Defects of mitochondria-lysosomes communication induce secretion of mitochondria-derived vesicles and drive chemoresistance in ovarian cancer cells.

BACKGROUND: Among the mechanisms of mitochondrial quality control (MQC), generation of mitochondria-derived vesicles (MDVs) is a process to avoid complete failure of mitochondria determining lysosomal degradation of mitochondrial damaged proteins. In this context, RAB7, a late endocytic small GTPase, controls delivery of MDVs to late endosomes for subsequent lysosomal degradation. We previously demonstrated that RAB7 has a pivotal role in response to cisplatin (CDDP) regulating resistance to the drug by extracellular vesicle (EVs) secretion. METHODS: Western blot and immunofluorescence analysis were used to analyze structure and function of endosomes and lysosomes in CDDP chemosensitive and chemoresistant ovarian cancer cell lines. EVs were purified from chemosensitive and chemoresistant cells by ultracentrifugation or immunoisolation to analyze their mitochondrial DNA and protein content. Treatment with cyanide m-chlorophenylhydrazone (CCCP) and RAB7 modulation were used, respectively, to understand the role of mitochondrial and late endosomal/lysosomal alterations on MDV secretion. Using conditioned media from chemoresistant cells the effect of MDVs on the viability after CDDP treatment was determined. Seahorse assays and immunofluorescence analysis were used to study the biochemical role of MDVs and the uptake and intracellular localization of MDVs, respectively. RESULTS: We observed that CDDP-chemoresistant cells are characterized by increased MDV secretion, impairment of late endocytic traffic, RAB7 downregulation, an increase of RAB7 in EVs, compared to chemosensitive cells, and downregulation of the TFEB-mTOR pathway overseeing lysosomal and mitochondrial biogenesis and turnover. We established that MDVs can be secreted rather than delivered to lysosomes and are able to deliver CDDP outside the cells. We showed increased secretion of MDVs by chemoresistant cells ultimately caused by the extrusion of RAB7 in EVs, resulting in a dramatic drop in its intracellular content, as a novel mechanism to regulate RAB7 levels. We demonstrated that MDVs purified from chemoresistant cells induce chemoresistance in RAB7-modulated process, and, after uptake from recipient cells, MDVs localize to mitochondria and slow down mitochondrial activity. CONCLUSIONS: Dysfunctional MQC in chemoresistant cells determines a block in lysosomal degradation of MDVs and their consequent secretion, suggesting that MQC is not able to eliminate damaged mitochondria whose components are secreted becoming effectors and potential markers of chemoresistance.

Mutant-Dependent Local Orientational Correlation in Biofilms of Vibrio campbellii Revealed through Digital Processing of Light Microscopy Images.

Biofilms are key bacterial communities in genetic and adaptive resistance to antibiotics as well as disease control strategies. The mature high-coverage biofilm formations of the Vibrio campbellii strains (wild type BB120 and isogenic derivatives JAF633, KM387, and JMH603) are studied here through the unstraightforward digital processing of morphologically complex images without segmentation or the unrealistic simplifications used to artificially simulate low-density formations. The main results concern the specific mutant- and coverage-dependent short-range orientational correlation as well as the coherent development of biofilm growth pathways over the subdomains of the image. These findings are demonstrated to be unthinkable based only on a visual inspection of the samples or on methods such as Voronoi tessellation or correlation analyses. The presented approach is general, relies on measured rather than simulated low-density formations, and could be employed in the development of a highly efficient screening method for drugs or innovative materials.

Rid7C, a Member of the YjgF/YER057c/UK114 (Rid) Protein Family, Is a Novel Endoribonuclease That Regulates the Expression of a Specialist RNA Polymerase Involved in Differentiation in Nonomuraea gerenzanensis.

The YjgF/YER057c/UK114 (Rid) is a protein family breadth conserved in all domains of life and includes the widely distributed archetypal RidA (YjgF) subfamily and seven other subfamilies (Rid1 to Rid7). Among these subfamilies, RidA is the only family to have been biochemically well characterized and is involved in the deamination of the reactive enamine/imine intermediates. In this study, we have characterized a protein of the Rid7 subfamily, named Rid7C, in Nonomuraea gerenzanensis, an actinomycete that is characterized by the presence of two types of RNA polymerases. This is due to the coexistence in its genome of two RNA polymerase (RNAP) ß chain-encoding genes, rpoB(S) (the wild-type rpoB gene) and rpoB(R) (a specialist, mutant-type rpoB gene) that controls A40926 antibiotic production and a wide range of metabolic adaptive behaviors. Here, we found that expression of rpoB(R) is regulated posttranscriptionally by RNA processing in the 5' untranslated region (UTR) of rpoB(R) mRNA and that the endoribonuclease activity of Rid7C is responsible for mRNA processing, thereby overseeing several tracts of morphological and biochemical differentiation. We also provide evidence that Rid7C may be associated with RNase P M1 RNA, although M1 RNA is not required for rpoB(R) mRNA processing in vitro, and that Rid7C endoribonuclease activity is inhibited by A40926, suggesting the existence of a negative feedback loop in A40926 production and a role of the endogenous synthesis of A40926 in the modulation of biochemical differentiation in this microorganism. IMPORTANCE The YjgF/YER057c/UK114 family includes many proteins with diverse functions involved in detoxification, RNA maturation, and control of mRNA translation. We found that Rid7C is an endoribonuclease that is involved in processing of rpoB(R) mRNA, coding for a specialized RNA polymerase beta subunit that oversees morphological differentiation and A40926 antibiotic production in Nonomuraea gerenzanensis. Rid7C-mediated processing promotes rpoB(R) mRNA translation and antibiotic production, while Rid7C endoribonuclease activity is inhibited by A40926, suggesting a role of the endogenous synthesis of A40926 in modulation of biochemical differentiation in this microorganism. Finally, we show that recombinant Rid7C copurified with M1 RNA (the RNA subunit of RNase P) from Escherichia coli extract, suggesting a functional interaction between Rid7C and M1 RNA activities.

HrpA anchors meningococci to the dynein motor and affects the balance between apoptosis and pyroptosis.

BACKGROUND: In Neisseria meningitidis the HrpA/HrpB two-partner secretion system (TPS) was implicated in diverse functions including meningococcal competition, biofilm formation, adherence to epithelial cells, intracellular survival and vacuolar escape. These diverse functions could be attributed to distinct domains of secreted HrpA. METHODS: A yeast two-hybrid screening, in vitro pull-down assay and immunofluorescence microscopy experiments were used to investigate the interaction between HrpA and the dynein light-chain, Tctex-type 1 (DYNLT1). In silico modeling was used to analyze HrpA structure. Western blot analysis was used to investigate apoptotic and pyroptotic markers. RESULTS: The HrpA carboxy-terminal region acts as a manganese-dependent cell lysin, while the results of a yeast two-hybrid screening demonstrated that the HrpA middle region has the ability to bind the dynein light-chain, Tctex-type 1 (DYNLT1). This interaction was confirmed by in vitro pull-down assay and immunofluorescence microscopy experiments showing co-localization of N. meningitidis with DYNLT1 in infected epithelial cells. In silico modeling revealed that the HrpA-M interface interacting with the DYNLT1 has similarity with capsid proteins of neurotropic viruses that interact with the DYNLT1. Indeed, we found that HrpA plays a key role in infection of and meningococcal trafficking within neuronal cells, and is implicated in the modulation of the balance between apoptosis and pyroptosis. CONCLUSIONS: Our findings revealed that N. meningitidis is able to effectively infect and survive in neuronal cells, and that this ability is dependent on HrpA, which establishes a direct protein-protein interaction with DYNLTI in these cells, suggesting that the HrpA interaction with dynein could be fundamental for N. meningitidis spreading inside the neurons. Moreover, we found that the balance between apoptotic and pyroptotic pathways is heavily affected by HrpA.

An Integrative, Multiparametric Approach for the Comprehensive Assessment of Microbial Quality and Pollution in Aquaculture Systems.

As the aquaculture sector significantly expanded worldwide in the past decades, the concept of sustainable aquaculture has developed with the challenge of not only maximizing benefits but also minimizing the negative impacts on the environment assuring, at the same time, food security. In this framework, monitoring and improving the microbiological water quality and animal health are a central topic. In the present study, we evaluated the seawater microbiological quality in a mariculture system located in a Mediterranean coastal area (Northern Ionian Sea, Italy). We furnished, for the first time, a microbial inventory based on conventional culture-based methods, integrated with the 16S rRNA gene metabarcoding approach for vibrios identification and diversity analyses, and further implemented with microbial metabolic profiling data obtained from the Biolog EcoPlate system. Microbiological pollution indicators, vibrios diversity, and microbial metabolism were determined in two different times of the year (July and December). All microbial parameters measured in July were markedly increased compared to those measured in December. The presence of potentially pathogenic vibrios is discussed concerning the risk of fish disease and human infections. Thus, the microbial inventory here proposed might represent a new multiparametric approach for the suitable surveillance of the microbial quality in a mariculture system. Consequently, it could be useful for ensuring the safety of both the reared species and the consumers in the light of sustainable, eco-friendly aquaculture management.

Evidence for a Negative Correlation between Human Reactive Enamine-Imine Intermediate Deaminase A (RIDA) Activity and Cell Proliferation Rate: Role of Lysine Succinylation of RIDA.

Reactive intermediate deaminase (Rid) proteins are enzymes conserved in all domains of life. UK114, a mammalian member of RidA subfamily, has been firstly identified as a component of liver perchloric acid-soluble proteins (L-PSP). Although still poorly defined, several functions have been attributed to the mammalian protein UK114/RIDA, including the reactive intermediate deamination activity. The expression of UK114/RIDA has been observed in some tumors, arousing interest in this protein as an evaluable tumor marker. However, other studies reported a negative correlation between UK114/RIDA expression, tumor differentiation degree and cell proliferation. This work addressed the question of UK114/RIDA expression in human non-tumor HEK293 cell lines and in some human tumor cell lines. Here we reported that human RIDA (hRIDA) was expressed in all the analyzed cell line and subjected to lysine (K-)succinylation. In HEK293, hRIDA K-succinylation was negatively correlated to the cell proliferation rate and was under the control of SIRT5. Moreover, K-succinylation clearly altered hRIDA quantification by immunoblotting, explaining, at least in part, some discrepancies about RIDA expression reported in previous studies. We found that hRIDA was able to deaminate reactive enamine-imine intermediates and that K-succinylation drastically reduced deaminase activity. As predicted by in silico analysis, the observed reduction of deaminase activity has been related to the drastic alterations of hRIDA structure inferred by K-succinylation. The role of hRIDA and the importance of its K-succinylation in cell metabolism, especially in cancer biology, have been discussed.

RhoTermPredict: an algorithm for predicting Rho-dependent transcription terminators based on Escherichia coli, Bacillus subtilis and Salmonella enterica databases.

BACKGROUND: In bacterial genomes, there are two mechanisms to terminate the DNA transcription: the "intrinsic" or Rho-independent termination and the Rho-dependent termination. Intrinsic terminators are characterized by a RNA hairpin followed by a run of 6-8 U residues relatively easy to identify using one of the numerous available prediction programs. In contrast, Rho-dependent termination is mediated by the Rho protein factor that, firstly, binds to ribosome-free mRNA in a site characterized by a C > G content and then reaches the RNA polymerase to induce its release. Conversely on intrinsic terminators, the computational prediction of Rho-dependent terminators in prokaryotes is a very difficult problem because the sequence features required for the function of Rho are complex and poorly defined. This is the reason why it still does not exist an exhaustive Rho-dependent terminators prediction program. RESULTS: In this study we introduce RhoTermPredict, the first published algorithm for an exhaustive Rho-dependent terminators prediction in bacterial genomes. RhoTermPredict identifies these elements based on a previously proposed consensus motif common to all Rho-dependent transcription terminators. It essentially searches for a 78 nt long RUT site characterized by a C > G content and with regularly spaced C residues, followed by a putative pause site for the RNA polymerase. We tested RhoTermPredict performances by using available genomic and transcriptomic data of the microorganism Escherichia coli K-12, both in limited-length sequences and in the whole-genome, and available genomic sequences from Bacillus subtilis 168 and Salmonella enterica LT2 genomes. We also estimated the overlap between the predictions of RhoTermPredict and those obtained by the predictor of intrinsic terminators ARNold webtool. Our results demonstrated that RhoTermPredict is a very performing algorithm both for limited-length sequences (F1-score obtained about 0.7) and for a genome-wide analysis. Furthermore the degree of overlap with ARNold predictions was very low. CONCLUSIONS: Our analysis shows that RhoTermPredict is a powerful tool for Rho-dependent terminators search in the three analyzed genomes and could fill this gap in computational genomics. We conclude that RhoTermPredict could be used in combination with an intrinsic terminators predictor in order to predict all the transcription terminators in bacterial genomes.

Virulence Traits of a Serogroup C Meningococcus and Isogenic cssA Mutant, Defective in Surface-Exposed Sialic Acid, in a Murine Model of Meningitis.

In serogroup C Neisseria meningitidis, the cssA (siaA) gene codes for an UDP-N-acetylglucosamine 2-epimerase that catalyzes the conversion of UDP-N-acetyl-α-d-glucosamine into N-acetyl-d-mannosamine and UDP in the first step in sialic acid biosynthesis. This enzyme is required for the biosynthesis of the (α2→9)-linked polysialic acid capsule and for lipooligosaccharide (LOS) sialylation. In this study, we have used a reference serogroup C meningococcal strain and an isogenic cssA knockout mutant to investigate the pathogenetic role of surface-exposed sialic acids in a model of meningitis based on intracisternal inoculation of BALB/c mice. Results confirmed the key role of surface-exposed sialic acids in meningococcal pathogenesis. The 50% lethal dose (LD50) of the wild-type strain 93/4286 was about four orders of magnitude lower than that of the cssA mutant. Compared to the wild-type strain, the ability of this mutant to replicate in brain and spread systemically was severely impaired. Evaluation of brain damage evidenced a significant reduction in cerebral hemorrhages in mice infected with the mutant in comparison with the levels in those challenged with the wild-type strain. Histological analysis showed the typical features of bacterial meningitis, including inflammatory cells in the subarachnoid, perivascular, and ventricular spaces especially in animals infected with the wild type. Noticeably, 80% of mice infected with the wild-type strain presented with massive bacterial localization and accompanying inflammatory infiltrate in the corpus callosum, indicating high tropism of meningococci exposing sialic acids toward this brain structure and a specific involvement of the corpus callosum in the mouse model of meningococcal meningitis.

G4PromFinder: an algorithm for predicting transcription promoters in GC-rich bacterial genomes based on AT-rich elements and G-quadruplex motifs.

BACKGROUND: Over the last few decades, computational genomics has tremendously contributed to decipher biology from genome sequences and related data. Considerable effort has been devoted to the prediction of transcription promoter and terminator sites that represent the essential "punctuation marks" for DNA transcription. Computational prediction of promoters in prokaryotes is a problem whose solution is far from being determined in computational genomics. The majority of published bacterial promoter prediction tools are based on a consensus-sequences search and they were designed specifically for vegetative σ70 promoters and, therefore, not suitable for promoter prediction in bacteria encoding a lot of σ factors, like actinomycetes. RESULTS: In this study we investigated the possibility to identify putative promoters in prokaryotes based on evolutionarily conserved motifs, and focused our attention on GC-rich bacteria in which promoter prediction with conventional, consensus-based algorithms is often not-exhaustive. Here, we introduce G4PromFinder, a novel algorithm that predicts putative promoters based on AT-rich elements and G-quadruplex DNA motifs. We tested its performances by using available genomic and transcriptomic data of the model microorganisms Streptomyces coelicolor A3(2) and Pseudomonas aeruginosa PA14. We compared our results with those obtained by three currently available promoter predicting algorithms: the σ70consensus-based PePPER, the σ factors consensus-based bTSSfinder, and PromPredict which is based on double-helix DNA stability. Our results demonstrated that G4PromFinder is more suitable than the three reference tools for both the genomes. In fact our algorithm achieved the higher accuracy (F1-scores 0.61 and 0.53 in the two genomes) as compared to the next best tool that is PromPredict (F1-scores 0.46 and 0.48). Consensus-based algorithms produced lower performances with the analyzed GC-rich genomes. CONCLUSIONS: Our analysis shows that G4PromFinder is a powerful tool for promoter search in GC-rich bacteria, especially for bacteria coding for a lot of σ factors, such as the model microorganism S. coelicolor A3(2). Moreover consensus-based tools and, in general, tools that are based on specific features of bacterial σ factors seem to be less performing for promoter prediction in these types of bacterial genomes.

Pirin: A novel redox-sensitive modulator of primary and secondary metabolism in Streptomyces.

Pirins are evolutionarily conserved iron-containing proteins that are found in all kingdoms of life, and have been implicated in diverse molecular processes, mostly associated with cellular stress. In the present study, we started from the evidence that the insertional inactivation of pirin-like gene SAM23877_RS18305 (pirA) by ΦC31 Att/Int system-based vectors in spiramycin-producing strain Streptomyces ambofaciens ATCC 23877 resulted in marked effects on central carbon and energy metabolism gene expression, high sensitivity to oxidative injury and repression of polyketide antibiotic production. By using integrated transcriptomic, proteomic and metabolite profiling, together with genetic complementation, we here show that most of these effects could be traced to the inability of the pirA-defective strain to modulate beta-oxidation pathway, leading to an unbalanced supply of precursor monomers for polyketide biosynthesis. Indeed, in silico protein-protein interaction modeling and in vitro experimental validation allowed us to demonstrate that PirA is a novel redox-sensitive negative modulator of very long-chain acyl-CoA dehydrogenase, which catalyzes the first committed step of the beta-oxidation pathway.

Aglaophenia octodonta (Cnidaria, Hydrozoa) and the Associated Microbial Community: a Cooperative Alliance?

Recently, genetic approaches have revealed a surprising bacterial world as well as a growing knowledge of the enormous distribution of animal-bacterial interactions. In the present study, the diversity of the microorganisms associated to the hydroid Aglaophenia octodonta was studied with epifluorescence, optical, and scanning electron microscopy. Small subunit ribosomal RNA gene sequencing with "universal" and taxon-specific primers allowed the assignment of the microalgae to Symbiodinium and the peritrich ciliates to Pseudovorticella, while the luminous vibrios were identified as Vibrio jasicida of the Harvey clade. To understand the possible relationships among Vibrio jasicida, Symbiodinium, A. octodonta, and Pseudovorticella, specific treatments were conducted in microcosm experiments, with the antibiotic ampicillin and other substances that interfere with bacterial and hydroid metabolism. Treatment of A. octodonta with ampicillin resulted in a decrease of bacterial luminescence followed by Pseudovorticella detachment and Symbiodinium expulsion and suggesting that these microorganisms form a "consortium" with beneficial metabolic interdependence. This hypothesis was reinforced by the evidence that low concentrations of hydrogen peroxide, which stimulate the bacterial oxidative metabolism and luminescence by releasing oxygen, were able to counteract the detrimental effect of ampicillin on the stability of the studied A. octodonta association. A model is proposed in which microalgae that release oxygen during photosynthesis are useful to luminous bacteria for their metabolism and for establishing/maintaining symbiosis leading to a close alliance and mutual benefit of the system A. octodonta-Vibrio jasicida-Pseudovorticella sp.-Symbiodinium sp.

Cultivable gut bacteria provide a pathway for adaptation of Chrysolina herbacea to Mentha aquatica volatiles.

BACKGROUND: A chemical cross-talk between plants and insects is required in order to achieve a successful co-adaptation. In response to herbivory, plants produce specific compounds, and feeding insects respond adequately7 to molecules produced by plants. Here we show the role of the gut microbial community of the mint beetle Chrysolina herbacea in the chemical cross-talk with Mentha aquatica (or watermint). RESULTS: By using two-dimensional gas chromatography-mass spectrometry we first evaluated the chemical patterns of both M. aquatica leaf and frass volatiles extracted by C. herbacea males and females feeding on plants, and observed marked differences between males and females volatiles. The sex-specific chemical pattern of the frass paralleled with sex-specific distribution of cultivable gut bacteria. Indeed, all isolated gut bacteria from females belonged to either α- or γ-Proteobacteria, whilst those from males were γ-Proteobacteria or Firmicutes. We then demonstrated that five Serratia marcescens strains from females possessed antibacterial activity against bacteria from males belonging to Firmicutes suggesting competition by production of antimicrobial compounds. By in vitro experiments, we lastly showed that the microbial communities from the two sexes were associated to specific metabolic patterns with respect to their ability to biotransform M. aquatica terpenoids, and metabolize them into an array of compounds with possible pheromone activity. CONCLUSIONS: Our data suggest that cultivable gut bacteria of Chrysolina herbacea males and females influence the volatile blend of herbivory induced Mentha aquatica volatiles in a sex-specific way.

Fitness Cost of Rifampin Resistance in Neisseria meningitidis: In Vitro Study of Mechanisms Associated with rpoB H553Y Mutation.

Rifampin chemoprophylaxis against Neisseria meningitidis infections led to the onset of rifampin resistance in clinical isolates harboring point mutations in the rpoB gene, coding for the RNA polymerase ß chain. These resistant strains are rare in medical practice, suggesting their decreased fitness in the human host. In this study, we isolated rifampin-resistant rpoB mutants from hypervirulent serogroup C strain 93/4286 and analyzed their different properties, including the ability to grow/survive in different culture media and in differentiated THP-1 human monocytes and to compete with the wild-type strain in vitro. Our results demonstrate that different rpoB mutations (H553Y, H553R, and S549F) may have different effects, ranging from low- to high-cost effects, on bacterial fitness in vitro. Moreover, we found that the S549F mutation confers temperature sensitivity, possibly explaining why it is observed very rarely in clinical isolates. Comparative high-throughput RNA sequencing analysis of bacteria grown in chemically defined medium demonstrated that the low-cost H553Y substitution resulted in global transcriptional changes that functionally mimic the stringent response. Interestingly, many virulence-associated genes, including those coding for meningococcal type IV pili, porin A, adhesins/invasins, IgA protease, two-partner secretion system HrpA/HrpB, enzymes involved in resistance to oxidative injury, lipooligosaccharide sialylation, and capsular polysaccharide biosynthesis, were downregulated in the H553Y mutant compared to their level of expression in the wild-type strain. These data might account for the reduced capacity of this mutant to grow/survive in differentiated THP-1 cells and explain the rarity of H553Y mutants among clinical isolates.

Serogroup-specific interaction of Neisseria meningitidis capsular polysaccharide with host cell microtubules and effects on tubulin polymerization.

We have previously shown that during late stages of the infectious process, serogroup B meningococci (MenB) are able to escape the phagosome of in vitro-infected human epithelial cells. They then multiply in the cytosolic environment and spread intracellularly and to surrounding cells by exploiting the microtubule cytoskeleton, as suggested by results of infections in the presence of microtubule inhibitors and evidence of nanotubes connecting neighboring cells. In this study, by using microtubule binding assays with purified microtubule asters and bundles and microtubule bundles synthesized in vitro, we demonstrate that the MenB capsule directly mediates the interaction between bacteria and microtubules. The direct interaction between the microtubules and the MenB capsular polysaccharide was confirmed by coimmunoprecipitation experiments. Unexpectedly, serogroup C meningococci (MenC), which have a capsular polysaccharide that differs from that of MenB only by its anomeric linkage, α(2→9) instead of α(2→8), were not able to interact with the microtubules, and the lack of interaction was not due to capsular polysaccharide O-acetylation that takes place in most MenC strains but not in MenB strains. Moreover, we demonstrate that the MenB capsular polysaccharide inhibits tubulin polymerization in vitro. Thus, at variance with MenC, MenB may interfere with microtubule dynamics during cell infection.

Comparative genomics revealed key molecular targets to rapidly convert a reference rifamycin-producing bacterial strain into an overproducer by genetic engineering.

Rifamycins are mainstay agents in treatment of many widespread diseases, but how an improved rifamycin producer can be created is still incompletely understood. Here, we describe a comparative genomic approach to investigate the mutational patterns introduced by the classical mutate-and-screen method in the genome of an improved rifamycin producer. Comparing the genome of the rifamycin B overproducer Amycolatopsis mediterranei HP-130 with those of the reference strains A. mediterranei S699 and U32, we identified 250 variations, affecting 227 coding sequences (CDS), 109 of which were HP-130-specific since they were absent in both S699 and U32. Mutational and transcriptional patterns indicated a series of genomic manipulations that not only proved the causative effect of mutB2 (coding for methylmalonyl-CoA mutase large subunit) and argS2 (coding for arginyl tRNA synthetase) mutations on the overproduction of rifamycin, but also constituted a rational strategy to genetically engineer a reference strain into an overproducer.

Characterization of vibrios diversity in the mucus of the polychaete Myxicola infundibulum (Annellida, Polichaeta).

Vibrios are among the most abundant culturable microbes in aquatic environments. They can be either free-living in the water column or associated with several marine organisms as mutualists, saprophytes, or parasites. In the present study we analysed vibrios abundance and diversity in the mucus of the polychaete Myxicola infundibulum, complementing culture-based with molecular methods. Vibrios reached 4.6 × 10(3) CFU mL(-1) thus representing a conspicuous component of the heterotrophic culturable bacteria. In addition, luminous vibrios accounted for about 60% of the total culturable vibrios in the mucus. The isolates were assigned to: Vibrio gigantis, Vibrio fischeri, Vibrio jasicida, Vibrio crassostreae, Vibrio kanaloae, and Vibrio xuii. Two Vibrio isolates (MI-13 and MI-15) may belong to a new species. We also tested the ability of the Vibrio isolates to grow on M. infundibulum mucus as the sole carbon source. All strains showed appreciable growth in the presence of mucus, leading us to conclude that this matrix, which is abundant and covers the animal entirely, may represent a microcosm and a food source for some bacteria, playing a crucial role in the structuring of a mucus-associated beneficial microbial community. Moreover, the trophic relationship between vibrios and M. infundibulum mucus could be enhanced by the protection that mucus offers to vibrios. The results of this study represent a contribution to the growing evidence for complex and dynamic invertebrate-microbe associations present in nature and highlight the importance of exploring relationships that Vibrio species establish with marine invertebrates.

Cyclic olefin copolymer (COC) as a promising biomaterial for affecting bacterial colonization: investigation on Vibrio campbellii.

Cyclic olefin copolymer (COC) has emerged as an interesting biocompatible material for Organ-on-a-Chip (OoC) devices monitoring growth, viability, and metabolism of cells. Despite ISO 10993 approval, systematic investigation of bacteria grown onto COC is a still not documented issue. This study discusses biofilm formations of the canonical wild type BB120 Vibrio campbellii strain on a native COC substrate and addresses the impact of the physico-chemical properties of COC compared to conventional hydroxyapatite (HA) and poly(dimethylsiloxane) (PDMS) surfaces. An interdisciplinary approach combining bacterial colony counting, light microscopy imaging and advanced digital image processing remarks interesting results. First, COC can reduce biomass adhesion with respect to common biopolymers, that is suitable for tuning biofilm formations in the biological and medical areas. Second, remarkably different biofilm morphology (dendritic complex patterns only in the case of COC) was observed among the examined substrates. Third, the observed biofilm morphogenesis was related to the interaction of COC with the conditioning layer of the planktonic biological medium. Fourth, Level Co-occurrence Matrix (CGLM)-based analysis enabled quantitative assessment of the biomass textural fractal development under different coverage conditions. All of this is of key practical relevance in searching innovative biocompatible materials for pharmaceutical, implantable and medical products.

Microbial consortium involved in ferromanganese and francolite biomineralization in an anchialine environment (Zinzulùsa Cave, Castro, Italy).

Tidally-influenced subterranean settings represent natural geomicrobiological laboratories, relatively unexplored, that facilitate the investigation of new biomineralization processes. The unusual water chemistry of Zinzulùsa Cave and its oligotrophic and aphotic conditions have allowed the development of a unique ecosystem in which complex bacterial activities induce rare biomineralization processes. A diversified microbial community develops on centimeter-thick crusts that form in the submerged part of the cave. The crusts are formed of Ca-phosphate minerals, mostly carbonate-fluoroapatite (francolite), covered by a black crust, few microns in thickness, composed of ferromanganiferous oxides (hematite and vernadite). Diffuse coccoidal and filamentous bacteria and amorphous organic matter are mixed with the minerals. The micromorphologies and comparative 16S rRNA gene-based metabarcoding analyses identify a "core microbiota" also common to other natural environments characterized by FeMn and Ca-phosphate mineralization. The microbiota is characterized by nitrifying, sulfide/sulfur/thiosulfate-oxidizing and sulfate/thiosulfate/sulfur-reducing bacteria. In addition, manganese-oxidizing bacteria include the recently described "Ca. Manganitrophus noduliformans" and an abundance of bacteria belonging to the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum, as well as Haliangiales (fruiting body-forming bacteria) and Hyphomicrobiales (stalked and budding bacteria) that are known to produce extracellular polymers that trap iron and manganese oxides. 16S rRNA gene metabarcoding analysis showed the presence of bacteria able to utilize many organic P substrates, including Ramlibacter, and SEM images revealed traces of fossilized microorganisms resembling "cable bacteria", which may play a role in Ca-phosphate biomineralization. Overall, the data indicate biomineralization processes induced by microbial metabolic activities for both ferromanganiferous oxide and francolite components of these crusts.

In silico evidence that substitution of glycine for valine (p.G8V) in a common variant of TMPRSS2 isoform 1 increases accessibility to an endocytic signal: Implication for SARS-cov-2 entry into host cells and susceptibility to COVID-19.

The TMPRSS2 protease plays a key role in the entry of the SARS-CoV-2 into cells. The TMPRSS2 gene is highly polymorphic in humans, and some polymorphisms may affect the susceptibility to COVID-19 or disease severity. rs75603675 (c.23G > T) is a missense variant that causes the replacement of glycine with valine at position 8 (p.G8V) in the TMPRSS2 isoform 1. According to GnomAD v4.0.0 database, the allele frequency of the rs75603675 on a global scale is 38.10 %, and range from 0.92 % in East Asian to 40.77 % in non-Finnish European (NFE) population. We analyzed the occurrence of the rs75603675 in two cohorts of patients, the first with severe/critical COVID-19 enrolled in a French hospital (42 patients), and the second with predominantly asymptomatic/pauci-symptomatic/mild COVID-19 enrolled in an Italian hospital (69 patients). We found that the TMPRSS2-c.23T minor allele frequency was similar in the two cohorts, 46.43 % and 46.38 %, respectively, and higher than the frequency in the NFE population (40.77 %). Chi-square test provided significant results (p < 0.05) when the genotype data (TMPRSS2-c.23T/c.23T homozygotes + TMPRSS2-c.23G/c.23T heterozygotes vs. TMPRSS2-c.23G/c.23G homozygotes) of the two patient groups were pooled and compared to the expected data for the NFE population, suggesting a possible pathogenetic mechanism of the p.G8V substitution. We explored the possible effects of the p.G8V substitution and found that the N-terminal region of the TMPRSS2 isoform 1 contains a signal for clathrin/AP-2-dependent endocytosis. In silico analysis predicted that the p.G8V substitution may increase the accessibility to the endocytic signal, which could help SARS-CoV-2 enter cells.

Dysregulation of a Subset of Circulating and Vesicle-Associated miRNA in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplasia, characterized by early metastasis, low diagnostic rates at early stages, resistance to drugs, and poor prognosis. There is an urgent need to better characterize this disease in order to identify efficient diagnostic/prognostic biomarkers. Since microRNAs (miRNAs) contribute to oncogenesis and metastasis formation in PDAC, they are considered potential candidates for fulfilling this task. In this work, the levels of two miRNA subsets (involved in chemoresistance or with oncogenic/tumor suppressing functions) were investigated in a panel of PDAC cell lines and liquid biopsies of a small cohort of patients. We used RT-qPCR and droplet digital PCR (ddPCR) to measure the amounts of cellular- and vesicle-associated, and circulating miRNAs. We found that both PDAC cell lines, also after gemcitabine treatment, and patients showed low amounts of cellular-and vesicle-associated miR-155-5p, compared to controls. Interestingly, we did not find any differences when we analyzed circulating miR-155-5p. Furthermore, vesicle-related miR-27a-3p increased in cancer patients compared to the controls, while circulating let-7a-5p, miR-221-3p, miR-23b-3p and miR-193a-3p presented as dysregulated in patients compared to healthy individuals. Our results highlight the potential clinical significance of these analyzed miRNAs as non-invasive diagnostic molecular tools to characterize PDAC.