observed against the fungus Aspergillusfumigatus, the bacteria (methicillin-resistant Staphylococcus aureus and Escherichia coli), the human liver cancer cell line HepG2 and the parasite Trypanosoma cruzi. Medium scale-up volume extracts confirmed anti-fungal activity by strains Proteus mirabilis #118_13 and Proteus sp. (JX006497) strain #118_20. Anti-parasitic activity was also confirmed in Enterococcus faecalis strain #118_3. Moreover, P. mirabilis #118_13 showed bioactivity in human melanoma cell line A2058 and the human hepatocellular carcinoma cell line HepG2. The dereplication of bioactive extracts showed the existence of a variety of secondary metabolites, with some unidentifiable molecules. This work shows that bacterial communities of sponges are indeed good candidates for drug discovery and, as far as we know, we describe anti-parasitic activity of a strain of E. faecalis and the presence of diketopiperazines in Proteus genus for the first time. This research was partially supported by the Strategic Funding UID/Multi/04423/2019 through national funds provided by FCT - Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020. The MEDINA authors disclosed the receipt of financial support from Fundación MEDINA, a public–private partnership of Merck Sharp & Dohme de España S.A./Universidad de Granada/Junta de Andaluc ́ıa. Moreover, some of the equipment used in this work was supported by the Ministerio de Ciencia e Innovación and the European Union (Grant INP-2011-0016-PCT-010000- ACT6).

Due to the unique biodiversity and the physical-chemical properties of their environment, marine microorganisms have evolved defense and signaling compounds that often have no equivalent in terrestrial habitats. The aim of this study was to screen extracts of the dinoflagellate Amphidinium carterae for possible bioactivities (i.e., anticancer, anti-inflammatory, anti-diabetes, antibacterial and antifungal properties) and identify bioactive compounds. Anticancer activity was evaluated on human lung adenocarcinoma (A549), human skin melanoma (A2058), human hepatocellular carcinoma (HepG2), human breast adenocarcinoma (MCF7) and human pancreas carcinoma (MiaPaca-2) cell lines. Antimicrobial activities were evaluated against Gram-positive bacteria (Staphylococcus aureus MRSA and MSSA), Gram-negative bacteria (i.e., Escherichia coli and Klebsiella pneumoniae), Mycobacterium tuberculosis and the fungus Aspergillus fumigatus. The results indicated moderate biological activities against all the cancer cells lines and microorganisms tested. Bioassay-guided fractionation assisted by HRMS analysis allowed the detection of one new and two known amphidinols that are potentially responsible for the antifungal and cytotoxic activities observed. Further isolation, purification and structural elucidation led to a new amphidinol, named amphidinol 22. The planar structure of the new compound was determined by analysis of its HRMS and 1D and 2D NMR spectra. Its biological activity was evaluated, and it displayed both anticancer and antifungal activities.

Parkinson's disease (PD) and Amyotrophic lateral sclerosis (ALS), are neurodegenerative disorders characterized by loss of dopaminergic or motor neurons, respectively. Although understanding of the PD and ALS pathogenesis remains incomplete, increasing evidence from human and animal studies has suggested that aberrant GSK3β, oxidative stress and mitochondrial damage are involved in their pathogenesis. Using two different molecular models, treatment with L-BMAA for ALS and rotenone for PD the effect of isolecanoric acid, a natural product isolated from a fungal culture, was evaluated. Pre-treatment with this molecule caused inhibition of GSK3β and CK1, and a decrease in oxidative stress, mitochondrial damage, apoptosis and cell death. Taken together, these results indicated that isolecanoric acid might have a protective effect against the development of these neurodegenerative disorders.

In the beginning of the twenty-first century, humanity faces great challenges regarding diseases and health-related quality of life. A drastic rise in bacterial antibiotic resistance, in the number of cancer patients, in the obesity epidemics and in chronic diseases due to life expectation extension are some of these challenges. The discovery of novel therapeutics is fundamental and it may come from underexplored environments, like marine habitats, and microbial origin. Actinobacteria are well-known as treasure chests for the discovery of novel natural compounds. In this study, eighteen Actinomycetales isolated from marine sponges of three Erylus genera collected in Portuguese waters were tested for bioactivities with the main goal of isolating and characterizing the responsible bioactive metabolites. The screening comprehended antimicrobial, anti-fungal, anti-parasitic, anti-cancer and anti-obesity properties. Fermentations of the selected strains were prepared using ten different culturing media. Several bioactivities against the fungus Aspergillus fumigatus, the bacteria Staphylococcus aureus methicillin-resistant (MRSA) and the human liver cancer cell line HepG2 were obtained in small volume cultures. Screening in higher volumes showed consistent anti-fungal activity by strain Dermacoccus sp. #91-17 and Micrococcus luteus Berg02-26. Gordonia sp. Berg02-22.2 showed anti-parasitic (Trypanosoma cruzi) and anti-cancer activity against several cell lines (melanoma A2058, liver HepG2, colon HT29, breast MCF7 and pancreatic MiaPaca). For the anti-obesity assay, Microbacterium foliorum #91-29 and #91-40 induced lipid reduction on the larvae of zebrafish (Danio rerio). Dereplication of the extracts from several bacteria showed the existence of a variety of secondary metabolites, with some undiscovered molecules. This work showed that Actinomycetales are indeed good candidates for drug discovery., This research was partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT – Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020, the EU H2020-TWINN-2015, BLUEandGREEN – Boosting scientific excellence and innovation capacity in biorefineries based on marine resources (Project No. 692419) and the European ERA-NET Marine Biotechnology project CYANOBESITY (ERA-MBT/0001/2015), financed by national funds through FCT (Foundation for Science and Technology, Portugal). Ralph Urbatzka was supported by a FCT postdoc grant (SFRH/BPD/112287/2015). The MEDINA authors disclosed the receipt of financial support from Fundación MEDINA, a public–private partnership of Merck Sharp & Dohme de España S.A./Universidad de Granada/Junta de Andalucía. Moreover, some of the equipment used in this work was supported by the Ministerio de Ciencia e Innovación and the European Union (Grant INP-2011-0016-PCT-010000-ACT6).

Cancer cells are known to inactivate tumor suppressor proteins by triggering their anomalous subcellular location. It has been well established that the aberrant location of FOXO proteins is linked to tumor formation, progression of the same, or resistance to anti-neoplastic treatment. Furthermore, the abnormal location of FOXO has also been considered a potential biomarker for diabetic complications or longevity in different organisms. Here, we describe the immunodetection of endogenous FOXO by confocal microscopy, which can be used as a chemical tool to quantify FOXO expression levels, its cellular location, and even its active/inactive forms with relevant antibodies.

One-third of top-selling drugs are derived from natural products. When only a fraction of the bioactive natural products diversity has been explored, huge opportunities still remain for discovering novel leads for the development of new drugs. Clear cell renal cell carcinoma (ccRCC) is a highly vascular tumour arising from epithelial elements. Mutations in the Von Hippel-Lindau (VHL) gene are responsible for VHL disease and arise in the majority of Renal Cell Carcinoma (RCC) as well as in other types of cancer. Renal carcinoma cell lines with naturally occurring VHL mutations (RCC4 VA) and their genetically matched wild-type VHL (RCC4 VHL) counterparts were seeded onto 96-well plates and allowed to attach overnight. Fungal extracts were tested on both cell lines. Clinically useful antitumor agents were used as positive controls and as reference points to establish the efficacy and selectivity of the new compounds. Renal cell carcinoma cell lines expressing VHL or not were treated with Carboxyfluorescein succinimidyl ester (CFSE). The day after cell inoculation, extracts were added and during the following days of incubation, fluorescence intensity was measured as a surrogate marker for cell viability. The most promising extracts selectively inhibited growth of pVHL-defi- cient cells but not of wild-type VHL cells. We used High Content Bio-imaging, a complete cellular imaging workflow that integrates instruments and software to acquire and analyze images, to evaluate their effect. Cell imaging can reveal effects that would be overlooked by other cell assay approaches. This target-based whole cell screen is a new strategy, which ensures cell permeability and target selectivity especially in natural product screening where natural product purification is a labour of extensive work. This approach permitted a dynamic study where fluorescence was measured without affecting cell viability and enabling a better detection of cytotoxic effects such as autophagy, senescence or late apoptosis.

FOXO factors are tumour suppressor proteins commonly inactivated in human tumours by posttranslational modifications. Furthermore, genetic variation within the FOXO3a gene is consistently associated with human longevity. Therefore, the pharmacological activation of FOXO proteins is considered as an attractive therapeutic approach to treat cancer and age-related diseases. In order to identify agents capable of activating FOXOs, we tested a collection of small chemical compounds using image-based high content screening technology. Here, we report the discovery of LOM612 (compound 1a), a newly synthesized isothiazolonaphthoquinone as a potent FOXO relocator. Compound 1a induces nuclear translocation of a FOXO3a reporter protein as well as endogenous FOXO3a and FOXO1 in U2OS cells in a dose-dependent manner. This activity does not affect the subcellular localization of other cellular proteins including NFkB or inhibit CRM1-mediated nuclear export. Furthermore, compound 1a shows a potent antiproliferative effect in human cancer cell lines.

Toxoplasmosis is a ubiquitous parasitic infection causing a wide spectrum of diseases. It is usually asymptomatic but can lead to severe ocular and neurological disorders. Among the small-animal models available to study factors that determine susceptibility to toxoplasmosis, the rat appears to be rather similar to humans, particularly in terms of resistance to acute infection. Here, we demonstrate that the Lewis (LEW) rat strain displays an unexpected refractoriness toToxoplasmainfection. Complete resistance was assessed by both negative anti-Toxoplasmaserology and lack of detection of the parasite during the course of infection. In this model, sex, age, major histocompatibility complex, and inoculum size had no effect on resistance. Interestingly, progeny from F1hybrid crosses between Fischer (F344) or Brown Norway susceptible rats and LEW resistant rats were also fully resistant, showing a dominant effect of the gene or set of genes. Furthermore, resistance of the LEW rat was shown to be dependent on hematopoietic cells and partially abrogated by neutralization of endogenous gamma interferon. To our knowledge, this is the first observation of a rodent strain that is refractory toToxoplasmainfection. This model is therefore an attractive and powerful tool to dissect host genetic factors involved in susceptibility to toxoplasmosis.

Differential cytokine production by T cells plays an important role in the outcome of the immune response. We show that the level of CD45RC expression differentiates rat CD8 T cells in two subpopulations, CD45RChigh and CD45RClow, that have different cytokine profiles and functions. Upon in vitro stimulation, in an Ag-presenting cell-independent system, CD45RChigh CD8 T cells produce IL-2 and IFN-γ while CD45RClow CD8 T cells produce IL-4, IL-10, and IL-13. In vitro, these subsets also exhibit different cytotoxic and suppressive functions. The CD45RChigh/CD45RClow CD8 T cell ratio was determined in Lewis (LEW) and Brown-Norway (BN) rats. These two rat strains differ with respect to the Th1/Th2 polarization of their immune responses and to their susceptibility to develop distinct immune diseases. The CD45RChigh/CD45RClow CD8 T cell ratio is higher in LEW than in BN rats, and this difference is dependent on hemopoietic cells. Linkage analysis in a F2(LEW × BN) intercross identified two quantitative trait loci on chromosomes 9 and 20 controlling the CD45RChigh/CD45RClow CD8 T cell ratio. This genetic control was confirmed in congenic rats. The region on chromosome 9 was narrowed down to a 1.2-cM interval that was found to also control the IgE response in a model of Th2-mediated disorder. Identification of genes that control the CD45RChigh/CD45RClow CD8 T cell subsets in these regions could be of great interest for the understanding of the pathophysiology of immune-mediated diseases.

High-content imaging with robotic microscopy has been widely used for phenotype-based cellular screening research. This technology is ideally suited to monitor intracellular translocation of macromolecules. Here, we describe in detail the procedures for screening microbial natural extracts for their capability to inhibit the general nuclear export machinery. The method is based on human cells that stably express a fluorescent-tagged reporter protein that contain a nuclear export signal capable of mediating its translocation from the nucleus to the cytoplasm through the nuclear pore. In the presence of a small molecule nuclear export inhibitor, the fluorescent signal is trapped to varying degrees within the nucleus. In order to analyze complex libraries of compounds, the assay has been scaled to 96- or 384-well formats and optimized for high-throughput screening (HTS). Active microbial extracts undergo confirmation screening, bioassay-guided fractionation, chemical dereplication, and compound purification. The active purified compound is characterized in secondary assays that monitor the nuclear export of disease-relevant proteins. Nuclear export inhibitors hold promise as potential cancer and antiviral drugs.

Throughout recent history, metabolites of microbial origin have had an extraordinary impact on the welfare of humanity. In fact, natural products have largely been--and still are--considered an exceedingly valuable platform for the discovery of new drugs against diverse pathologies. Such value is partly due to their higher complexity and chemical diversity as compared to those of synthetic and combinatorial compounds. Mutations in the Von Hippel-Lindau (vhl) gene are responsible for VHL disease, congenital polycythemia, and are found in many sporadic tumor types. The primary cause of morbidity and mortality for these patients arises from progression of Renal Cell Carcinoma (RCC) or end-stage renal disease. Inactivation of the Von Hippel-Lindau (vhl) tumor suppressor gene arises in the majority of Renal Cell Carcinoma (RCC) as well as in other types of cancer and is associated with a high degree of vascularization and poor prognosis. Loss of pVHL function thus represents a pathognomonic molecular defect for therapeutic exploitation. In this study, renal carcinoma cell lines with naturally occurring vhl mutations (RCC4 VA) and their genetically matched wild-type vhl (RCC4 VHL) counterparts were seeded onto 96-well plates and treated with a collection of 1,040 organic extracts obtained from 130 bacterial strains belonging to at least 25 genera of the phyla Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes. This strategy allowed us to identify several extracts obtained from bacterial strain F-278,770T, the type strain of the recently proposed new species Pseudomonas granadensis, showing biological activities not associated with previously known bioactive metabolites. The fractionation and structural elucidation of one of these extracts led to the discovery of a new lipodepsipeptide (MDN-0066) with specific toxicity in pVHL deficient cells that is not detectable in cells with pVHL expression rescue. This specific toxicity is associated with apoptosis induction in VHL deficient cell line as demonstrated with PARP activation and Annexin V staining. Our study demonstrated the feasibility of selectively targeting the loss of the vhl tumor suppressor gene for potential clinical benefit. Our results may have great impact on the development of new targeted therapies from natural products for the treatment of cancer and other genetic diseases.

Summary: The understanding of the mechanisms of immune tolerance and the unravelling of the pathophysiology of autoimmune diseases rely on animal models. In this respect, BN and LEW rats represent models of choice to study immune-mediated diseases from the cellular and genetic points of view. Indeed, BN and LEW rats are extremes with respect to their polarisation of the immune response as well as their susceptibility to autoimmune diseases. LEW rats are susceptible to Th1-mediated autoimmune diseases while BN rats are highly susceptible to Th2-mediated autoimmune disease. Comparison of the T cell compartment between LEW and BN rats revealed several important differences. 1) A MHC-dependent quantitative difference that is due to a defect in the CD8 T cell compartment in BN rats. 2) A qualitative MHC-independent difference that is related to a high frequency of CD45RClow CD4 and CD8 T cell subsets, producing IL-4, IL-13, IL-10 and TGF-β in BN rats as compared to LEW rats. 3) Interestingly, the genetic studies showed that susceptibility to Th1-mediated experimental autoimmune encephalomyelitis, and to Th2-mediated disorders triggered by gold salts as well as the difference in the CD45RChigh/CD45RClow ratio between LEW and BN rats are genetically determined by regions on chromosomes 9, 10 and 20.

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated autoimmune disease induced in susceptible rat strains by a single immunization with myelin basic protein (MBP). The Lewis (LEW) strain is susceptible to disease induction while the Brown Norway (BN) strain is resistant. This resistance involves non-MHC genes since congenic BN-1L rats, with LEW MHC on a BN-derived background, are also resistant. In the present study we show that, upon immunization with MBP, the non-MHC-encoded resistance to develop clinical EAE in BN-1L rats is associated with a decreased production of IFN-γ. This may be due to a difference between LEW and BN-1L rats in their ability to produce regulatory cytokines such as IL-4, IL-10 and TGF-β. In comparison to LEW rats, immune lymph node cells from BN-1L rats express an increased amount of IL-4 mRNA but produce less IL-10. Furthermore, the sera from BN-1L rats contain higher amounts of active TGF-β1. Therefore, we have investigated the involvement of IL-4 and TGF-β in the resistance of BN-1L rats to develop EAE using neutralizing mAb. Neutralization of TGF-β, but not IL-4, renders BN-1L rats susceptible to clinical EAEwithout affecting the proliferation or the cytokine repertoire of immune lymph node cells. With respect to the origin of the endogenous TGF-β production, we excluded the involvement of CD8 T cells and discuss a possible role of platelets and of CD4 T cells exhibiting the CD45RClow phenotype.

// Richard Hill 1,2,* , Bastien Cautain 3,* , Nuria de Pedro 3 and Wolfgang Link 1,2,* 1 Regenerative Medicine Program, Departamento de Ciencias Biomedicas e Medicina, Universidade do Algarve, Portugal 2 IBB-Institute for Biotechnology and Bioengineering, Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve, Campus de Gambelas, Faro, Portugal 3 Fundacion MEDINA Parque tecnologico ciencias de la salud, Avd. Conocimiento 3, Granada, Spain * These authors contributed equally to this work. Correspondence: Wolfgang Link, email: // Keywords : Tumor suppressors, Cancer, Anti-cancer therapy, Nuclear export, nuclear import Received : September 30, 2013 Accepted : November 22, 2013 Published : November 24, 2013 Abstract The intracellular location and regulation of proteins within each cell is critically important and is typically deregulated in disease especially cancer. The clinical hypothesis for inhibiting the nucleo-cytoplasmic transport is based on the dependence of certain key proteins within malignant cells. This includes a host of well-characterized tumor suppressor and oncoproteins that require specific localization for their function. This aberrant localization of tumour suppressors and oncoproteins results in their their respective inactivation or over-activation. This incorrect localization occurs actively via the nuclear pore complex that spans the nuclear envelope and is mediated by transport receptors. Accordingly, given the significant need for novel, specific disease treatments, the nuclear envelope and the nuclear transport machinery have emerged as a rational therapeutic target in oncology to restore physiological nucleus/cytoplasmic homeostasis. Recent evidence suggests that this approach might be of substantial therapeutic use. This review summarizes the mechanisms of nucleo-cytoplasmic transport, its role in cancer biology and the therapeutic potential of targeting this critical cellular process

Natural products are considered an extremely valuable source for the discovery of new drugs against diverse pathologies. As yet, we have only explored a fraction of the diversity of bioactive compounds, and opportunities for discovering new natural products leading to new drugs are huge. In the present study, U2nesRELOC, a previously established cell-based imaging assay, was employed to screen a collection of extracts of microbial origin for nuclear export inhibition activity. The fluorescent signal of untreated U2nesRELOC cells localizes predominantly to the cytoplasm. Upon treatment with the nuclear export inhibitor leptomycin B, the fluorescent-tagged reporter proteins appear as speckles in the nucleus. A proprietary collection of extracts from fungi, actinomycetes, and unicellular bacteria that covers an uncommonly broad chemical space was used to interrogate this nuclear export assay system. A two-step image-based analysis allowed us to identify 12 extracts with biological activities that are not associated with previously known active metabolites. The fractionation and structural elucidation of active compounds revealed several chemical structures with nuclear export inhibition activity. Here we show that substrates of the nuclear export receptor CRM1, such as Rev, FOXO3a and NF-B, accumulate in the nucleus in the presence of the fungal metabolite MDN-0105 with an IC50 value of 3.4 mu M. Many important processes in tumor formation and progression, as well as in many viral infections, critically depend on the nucleocytoplasmic trafficking of proteins and RNA molecules. Therefore, the disruption of nuclear export is emerging as a novel therapeutic approach with enormous clinical potential. Our work highlights the potential of applying high-throughput phenotypic imaging on natural product extracts to identify novel nuclear export inhibitors. Fundacion MEDINA; MICINN [PCT-010000-2010-3] info:eu-repo/semantics/publishedVersion

The development of new anti-neoplastic drugs is a key issue for cancer chemotherapy due to the reality that, most likely, certain cancer cells are resistant to current chemotherapy. The past two decades have witnessed tremendous advances in our understanding of the pathogenesis of cancer. These advances have allowed identification new targets as oncogenes, tumor supressor genes and the possible implication of the mitochondria (Fulda et al. Nat Rev Drug Discov 9:447-464, 2010). Annonaceous Acetogenins (ACGs) have been described as the most potent inhibitors of the respiratory chain because of their interaction with mitochondrial Complex I (Degli Esposti and Ghelli Biochim Biophys Acta 1187:116-120, 1994; Zafra-Polo et al. Phytochemistry 42:253-271, 1996; Miyoshi et al. Biochim Biophys Acta 1365:443-452, 1998; Tormo et al. Arch Biochem Biophys 369:119-126, 1999; Motoyama et al. Bioorg Med Chem Lett 12:2089-2092, 2002). To explore a possible application of natural products from Annonaceous plants to cancer treatment, we have selected four bis-tetrahydrofuranic ACGs, three from Annona cherimolia (cherimolin-1, motrilin and laherradurin) and one from Rollinia mucosa (rollinianstatin-1) in order to fully describe their mechanisms responsible within the cell (Fig. 1). In this study, using a hepato-carcinoma cell line (HepG2) as a model, we showed that the bis-THF ACGs caused cell death through the induction of the apoptotic mitochondrial pathway. Their potency and behavior were compared with the classical mitochondrial respiratory chain Complex I inhibitor rotenone in every apoptotic pathway step.

e13523 Background: The phosphatidylinositol 3-kinase (PI3K) pathway couples receptor-mediated signaling to essential cellular functions by generating the lipid second messenger phosphatidylinositol...

Autoreactive T cells exist in healthy individuals and represent a potential reservoir of pathogenic effectors which, when stimulated by microbial adjuvants, could trigger an autoimmune disease. Experimental studies have indicated that xenobiotics, well defined from a chemical point of view, could promote the differentiation of autoreactive T cells towards a pathogenic pathway. It is therefore theoretically possible that compounds present in vaccines such as thiomersal or aluminium hydroxyde can trigger autoimmune reactions through bystander effects. Mercury and gold in rodents can induce immunological disorders with autoimmune reactions. In vitro, both activate signal transduction pathways that result in the expression of cytokines, particularly of IL-4 and IFNgamma. In a suitable microenvironment heavy metals could therefore favour the activation of autoreactive T cells. In that respect, genetic background is of major importance. Genome-wide searches in the rat have shown that overlapping chromosomal regions control the immunological disorders induced by gold salt treatment, the development of experimental autoimmune encephalomyelitis and the CD45RC(high)/CD45RC(low)CD4(+)T cells balance. The identification and functional characterization of genes controlling these phenotypes may shed light on key regulatory mechanisms of immune responses. This should help to improve efficacy and safety of vaccines.

The level of CD45RC expression differentiates rat CD4 T cells in two subpopulations, CD45RChigh and CD45RClow, that have different cytokine profiles and functions. Interestingly, Lewis (LEW) and Brown Norway (BN) rats, two strains that differ in their ability to mount type 1 and type 2 immune responses and in their susceptibility to autoimmune diseases, exhibit distinct CD45RChigh/CD45RClow CD4 T cell ratios. The CD45RChigh subpopulation predominates in LEW rats, and the CD45RClow subpopulation in BN rats. In this study, we found that the antiinflammatory cytokines, IL-4, IL-10, and IL-13, are exclusively produced by the CD45RClow CD4 T cells. Using bone marrow chimeras, we showed that the difference in the CD45RChigh/CD45RClow CD4 T cell ratio between naive LEW and BN rats is intrinsic to hemopoietic cells. Furthermore, a genome-wide search for loci controlling the balance between T cell subpopulations was conducted in a (LEW × BN) F2 intercross. Genome scanning identified one quantitative trait locus on chromosome 9 (∼17 centiMorgan (cM); log of the odds ratio (LOD) score 3.9). In addition, two regions on chromosomes 10 (∼28 cM; LOD score 3.1) and 20 (∼40 cM; LOD ratio score 3) that contain, respectively, a cytokine gene cluster and the MHC region were suggestive for linkage. Interestingly, overlapping regions on these chromosomes have been implicated in the susceptibility to various immune-mediated disorders. The identification and functional characterization of genes in these regions controlling the CD45RChigh/CD45RClow Th cell subpopulations may shed light on key regulatory mechanisms of pathogenic immune responses.

Mesopelagic organisms form huge biomass aggregations, supporting important pelagic trophic webs and several top predators. Although some studies on the occurrence, biology and ecology of these organisms are available, to date there are no investigations on their potential use for anticancer and antimicrobial biotechnological applications. The aim of this study was to screen extracts of seven mesopelagic species for possible anticancer (Lung cell line A549, skin cell line A2058, liver cell line HepG2, breast cell line MCF7 and pancreas cell line MiaPaca-2) and antibacterial (Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae, the Gram-positive bacteria methicillin resistant/sensitive Staphylococcus aureus, and Mycobacterium tuberculosis) activities. Results showed that only two species were active, the lanternfish Myctophum punctatum and the Mediterranean krill Meganyctiphanes norvegica. In particular, M. punctatum showed strong activity against the A549 and MCF7 cells, while M. norvegica was more active against HepG2 cells. Regarding antibacterial assays, both species were active against methicillin resistant S. aureus. Fractionation and LC/MS dereplication of the fractions showed that the main compounds found in extracts of both species were EPA, DHA and ETA. For some of the detected compounds anticancer and/or antibacterial activity are already known, but this is the first time that such activities have been found for mesopelagic species.