Tisochrysis lutea F&M-M36 Mitigates Risk Factors of Metabolic Syndrome and Promotes Visceral Fat Browning through ß3-Adrenergic Receptor/UCP1 Signaling.

Pre-metabolic syndrome (pre-MetS) may represent the best transition phase to start treatments aimed at reducing cardiometabolic risk factors of MetS. In this study, we investigated the effects of the marine microalga Tisochrysis lutea F&M-M36 (T. lutea) on cardiometabolic components of pre-MetS and its underlying mechanisms. Rats were fed a standard (5% fat) or a high-fat diet (20% fat) supplemented or not with 5% of T. lutea or fenofibrate (100 mg/Kg) for 3 months. Like fenofibrate, T. lutea decreased blood triglycerides (p < 0.01) and glucose levels (p < 0.01), increased fecal lipid excretion (p < 0.05) and adiponectin (p < 0.001) without affecting weight gain. Unlike fenofibrate, T. lutea did not increase liver weight and steatosis, reduced renal fat (p < 0.05), diastolic (p < 0.05) and mean arterial pressure (p < 0.05). In visceral adipose tissue (VAT), T. lutea, but not fenofibrate, increased the ß3-adrenergic receptor (ß3ADR) (p < 0.05) and Uncoupling protein 1 (UCP-1) (p < 0.001) while both induced glucagon-like peptide-1 receptor (GLP1R) protein expression (p < 0.001) and decreased interleukin (IL)-6 and IL-1ß gene expression (p < 0.05). Pathway analysis on VAT whole-gene expression profiles showed that T. lutea up-regulated energy-metabolism-related genes and down-regulated inflammatory and autophagy pathways. The multitarget activity of T. lutea suggests that this microalga could be useful in mitigating risk factors of MetS.

Changes in Faecal Microbiota Profile and Plasma Biomarkers following the Administration of an Antioxidant Oleuropein-Rich Leaf Extract in a Rat Model Mimicking Colorectal Cancer.

Oleuropein (OLE), a phenolic compound particularly abundant in the olive leaves, has been reported to have beneficial activities against colorectal cancer (CRC). In vitro studies suggested that these latter could be due to a modulation of the intestinal microbiota. Aiming to evaluate if OLE could affect the intestinal microbiota and the plasma metabolome, an antioxidant oleuropein-rich leaf extract (ORLE) was administered for one week to PIRC rats (F344/NTac-Apcam1137), a genetic model mimicking CRC. ORLE treatment significantly modulated the gut microbiota composition. Plasma metabolomic profiles revealed a significant predictive ability for amino acids, medium-chain fatty acids, and aldehydes. Pathway analysis revealed a significant decrease in phosphatidylcholine accumulation (LogFC = -1.67) in PIRC rats. These results suggest a significant effect of ORLE administration on faecal microbiota profiles and plasma metabolomes, thereby offering new omics-based insights into its protective role in CRC progression.

Fish Consumption and Colorectal Cancer Risk: Meta-Analysis of Prospective Epidemiological Studies and Review of Evidence from Animal Studies.

Background: Epidemiological studies on the association between fish consumption and colorectal cancer (CRC) risk have yielded inconsistent results, despite evidence from preclinical studies that long-chain ω-3 polyunsaturated fatty acids inhibit colorectal carcinogenesis. We conducted a meta-analysis of prospective epidemiological studies investigating the association between fish consumption and CRC risk among humans and reviewed studies examining the link between fish components and colorectal carcinogenesis in animal models. Methods: We included studies published until November 2020. We calculated the summary risk ratio (SRR) and 95% confidence intervals (CI) through random effects meta-analysis models in order to summarize evidence from studies among humans. Results: Twenty-five prospective epidemiological studies encompassing 25,777 CRC cases were included. Individuals in the highest (vs. lowest) category of fish consumption had a significantly reduced risk of CRC (SRR 0.94, 95%CI 0.89-0.99). In dose-response meta-analysis, a 50-g increment in the daily consumption of fish was associated with a statistically significant 4% reduction in CRC risk (SRR 0.96, 95%CI 0.92-0.99). Preclinical studies (n = 25) identified multiple mechanisms of action of fish and fish components on colorectal carcinogenesis. Conclusions: Dietary recommendations for cancer prevention should take into account the evidence from epidemiological and preclinical studies that increasing fish consumption may be effective in preventing CRC.

Endophytic Bacteria and Essential Oil from Origanum vulgare ssp. vulgare Share Some VOCs with an Antibacterial Activity.

Medicinal aromatic plants' essential oils (EOs) are mixtures of volatile compounds showing antimicrobial activity, which could be exploited to face the emerging problem of multi-drug resistance. Their chemical composition can depend on the interactions between the plant and its endophytic microbiota, which is known to synthesize volatile organic compounds (VOCs). However, it is still not clear whether those volatile metabolites can contribute to the composition of the aroma profile of plants' EOs. The aims of this study were to characterize medicinal plant O. vulgare ssp. vulgare bacterial endophyte VOCs, evaluating their ability to antagonize the growth of opportunistic human pathogens belonging to the Burkholderia cepacia complex (Bcc) and compare them with O. vulgare EO composition. Many of the tested endophytic strains showed (i) a bactericidal and/or bacteriostatic activity against most of Bcc strains and (ii) the production of VOCs with widely recognized antimicrobial properties, such as dimethyl disulfide, dimethyl trisulfide, and monoterpenes. Moreover, these monoterpenes were also detected in the EOs extracted from the same O. vulgare plants from which endophytes were isolated. Obtained results suggest that endophytes could also play a role in the antibacterial properties of O. vulgare ssp. vulgare and, potentially, in determining its aromatic composition.

Paper-based genetic assays with bioconjugated gold nanorods and an automated readout pipeline.

Paper-based biosensors featuring immunoconjugated gold nanoparticles have gained extraordinary momentum in recent times as the platform of choice in key cases of field applications, including the so-called rapid antigen tests for SARS-CoV-2. Here, we propose a revision of this format, one that may leverage on the most recent advances in materials science and data processing. In particular, we target an amplifiable DNA rather than a protein analyte, and we replace gold nanospheres with anisotropic nanorods, which are intrinsically brighter by a factor of ~ 10, and multiplexable. By comparison with a gold-standard method for dot-blot readout with digoxigenin, we show that gold nanorods entail much faster and easier processing, at the cost of a higher limit of detection (from below 1 to 10 ppm in the case of plasmid DNA containing a target transgene, in our current setup). In addition, we test a complete workflow to acquire and process photographs of dot-blot membranes with custom-made hardware and regression tools, as a strategy to gain more analytical sensitivity and potential for quantification. A leave-one-out approach for training and validation with as few as 36 sample instances already improves the limit of detection reached by the naked eye by a factor around 2. Taken together, we conjecture that the synergistic combination of new materials and innovative tools for data processing may bring the analytical sensitivity of paper-based biosensors to approach the level of lab-grade molecular tests.

The Histidine Biosynthetic Genes in the Superphylum Bacteroidota-Rhodothermota-Balneolota-Chlorobiota: Insights into the Evolution of Gene Structure and Organization.

One of the most studied metabolic routes is the biosynthesis of histidine, especially in enterobacteria where a single compact operon composed of eight adjacent genes encodes the complete set of biosynthetic enzymes. It is still not clear how his genes were organized in the genome of the last universal common ancestor community. The aim of this work was to analyze the structure, organization, phylogenetic distribution, and degree of horizontal gene transfer (HGT) of his genes in the Bacteroidota-Rhodothermota-Balneolota-Chlorobiota superphylum, a group of phylogenetically close bacteria with different surviving strategies. The analysis of the large variety of his gene structures and organizations revealed different scenarios with genes organized in more or less compact-heterogeneous or homogeneous-operons, in suboperons, or in regulons. The organization of his genes in the extant members of the superphylum suggests that in the common ancestor of this group, genes were scattered throughout the chromosome and that different forces have driven the assembly of his genes in compact operons. Gene fusion events and/or paralog formation, HGT of single genes or entire operons between strains of the same or different taxonomic groups, and other molecular rearrangements shaped the his gene structure in this superphylum.

Oleuropein-Rich Leaf Extract as a Broad Inhibitor of Tumour and Macrophage iNOS in an Apc Mutant Rat Model.

Oleuropein, the major compound found in olive leaves, has been reported to exert numerous pharmacological properties, including anti-inflammatory, anti-diabetic and anti-cancer effects. The purpose of this study was to evaluate, for the first time, the effect of oleuropein-rich leaf extracts (ORLE) in already-developed colon tumours arising in Apc (adenomatous polyposis coli) mutated PIRC rats (F344/NTac-Apcam1137). Here, we were able to investigate in parallel the anti-cancer effect of ORLE, both in vivo and in vitro, and its anti-inflammatory effect on macrophages, representing a critical and abundant population in most solid tumour microenvironment. We found that in vivo ORLE treatment promoted apoptosis and attenuated iNOS activity both in colon tumours as in peritoneal macrophages of PIRC rats. We this confirmed in vitro using primary RAW264.7 cells: ORLE reduced iNOS activity in parallel with COX-2 and pro-inflammatory cytokines, such as IL-1ß, IL-6 and TGF-ß. These findings suggest that ORLE possess a strong anti-inflammatory activity, which could be crucial for dampening the pro-tumourigenic activity elicited by a chronic inflammatory state generated by either tumour cells or tumour-associated macrophages.

Antibacterial activity of bacteria isolated from Phragmites australis against multidrug-resistant human pathogens.

Background: Rising number of multidrug-resistant human pathogens demands novel antibiotics: to this aim, unexplored natural sources are investigated to find new compounds. In this context, bacteria associated to medicinal plants, including Phragmites australis, might represent an important source of antimicrobial compounds. Materials & methods: In the present work, 21 bacterial endophytes isolated from P. australis roots were tested, by cross-streaking, for their inhibitory activity against 36 multidrug-resistant pathogens isolated from food, clinical patients and hospitals. Results & conclusion: Seven endophytes, belonging to Pseudomonas and Stenotrophomonas, were able to inhibit the growth of most of the target strains. In conclusion, this preliminary work could pave the way for the discovery of new antibiotics against superbugs.

Lay abstract In the present work, 21 bacteria associated to Phragmites australis roots were tested for their inhibitory activity against 36 human pathogens isolated from food, clinical patients and/or hospitals, which have the ability to escape several commonly used antibiotics. Seven out of 21 bacteria associated to P. australis were able to inhibit the growth of most of target pathogens. This preliminary work could pave the way to the discovery of new antimicrobial compounds active against bacterial pathogens that cannot be killed using several antibiotics.

Exploring the role of the histidine biosynthetic hisF gene in cellular metabolism and in the evolution of (ancestral) genes: from LUCA to the extant (micro)organisms.

Histidine biosynthesis is an ancestral pathway that was assembled before the appearance of the Last Universal Common Ancestor; afterwards, it remained unaltered in all the extant histidine-synthesizing (micro)organisms. It is a metabolic cross-road interconnecting histidine biosynthesis to nitrogen metabolism and the de novo synthesis of purines. This interconnection is due to the reaction catalyzed by the products of hisH and hisF genes. The latter gene is an excellent model to study which trajectories have been followed by primordial cells to build the first metabolic routes, since its evolution is the result of different molecular rearrangement events, i.e. gene duplication, gene fusion, gene elongation, and domain shuffling. Additionally, this review summarizes data concerning the involvement of hisF and its product in other different cellular processes, revealing that HisF very likely plays a role also in cell division control and involvement in virulence and nodule development in different bacteria. From the metabolic viewpoint, these results suggest that HisF plays a central role in cellular metabolism, highlighting the interconnections of different metabolic pathways.

The Role of Gene Elongation in the Evolution of Histidine Biosynthetic Genes.

Gene elongation is a molecular mechanism consisting of an in-tandem duplication of a gene and divergence and fusion of the two copies, resulting in a gene constituted by two divergent paralogous modules. The aim of this work was to evaluate the importance of gene elongation in the evolution of histidine biosynthetic genes and to propose a possible evolutionary model for some of them. Concerning the genes hisA and hisF, which code for two homologous (ß/α)8-barrels, it has been proposed that the two extant genes could be the result of a cascade of gene elongation/domain shuffling events starting from an ancestor gene coding for just one (ß/α) module. A gene elongation event has also been proposed for the evolution of hisB and hisD; structural analyses revealed the possibility of an early elongation event, resulting in the repetition of modules. Furthermore, it is quite possible that the gene elongations responsible for the evolution of the four proteins occurred before the earliest phylogenetic divergence. In conclusion, gene elongation events seem to have played a crucial role in the evolution of the histidine biosynthetic pathway, and they may have shaped the structures of many genes during the first steps of their evolution.

In vivo evaluation of the interaction between the Escherichia coli IGP synthase subunits using the Bacterial Two-Hybrid system.

Histidine biosynthesis is one of the most characterized metabolic routes for its antiquity and its central role in cellular metabolism; indeed, it represents a cross-road between nitrogen metabolism and de novo synthesis of purines. This interconnection is due to the activity of imidazole glycerol phosphate synthase, a heterodimeric enzyme constituted by the products of two his genes, hisH and hisF, encoding a glutamine amidotransferase and a cyclase, respectively. Despite their interaction was suggested by several in vitro experiments, their in vivo complex formation has not been demonstrated. On the contrary, the analysis of the entire Escherichia coli interactome performed using the yeast two hybrid system did not suggest the in vivo interaction of the two IGP synthase subunits. The aim of this study was to demonstrate the interaction of the two proteins using the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system. Data obtained demonstrated the in vivo interaction occurring between the proteins encoded by the E. coli hisH and hisF genes; this finding might also open the way to pharmaceutical applications through the design of selective drugs toward this enzyme.

Temporal Evolution of Bacterial Endophytes Associated to the Roots of Phragmites australis Exploited in Phytodepuration of Wastewater.

Improvement of industrial productions through more environment-friendly processes is a hot topic. In particular, land and marine environment pollution is a main concern, considering that recalcitrant compounds can be spread and persist for a long time. In this context, an efficient and cost-effective treatment of wastewater derived from industrial applications is crucial. Phytodepuration has been considered as a possible solution and it is based on the use of plants and their associated microorganisms to remove and/or transform pollutants. In this work we investigated the culturable microbiota of Phragmites australis roots, sampled from the constructed wetlands (CWs) pilot plant in the G.I.D.A. SpA wastewater treatment plant (WWTP) of Calice (Prato, Tuscany, Italy) before and after the CW activation in order to check how the influx of wastewater might affect the resident bacterial community. P. australis specimens were sampled and a panel of 294 culturable bacteria were isolated and characterized. This allowed to identify the dynamics of the microbiota composition triggered by the presence of wastewater. 27 out of 37 bacterial genera detected were exclusively associated to wastewater, and Pseudomonas was constantly the most represented genus. Moreover, isolates were assayed for their resistance against eight different antibiotics and synthetic wastewater (SWW). Data obtained revealed the presence of resistant phenotypes, including multi-drug resistant bacteria, and a general trend regarding the temporal evolution of resistance patterns: indeed, a direct correlation linking the appearance of antibiotic- and SWW-resistance with the time of exposure to wastewater was observed. In particular, nine isolates showed an interesting behavior since their growth was positively affected by the highest concentrations of SWW. Noteworthy, this study is among the few investigating the P. australis microbiota prior to the plant activation.

The Cultivable Bacterial Microbiota Associated to the Medicinal Plant Origanum vulgare L.: From Antibiotic Resistance to Growth-Inhibitory Properties.

The insurgence of antibiotic resistance and emergence of multidrug-resistant (MDR) pathogens prioritize research to discover new antimicrobials. In this context, medicinal plants produce bioactive compounds of pharmacological interest: some extracts have antimicrobial properties that can contrast different pathogens. For such a purpose, Origanum vulgare L. (Lamiaceae family) is a medicinal aromatic plant, whose essential oil (EO) is recognized for its antiseptic, antimicrobial and antiviral activities. The cultivable bacteria from different compartments (i.e., flower, leaf, stem and soil) were isolated in order to: (i) characterize the bacterial microbiota associated to the plant, determining the forces responsible for the structuring of its composition (by evaluation of cross inhibition); (ii) investigate if bacterial endophytes demonstrate antimicrobial activities against human pathogens. A pool of plants belonging to O. vulgare species was collected and the specimen chemotype was defined by hydrodistillation of its essential oil. The isolation of plant associated bacteria was performed from the four compartments. Microbiota was further characterized through a culture-independent approach and next-generation sequencing analysis, as well. Isolates were molecularly typed by Random Amplified Polymorphic DNA (RAPD) profiling and taxonomically assigned by 16S rRNA gene sequencing. Antibiotic resistance profiles of isolates and pairwise cross-inhibition of isolates on agar plates (i.e., antagonistic interactions) were also assessed. High level of diversity of bacterial isolates was detected at both genus and strain level in all different compartments. Most strains were tolerant against common antibiotics; moreover, they produced antagonistic patterns of interactions mainly with strains from different compartments with respect to that of original isolation. Strains that exhibited high inhibitory properties were further tested against human pathogens, revealing a strong capacity to inhibit the growth of strains resistant to several antibiotics. In conclusion, this study regarded the characterization of O. vulgare L. chemotype and of the bacterial communities associated to this medicinal plant, also allowing the evaluation of antibiotic resistance and antagonistic interactions. This study provided the bases for further analyses on the possible involvement of endophytic bacteria in the production of antimicrobial molecules that could have an important role in clinical and therapeutic applications.