Extracellular Self-DNA Effects on Yeast Cell Cycle and Transcriptome during Batch Growth.

The cell cycle and the transcriptome dynamics of yeast exposed to extracellular self-DNA during an aerobic batch culture on glucose have been investigated using cytofluorimetric and RNA-seq analyses. In parallel, the same study was conducted on yeast cells growing in the presence of (heterologous) nonself-DNA. The self-DNA treatment determined a reduction in the growth rate and a major elongation of the diauxic lag phase, as well as a significant delay in the achievement of the stationary phase. This was associated with significant changes in the cell cycle dynamics, with slower exit from the G0 phase, followed by an increased level of cell percentage in the S phase, during the cultivation. Comparatively, the exposure to heterologous DNA did not affect the growth curve and the cell cycle dynamics. The transcriptomic analysis showed that self-DNA exposure produced a generalized downregulation of transmembrane transport and an upregulation of genes associated with sulfur compounds and the pentose phosphate pathway. Instead, in the case of the nonself treatment, a clear response to nutrient deprivation was detected. Overall, the presented findings represent further insights into the complex functional mechanisms of self-DNA inhibition.

RAF1 gene fusions are recurrent driver events in infantile fibrosarcoma-like mesenchymal tumors.

Infantile fibrosarcomas (IFS) and congenital mesoblastic nephroma (CMN) are rare myofibroblastic tumors of infancy and early childhood commonly harboring the ETV6::NTRK3 gene fusion. IFS/CMN are considered as tumors with an 'intermediate prognosis' as they are locally aggressive, but rarely metastasize, and generally have a favorable outcome. A fraction of IFS/CMN-related neoplasms are negative for the ETV6::NTRK3 gene rearrangement and are characterized by other chimeric proteins promoting MAPK signaling upregulation. In a large proportion of these tumors, which are classified as IFS-like mesenchymal neoplasms, the contributing molecular events remain to be identified. Here, we report three distinct rearrangements involving RAF1 among eight ETV6::NTRK3 gene fusion-negative tumors with an original histological diagnosis of IFS/CMN. The three fusion proteins retain the entire catalytic domain of the kinase. Two chimeric products, GOLGA4::RAF1 and LRRFIP2::RAF1, had previously been reported as driver events in different cancers, whereas the third, CLIP1::RAF1, represents a novel fusion protein. We demonstrate that CLIP1::RAF1 acts as a bona fide oncoprotein promoting cell proliferation and migration through constitutive upregulation of MAPK signaling. We show that the CLIP1::RAF1 hyperactive behavior does not require RAS activation and is mediated by constitutive 14-3-3 protein-independent dimerization of the chimeric protein. As previously reported for the ETV6::NTRK3 fusion protein, CLIP1::RAF1 similarly upregulates PI3K-AKT signaling. Our findings document that RAF1 gene rearrangements represent a recurrent event in ETV6::NTRK3-negative IFS/CMN and provide a rationale for the use of inhibitors directed to suppress MAPK and PI3K-AKT signaling in these cancers. © 2024 The Pathological Society of Great Britain and Ireland.

Mycobacterium tuberculosis antigen 85B modifies BCG-induced antituberculosis immunity and favors pathogen survival.

Tuberculosis is one of the deadliest infectious diseases worldwide. Mycobacterium tuberculosis has developed strategies not only to evade host immunity but also to manipulate it for its survival. We investigated whether Mycobacterium tuberculosis exploited the immunogenicity of Ag85B, one of its major secretory proteins, to redirect host antituberculosis immunity to its advantage. We found that administration of Ag85B protein to mice vaccinated with Bacillus Calmette-Guérin impaired the protection elicited by vaccination, causing a more severe infection when mice were challenged with Mycobacterium tuberculosis. Ag85B administration reduced Bacillus Calmette-Guérin-induced CD4 T-cell activation and IFN-γ, CCL-4, and IL-22 production in response to Mycobacterium tuberculosis-infected cells. On the other hand, it promoted robust Ag85B-responsive IFN-γ-producing CD4 T cells, expansion of a subset of IFN-γ/IL-10-producing CD4+FOXP3+Treg cells, differential activation of IL-17/IL-22 responses, and activation of regulatory and exhaustion pathways, including programmed death ligand 1 expression on macrophages. All this resulted in impaired intracellular Mycobacterium tuberculosis growth control by systemic immunity, both before and after the Mycobacterium tuberculosis challenge. Interestingly, Mycobacterium tuberculosis infection itself generated Ag85B-reactive inflammatory immune cells incapable of clearing Mycobacterium tuberculosis in both unvaccinated and Bacillus Calmette-Guérin-vaccinated mice. Our data suggest that Mycobacterium tuberculosis can exploit the strong immunogenicity of Ag85B to promote its own survival and spread. Since Ag85B is normally secreted by replicating bacteria and is commonly found in the lungs of the Mycobacterium tuberculosis-infected host, our findings may advance the understanding on the mechanisms of Mycobacterium tuberculosis pathogenesis and immune evasion.

CD8+CD103+PD1+TIM3+ T cells in glioblastoma microenvironment correlate with prognosis.

Glioblastoma, isocitrate dehydrogenase-wildtype (GB), is the most common and aggressive primary brain malignancy with poor outcome. Immune checkpoint inhibitors (ICIs) have been tested in GB and, despite disappointing results, the identification of a small subgroup of responders underlies the need to improve our understanding of the tumour microenvironment (TME) immunity. This study aimed to determine whether the expression of selected immune checkpoints on tissue-resident memory T cells (Trm) may predict patient outcome. We conducted a single cohort observational study. Tumour samples were collected from 45 patients with histologically confirmed GB (WHO grade 4) and processed to obtain single-cell suspensions. Patients were assessed for the correlation of Trm phenotype with overall survival (OS) or progression-free survival (PFS) using multiparametric flow cytometry and uni/multivariate analyses. Levels of Trm expressing programmed cell death protein 1 (PD1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) were found to be linked to clinical outcome. Low frequency of Trm expressing PD1 or TIM3 or both markers defined subgroups as independent positive prognostic factors for patient survival. On multivariate analysis, low CD8+CD103+PD1+TIM3+ Trm and Karnofsky performance status (KPS) ≥70 were confirmed to be the most predictive independent factors associated with longer OS (hazard ratios-HR [95%CI]: 0.14 [0.04-0.52] p < 0.001, 0.39 [0.16-0.96] p = 0.04, respectively). The CD8+CD103+ Trm subgroups were also age-related predictors for survival in GB.

Metabolic labelling of a subpopulation of small extracellular vesicles using a fluorescent palmitic acid analogue.

Exosomes are among the most puzzling vehicles of intercellular communication, but several crucial aspects of their biogenesis remain elusive, primarily due to the difficulty in purifying vesicles with similar sizes and densities. Here we report an effective methodology for labelling small extracellular vesicles (sEV) using Bodipy FL C16, a fluorescent palmitic acid analogue. In this study, we present compelling evidence that the fluorescent sEV population derived from Bodipy C16-labelled cells represents a discrete subpopulation of small exosomes following an intracellular pathway. Rapid cellular uptake and metabolism of Bodipy C16 resulted in the incorporation of fluorescent phospholipids into intracellular organelles specifically excluding the plasma membrane and ultimately becoming part of the exosomal membrane. Importantly, our fluorescence labelling method facilitated accurate quantification and characterization of exosomes, overcoming the limitations of nonspecific dye incorporation into heterogeneous vesicle populations. The characterization of Bodipy-labelled exosomes reveals their enrichment in tetraspanin markers, particularly CD63 and CD81, and in minor proportion CD9. Moreover, we employed nanoFACS sorting and electron microscopy to confirm the exosomal nature of Bodipy-labelled vesicles. This innovative metabolic labelling approach, based on the fate of a fatty acid, offers new avenues for investigating exosome biogenesis and functional properties in various physiological and pathological contexts.

Extracellular DNA secreted in yeast cultures is metabolism-specific and inhibits cell proliferation.

Extracellular DNA (exDNA) can be actively released by living cells and different putative functions have been attributed to it. Further, homologous exDNA has been reported to exert species-specific inhibitory effects on several organisms. Here, we demonstrate by different experimental evidence, including 1H-NMR metabolomic fingerprint, that the growth rate decline in Saccharomyces cerevisiae fed-batch cultures is determined by the accumulation of exDNA in the medium. Sequencing of such secreted exDNA represents a portion of the entire genome, showing a great similarity with extrachromosomal circular DNA (eccDNA) already reported inside yeast cells. The recovered DNA molecules were mostly single strands and specifically associated to the yeast metabolism displayed during cell growth. Flow cytometric analysis showed that the observed growth inhibition by exDNA corresponded to an arrest in the S phase of the cell cycle. These unprecedented findings open a new scenario on the functional role of exDNA produced by living cells.

A cellular screening platform, stably expressing DENV2 NS5, defines a novel anti-DENV mechanism of action of Apigenin based on STAT2 activation.

Type I interferon (IFN-I) evasion by Dengue virus (DENV) is key in DENV pathogenesis. The non-structural protein 5 (NS5) antagonizes IFN-I response through the degradation of the signal transducer and activator of transcription 2 (STAT2). We developed a K562 cell-based platform, for high throughput screening of compounds potentially counteracting the NS5-mediated antagonism of IFN-I signaling. Upon a screening with a library of 1220 approved drugs, 3 compounds previously linked to DENV inhibition (Apigenin, Chrysin, and Luteolin) were identified. Luteolin and Apigenin determined a significant inhibition of DENV2 replication in Huh7 cells and the restoration of STAT2 phosphorylation in both cell systems. Apigenin and Luteolin were able to stimulate STAT2 even in the absence of infection. Despite the "promiscuous" and "pan-assay-interfering" nature of Luteolin, Apigenin promotes STAT2 Tyr 689 phosphorylation and activation, highlighting the importance of screening for compounds able to interact with host factors, to counteract viral proteins capable of dampening innate immune responses.

Differential Effects of Alarmins on Human and Mouse Basophils.

Epithelial-derived alarmins (IL-33, TSLP, and IL-25) play an upstream role in the pathogenesis of asthma. Basophil-derived cytokines are a pivotal component of allergic inflammation. We evaluated the in vitro effects of IL-33, TSLP, and IL-25, alone and in combination with IL-3 on purified peripheral blood human basophils (hBaso) and bone marrow-derived mouse basophils (mBaso) in modulating the production of IL-4, IL-13, CXCL8 or the mouse CXCL8 equivalents CXCL1 and CXCL2. IL-3 and IL-33, but not TSLP and IL-25, concentration-dependently induced IL-4, IL-13, and CXCL8 release from hBaso. IL-3 synergistically potentiated the release of cytokines induced by IL-33 from hBaso. In mBaso, IL-3 and IL-33 rapidly induced IL-4 and IL-13 mRNA expression and protein release. IL-33, but not IL-3, induced CXCL2 and CXCL1 from mBaso. Differently from hBaso, TSLP induced IL-4, IL-13, CXCL1 and CXCL2 mRNA expression and protein release from mBaso. IL-25 had no effect on IL-4, IL-13, and CXCL1/CXCL2 mRNA expression and protein release even in the presence of IL-3. No synergism was observed between IL-3 and either IL-25 or TSLP. IL-3 inhibited both TSLP- and IL-33-induced CXCL1 and CXCL2 release from mBaso. Our results highlight some similarities and marked differences between the effects of IL-3 and alarmins on the release of cytokines from human and mouse basophils.

Generation, Characterization, and Count of Fluorescent Extracellular Vesicles.

Extracellular vesicles (EVs) are membranous particles released by all cells in the external milieu. Depending on their origin, they are given different names: exosomes are nanovesicles that originate from the endosomal compartment, whereas microvesicles bud from plasma membrane. Both contain molecules that are crucial for the onset and spreading of different pathologies, from neurodegenerative diseases to cancer, and are considered promising disease markers. On the other hand, EVs are often used as therapeutic tools, and can be engineered to carry drugs and chemicals. This chapter describes a method to produce EVs, mainly exosomes, containing the green fluorescent protein (GFP) linked to an exosome anchoring protein (Nefmut). This enables counting and tracing of fluorescent EVs by different methods, including conventional flow cytometry.

Different Susceptibilities of Human Melanoma Cell Lines to G2/M Blockage and Cell Death Activation in Response to the Estrogen Receptor ß agonist LY500307.

Background: Gender differences in melanoma incidence, metastasis formation and disease progression are increasingly evident in epidemiological studies, with women showing significantly better survival than men. Among factors possibly underlying the disparities, sex hormones seem to play a key role. Nonetheless, functional mechanisms are still unclear, except for the antitumor ability of Estrogen Receptor (ER) ß, whose expression determination has often been suggested for melanoma prognosis. In this study, we aimed at evaluating the molecular mechanisms and functional effects associated with ERß signaling by using its agonist LY500307. Methods: We evaluated the antitumor effect of the specific synthetic ERß agonist LY500307 on some human melanoma cell lines, selected for different genetic background, expression levels of ERs and tumor progression. The expression of α and ß estrogen receptors was investigated taking advantage of The Cancer Genome Atlas database and confirmed on some selected melanoma cell lines. The biological effects of LY500307 were determined in vitro looking at melanoma cell proliferation, cell cycle profiles and migration demonstrating by western blot the involvement of some pathway specific markers. The LY500307-dependent induction of cell death was also analyzed by flow cytometry and western blot analysis of caspase 3 and poly adenosine diphosphate-ribose polymerase (PARP). Results: A significant decrease in the expression of both ERs, even more pronounced for ERα, has been found in patients with metastatic NRAS mutation. Treatment with LY500307 significantly reduced the proliferation of melanoma cells showing a cell cycle arrest at the G2/M boundary phase and promoting apoptosis with different sensitivities associated with disease stage and mutation. Indeed, the ERß agonist affects melanoma migration, inducing a reversion of the epithelial-mesenchymal transition, more evident in a low aggressive primary melanoma cell line. Conclusion: These results demonstrate the capability of LY500307 to reduce melanoma malignancy, counteracting cell viability and dissemination, overall suggesting a possible future use of LY500307 in personalized combined therapy.

Hyperactive HRAS dysregulates energetic metabolism in fibroblasts from patients with Costello syndrome via enhanced production of reactive oxidizing species.

Germline-activating mutations in HRAS cause Costello syndrome (CS), a cancer prone multisystem disorder characterized by reduced postnatal growth. In CS, poor weight gain and growth are not caused by low caloric intake. Here, we show that constitutive plasma membrane translocation and activation of the GLUT4 glucose transporter, via reactive oxygen species-dependent AMP-activated protein kinase α and p38 hyperactivation, occurs in primary fibroblasts of CS patients, resulting in accelerated glycolysis and increased fatty acid synthesis and storage as lipid droplets. An accelerated autophagic flux was also identified as contributing to the increased energetic expenditure in CS. Concomitant inhibition of p38 and PI3K signaling by wortmannin was able to rescue both the dysregulated glucose intake and accelerated autophagic flux. Our findings provide a mechanistic link between upregulated HRAS function, defective growth and increased resting energetic expenditure in CS, and document that targeting p38 and PI3K signaling is able to revert this metabolic dysfunction.

pH-responsive oleic acid based nanocarriers: Melanoma treatment strategies.

Numerous clinical observations indicate that, despite novel therapeutic approaches, a high percentage of melanoma patients is non-responder or suffers of severe drug-related toxicity. To overcome these problems, we considered the option of designing, preparing and characterizing nanoemulsions and niosomes containing oleic acid, a pH-sensitive monounsaturated fatty acid holding per se an antimetastatic and anti-inflammatory role in melanoma. These new nanostructures will allow in vivo administration of oleic acid, otherwise toxic in its free form. For pulmonary route chitosan, a mucoadhesive agent, was enclosed in these nanocarriers to improve residence time at the lung site. A deep physical and chemical characterization was carried out evaluating size, ζ -potential, microviscosity, polarity as well as stability over time and in culture media. Moreover, their pH-sensitivity was evaluated by fluorometric assay. Cytotoxicity and cellular uptake were assessed in cultured normal fibroblasts and human melanoma cell lines. Interestingly, results obtained confirm nanocarrier stability and pH-sensitivity, associated to absence of cell toxicity, efficient cellular uptake and retention. Therefore, these new pH-sensitive oleic acid-based nanostructures could represent, by combining drug delivery in a pH-dependent manner with the antimetastatic potential of this fatty acid, a powerful strategy for more specific medicine against metastatic melanoma.

Structural organization of erythrocyte membrane microdomains and their relation with malaria susceptibility.

Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and protein composition. These dynamic assemblies are involved in several biological processes, including infection by intracellular pathogens. This work provides a comprehensive analysis of the composition of human erythrocyte membrane microdomains. Based on their floating properties, we also categorized the microdomain-associated proteins into clusters. Interestingly, erythrocyte microdomains include the vast majority of the proteins known to be involved in invasion by the malaria parasite Plasmodium falciparum. We show here that the Ecto-ADP-ribosyltransferase 4 (ART4) and Aquaporin 1 (AQP1), found within one specific cluster, containing the essential host determinant CD55, are recruited to the site of parasite entry and then internalized to the newly formed parasitophorous vacuole membrane. By generating null erythroid cell lines, we showed that one of these proteins, ART4, plays a role in P. falciparum invasion. We also found that genetic variants in both ART4 and AQP1 are associated with susceptibility to the disease in a malaria-endemic population.

Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein-Protein Interactions.

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2-20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein-protein interactions in the function of SHP2.

A cytofluorimetric analysis of a Saccharomyces cerevisiae population cultured in a fed-batch bioreactor.

The yeast Saccharomyces cerevisiae is a reference model system and one of the widely used microorganisms in many biotechnological processes. In industrial yeast applications, combined strategies aim to maximize biomass/product yield, with the fed-batch culture being one of the most frequently used. Flow cytometry (FCM) is widely applied in biotechnological processes and represents a key methodology to monitor cell population dynamics. We propose here an application of FCM in the analysis of yeast cell cycle along the time course of a typical S. cerevisiae fed-batch culture. We used two different dyes, SYTOX Green and SYBR Green, with the aim to better define each stage of cell cycle during S. cerevisiae fed-batch culture. The results provide novel insights in the use of FCM cell cycle analysis for the real-time monitoring of S. cerevisiae bioprocesses.

Characterization of the erythrocyte GTPase Rac1 in relation to Plasmodium falciparum invasion.

Malaria is still a devastating disease with 228 million cases globally and 405,000 lethal outcomes in 2018, mainly in children under five years of age. The threat of emerging malaria strains resistant to currently available drugs has made the search for novel drug targets compelling. The process by which Plasmodium falciparum parasites invade the host cell has been widely studied, but only a few erythrocyte proteins involved in this process have been identified so far. The erythrocyte protein Rac1 is a GTPase that plays an important role in host cell invasion by many intracellular pathogens. Here we show that Rac1 is recruited in proximity to the site of parasite entry during P. falciparum invasion process and that subsequently localizes to the parasitophorous vacuole membrane. We also suggest that this GTPase may be involved in erythrocyte invasion by P. falciparum, by testing the effect of specific Rac1 inhibitory compounds. Finally, we suggest a secondary role of the erythrocyte GTPase also in parasite intracellular development. We here characterize a new erythrocyte protein potentially involved in P. falciparum invasion of the host cell and propose the human GTPase Rac1 as a novel and promising antimalarial drug target.

Corrigendum to "Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring".

[This corrects the article DOI: 10.1155/2020/1938704.].

Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring.

BACKGROUND: Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. METHODS: The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. RESULTS: Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. CONCLUSION: Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.

Zooplankton in Adriatic port environments: Indigenous communities and non-indigenous species.

The zooplankton community was analyzed in ten Adriatic ports as part of the port biological baseline surveys carried out within the framework of the BALMAS project. We provide the first inventory of resident zooplankton taxa and five detected non-indigenous zooplankton species (NIS), and their spatial and seasonal distribution patterns. Copepoda and meroplankton larvae, particularly of Mollusca, dominated the zooplankton in all sampled ports. We recorded a total of 76 indigenous copepod species and five NIS, among which Parvocalanus crassirostris detected in Sibenik and Rijeka ports and Oithona davisae in Venice port, are new for the Adriatic. All detected NIS were widely distributed within the recipient ports. Co-occurrences of NIS were observed in the ports of Venice, Bari, Ancona and Trieste. The results are expected to contribute to the quality of practical monitoring of zooplankton NIS and facilitate the synchronization of efforts in creating NIS-related policies for the Adriatic sub-region.

Tetanus-diphtheria vaccination in adults: the long-term persistence of antibodies is not dependent on polyclonal B-cell activation and the defective response to diphtheria toxoid re-vaccination is associated to HLADRB1∗01.

Cellular and humoral immune responses to tetanus-diphtheria vaccine (Td) were assessed in human leukocyte antigen (HLA)-typed Italian military personnel who received multiple concomitant vaccines. Td-specific antibodies and T-lymphocytes were measured in individuals with one (group-1) and more than one (group-2) Td boosters. A third group (group-3), who received several vaccines, but not Td, was studied to verify the hypothesis of the polyclonal B-cell activation as mechanism for antibody persistence. The antibody response to Td toxoids was higher in group-1, who showed lower baseline antibody levels, than in group-2 subjects. The antibody response to tetanus was higher than to diphtheria toxoid in both groups. No correlation between antibody and cellular response, and no interference in the response to Td by co-administration of different vaccines were observed. HLA-DRB1∗01 allele was detected at significant higher frequency in subjects unable to double the baseline anti-diphtheria antibody levels after the vaccination. Anti-tetanus and diphtheria antibodies half-lives were assessed and the long-lasting persistence above the threshold for protection (0.1 IU/ml) was estimated in over 65 and 20 years, respectively. No significant increase of anti-diphtheria antibodies was observed in consequence of polyclonal B-cell activation. This study emphasizes the duration of Td vaccination-induced seroprotection, suggesting that re-vaccination should probably be performed at intervals longer than 10 years. No reciprocal interference by concomitantly administered vaccines has been observed. HLA-DRB1∗01 allele was significantly associated with anti-diphtheria defective response. Finally, this study does not confirm that anti-diphtheria antibody levels are maintained by polyclonal B-cell activation. Clinical trial registry: The study was registered with NCT01807780.