Microbiota-Liver Diseases Interactions.

Gut microbiota regulates essential processes of host metabolism and physiology: synthesis of vitamins, digestion of foods non-digestible by the host (such as fibers), and-most important-protects the digestive tract from pathogens. In this study, we focus on the CRISPR/Cas9 technology, which is extensively used to correct multiple diseases, including liver diseases. Then, we discuss the non-alcoholic fatty liver disease (NAFLD), affecting more than 25% of the global population; colorectal cancer (CRC) is second in mortality. We give space to rarely discussed topics, such as pathobionts and multiple mutations. Pathobionts help to understand the origin and complexity of the microbiota. Since several types of cancers have as target the gut, it is vital extending the research of multiple mutations to the type of cancers affecting the gut-liver axis.

Polystyrene Microplastics Exacerbate Candida albicans Infection Ability In Vitro and In Vivo.

Plastic pollution is an important environmental problem, and microplastics have been shown to have harmful effects on human and animal health, affecting immune and metabolic physiological functions. Further, microplastics can interfere with commensal microorganisms and exert deleterious effects on exposure to pathogens. Here, we compared the effects of 1 µm diameter polystyrene microplastic (PSMPs) on Candida albicans infection in both in vitro and in vivo models by using HT29 cells and Galleria mellonella larvae, respectively. The results demonstrated that PSMPs could promote Candida infection in HT29 cells and larvae of G. mellonella, which show immune responses similar to vertebrates. In this study, we provide new experimental evidence for the risk to human health posed by PSMPs in conjunction with Candida infections.

Epigenetics and Helicobacter pylori.

Epigenetics regulates gene expression, cell type development during differentiation, and the cell response to environmental stimuli. To survive, bacteria need to evade the host immune response. Bacteria, including Helicobacter pylori (Hp), reach this target epigenetically, altering the chromatin of the host cells, in addition to several more approaches, such as DNA mutation and recombination. This review shows that Hp prevalently silences the genes of the human gastric mucosa by DNA methylation. Epigenetics includes different mechanisms. However, DNA methylation persists after DNA replication and therefore is frequently associated with the inheritance of repressed genes. Chromatin modification can be transmitted to daughter cells leading to heritable changes in gene expression. Aberrant epigenetic alteration of the gastric mucosa DNA remains the principal cause of gastric cancer. Numerous methylated genes have been found in cancer as well as in precancerous lesions of Hp-infected patients. These methylated genes inactivate tumor-suppressor genes. It is time for us to complain about our genetic and epigenetic makeups for our diseases.

Role of Branched-Chain Amino Acid Metabolism in Type 2 Diabetes, Obesity, Cardiovascular Disease and Non-Alcoholic Fatty Liver Disease.

Branched-chain amino acids (BCAAs) include leucine, isoleucine, and valine. Mammalians cannot synthesize these amino acids de novo and must acquire them through their diet. High levels of BCAAs are associated with insulin resistance; type 2 diabetes; obesity; and non-metabolic diseases, including several forms of cancer. BCAAs-in particular leucine-activate the rapamycin complex1 mTORC1, which regulates cell growth and metabolism, glucose metabolism and several more essential physiological processes. Diets rich in BCAAs are associated with metabolic diseases (listed above), while diets low in BCAAs are generally reported to promote metabolic health. As for the dysregulation of the metabolism caused by high levels of BCAAs, recent studies propose that the accumulation of acyl-carnitine and diacyl-CoA in muscles alters lipid metabolism. However, this suggestion is not broadly accepted. On clinical grounds, pre- and post-operative metabolic profiles of candidate patients for bariatric surgery are being used to select the optimal procedure for each individual patient.

Gut Microbiota Host-Gene Interaction.

Studies carried out in the last ten years have shown that the metabolites made up from the gut microbiota are essential for multiple functions, such as the correct development of the immune system of newborns, interception of pathogens, and nutritional enrichment of the diet. Therefore, it is not surprising that alteration of the gut microbiota is the starting point of gastrointestinal infection, obesity, type 2 diabetes, inflammatory bowel disease, colorectal cancer, and lung cancer. Diet changes and antibiotics are the major factors damaging the gut microbiota. Early exposure of the newborns to antibiotics may prevent their correct development of the immune system, exposing them to pathogen infections, allergies, and chronic inflammatory diseases. We already know much on how host genes, microbiota, and the environment interact, owing to experiments in several model animals, especially in mice; advances in molecular technology; microbiota transplantation; and comparative metagenomic analysis. However, much more remains to be known. Longitudinal studies on patients undergoing to therapy, along with the identification of bacteria prevalent in responding patients may provide valuable data for improving therapies.

Chemical Analysis and Antimicrobial Activity of Moringa oleifera Lam. Leaves and Seeds.

Moringa oleifera is a traditional food crop widespread in Asiatic, African, and South American continents. The plant, able to grow in harsh conditions, shows a high nutritional value and medicinal potential evidencing cardioprotective, anti-inflammatory, antioxidant, and antimicrobial properties. The purpose of this study was the phytochemical analysis of M. oleifera and the identification of the antimicrobial compounds by combining a chemical approach with in vitro tests. The metabolite profile of M. oleifera polar and apolar extracts of leaves and seeds were investigated by using Nuclear Magnetic Resonance spectroscopy and Gas Chromatography-Mass Spectrometry. The antimicrobial activity of all of the obtained extract was evaluated against four bacterial pathogens (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Salmonella enterica). The chemical analysis provided a wide set of metabolites that were identified and quantified. Moreover, apolar extracts from seeds showed a significant concentration-dependent antimicrobial activity against S. aureus and S. epidermidis, (4 mg/mL reduced the viability up to 50%) that was associated to the content of specific fatty acids. Our results remarked the advantages of an integrated approach for the identification of plant metabolites and its use in association with biological tests to recognize the compounds responsible for bioactivity without compounds purification.

Genetics of Host Protection against Helicobacter pylori Infections.

This narrative review discusses the genetics of protection against Helicobacter pylori (Hp) infection. After a brief overview of the importance of studying infectious disease genes, we provide a detailed account of the properties of Hp, with a view to those relevant for our topic. Hp displays a very high level of genetic diversity, detectable even between single colonies from the same patient. The high genetic diversity of Hp can be evaded by stratifying patients according to the infecting Hp strain. This approach enhances the power and replication of the study. Scanning for single nucleotide polymorphisms is generally not successful since genes rarely work alone. We suggest selecting genes to study from among members of the same family, which are therefore inclined to cooperate. Further, extending the analysis to the metabolism would significantly enhance the power of the study. This combined approach displays the protective role of MyD88, TIRAP, and IL1RL1 against Hp infection. Finally, several studies in humans have demonstrated that the blood T cell levels are under the genetic control of the CD39+ T regulatory cells (TREGS).

The Role of Formyl Peptide Receptors in Permanent and Low-Grade Inflammation: Helicobacter pylori Infection as a Model.

Formyl peptide receptors (FPRs) are cell surface pattern recognition receptors (PRRs), belonging to the chemoattractant G protein-coupled receptors (GPCRs) family. They play a key role in the innate immune system, regulating both the initiation and the resolution of the inflammatory response. FPRs were originally identified as receptors with high binding affinity for bacteria or mitochondria N-formylated peptides. However, they can also bind a variety of structurally different ligands. Among FPRs, formyl peptide receptor-like 1 (FPRL1) is the most versatile, recognizing N-formyl peptides, non-formylated peptides, and synthetic molecules. In addition, according to the ligand nature, FPRL1 can mediate either pro- or anti-inflammatory responses. Hp(2-20), a Helicobacter pylori-derived, non-formylated peptide, is a potent FPRL1 agonist, participating in Helicobacter pylori-induced gastric inflammation, thus contributing to the related site or not-site specific diseases. The aim of this review is to provide insights into the role of FPRs in H. pylori-associated chronic inflammation, which suggests this receptor as potential target to mitigate both microbial and sterile inflammatory diseases.

Lactoferrin, Quercetin, and Hydroxyapatite Act Synergistically against Pseudomonas fluorescens.

Pseudomonas fluorescens is an opportunistic, psychotropic pathogen that can live in different environments, such as plant, soil, or water surfaces, and it is associated with food spoilage. Bioactive compounds can be used as antimicrobials and can be added into packaging systems. Quercetin and lactoferrin are the best candidates for the development of a complex of the two molecules absorbed on bio combability structure as hydroxyapatite. The minimum inhibiting concentration (MIC) of single components and of the complex dropped down the single MIC value against Pseudomonas fluorescens. Characterization analysis of the complex was performed by means SEM and zeta-potential analysis. Then, the synergistic activity (Csyn) of single components and the complex was calculated. Finally, the synergistic activity was confirmed, testing in vitro its anti-inflammatory activity on U937 macrophage-like human cell line. In conclusion, the peculiarity of our study consists of optimizing the specific propriety of each component: the affinity of lactoferrin for LPS; that of quercetin for the bacterial membrane. These proprieties make the complex a good candidate in food industry as antimicrobial compounds, and as functional food.

Caulerpin Mitigates Helicobacter pylori-Induced Inflammation via Formyl Peptide Receptors.

The identification of novel strategies to control Helicobacter pylori (Hp)-associated chronic inflammation is, at present, a considerable challenge. Here, we attempt to combat this issue by modulating the innate immune response, targeting formyl peptide receptors (FPRs), G-protein coupled receptors that play key roles in both the regulation and the resolution of the innate inflammatory response. Specifically, we investigated, in vitro, whether Caulerpin-a bis-indole alkaloid isolated from algae of the genus Caulerpa-could act as a molecular antagonist scaffold of FPRs. We showed that Caulerpin significantly reduces the immune response against Hp culture filtrate, by reverting the FPR2-related signaling cascade and thus counteracting the inflammatory reaction triggered by Hp peptide Hp(2-20). Our study suggests Caulerpin to be a promising therapeutic or adjuvant agent for the attenuation of inflammation triggered by Hp infection, as well as its related adverse clinical outcomes.

Screen Printed Based Impedimetric Immunosensor for Rapid Detection of Escherichia coli in Drinking Water.

The development of a simple and low cost electrochemical impedance immunosensor based on screen printed gold electrode for rapid detection of Escherichia coli in water is reported. The immunosensor is fabricated by immobilizing anti-E. coli antibodies onto a gold surface in a covalent way by the photochemical immobilization technique, a simple procedure able to bind antibodies upright onto gold surfaces. Impedance spectra are recorded in 0.01 M phosphate buffer solution (PBS) containing 10 mM Fe(CN)63-/Fe(CN)64- as redox probe. The Nyquist plots can be modelled with a modified Randles circuit, identifying the charge transfer resistance Rct as the relevant parameter after the immobilization of antibodies, the blocking with BSA and the binding of E. coli. The introduction of a standard amplification procedure leads to a significant enhancement of the impedance increase, which allows one to measure E. coli in drinking water with a limit of detection of 3 × 101 CFU mL-1 while preserving the rapidity of the method that requires only 1 h to provide a "yes/no" response. Additionally, by applying the Langmuir adsorption model, we are able to describe the change of Rct in terms of the "effective" electrode, which is modified by the detection of the analyte whose microscopic conducting properties can be quantified.

An In Vitro Model to Investigate the Role of Helicobacter pylori in Type 2 Diabetes, Obesity, Alzheimer's Disease and Cardiometabolic Disease.

Helicobacter pylori (Hp) is a Gram-negative bacterium colonizing the human stomach. Nuclear Magnetic Resonance (NMR) analysis of intracellular human gastric carcinoma cells (MKN-28) incubated with the Hp cell filtrate (Hpcf) displays high levels of amino acids, including the branched chain amino acids (BCAA) isoleucine, leucine, and valine. Polymerase chain reaction (PCR) Array Technology shows upregulation of mammalian Target Of Rapamycin Complex 1 (mTORC1), inflammation, and mitochondrial dysfunction. The review of literature indicates that these traits are common to type 2 diabetes, obesity, Alzheimer's diseases, and cardiometabolic disease. Here, we demonstrate how Hp may modulate these traits. Hp induces high levels of amino acids, which, in turn, activate mTORC1, which is the complex regulating the metabolism of the host. A high level of BCAA and upregulation of mTORC1 are, thus, directly regulated by Hp. Furthermore, Hp modulates inflammation, which is functional to the persistence of chronic infection and the asymptomatic state of the host. Finally, in order to induce autophagy and sustain bacterial colonization of gastric mucosa, the Hp toxin VacA localizes within mitochondria, causing fragmentation of these organelles, depletion of ATP, and oxidative stress. In conclusion, our in vitro disease model replicates the main traits common to the above four diseases and shows how Hp may potentially manipulate them.

Role of phage ϕ1 in two strains of Salmonella Rissen, sensitive and resistant to phage ϕ1.

BACKGROUND: The study describes the Salmonella Rissen phage ϕ1 isolated from the ϕ1-sensitive Salmonella Rissen strain RW. The same phage was then used to select the resistant strain RRϕ1+, which can harbour or not ϕ1. RESULTS: Following this approach, we found that ϕ1, upon excision from RW cells with mitomycin, behaves as a temperate phage: lyses host cells and generates phage particles; instead, upon spontaneous excision from RRϕ1+ cells, it does not generate phage particles; causes loss of phage resistance; switches the O-antigen from the smooth to the rough phenotype, and favors the transition of Salmonella Rissen from the planktonic to the biofilm growth. The RW and RRϕ1+ strains differ by 10 genes; of these, only two (phosphomannomutase_1 and phosphomannomutase_2; both involved in the mannose synthesis pathway) display significant differences at the expression levels. This result suggests that phage resistance is associated with these two genes. CONCLUSIONS: Phage ϕ1 displays the unusual property of behaving as template as well as lytic phage. This feature was used by the phage to modulate several phases of Salmonella Rissen lifestyle.

Milk microRNA-146a as a potential biomarker in bovine tuberculosis.

In this research communication we exploited the potential use of milk microRNAs (miRs) as biomarkers for bovine tuberculosis (bTB). bTB is a zoonotic disease caused by Mycobacterium bovis which affects animal health, influencing herd economic sustainability. Diagnosis is based on skin delayed-type hypersensitivity reaction and quantification of interferon gamma but both techniques are influenced by several confounding factors. Thus, new methods for early diagnosis are required. In this context, microRNAs have been used as promising biomarkers for both infectious and non-infectious diseases. To determine the possible involvement of microRNAs in bTB, we analysed the expression of four immune-related miRs in 200 cows grouped in cases and controls with respect to positivity to tuberculosis. The analysis showed a different magnitude of expression in the groups indicating that active tuberculosis could influence miRs expression. We used expression values of miR-146a, the highest differentially expressed miR, for Receiver operating characteristic (ROC) curve analysis. In order to determine a test cut-off value for miR-146a expression that would differentiate cases and controls, a value for the miR-146a expression higher than 8 was selected as this gave a test specificity and sensitivity of 80·0% and 86·0% respectively. These values confirm the possibility of using miR-146a as a milk prognostic biomarker for bovine tuberculosis.

Molecular characterisation, genetic variability and detection of a functional polymorphism influencing the promoter activity of OXT gene in goat and sheep.

The purpose of the study described in this Research Communication was to report the full characterisation of the goat and sheep oxytocin-neurophysin I gene (OXT), their promoters and amino acid sequences. Using the genomic DNA as template, we sequenced and compared the whole OXT gene (3 exons), plus 958/960 nucleotides at the 5' flanking region and 478/477 nucleotides at the 3' flanking region, in 46 sheep and 24 goats belonging to different breeds/genetic types reared in Italy, Greece and Germany. The comparison of the obtained sequences showed a high degree of genetic variability at these loci. In particular, we focused on the SNP g.438T > C as possible example of trans-specific polymorphism. This SNP alters a putative binding site of the transcription factor Oct-1. The set-up of a luciferase assay confirmed that the C variant of this SNP negatively affects the promoter activity of the sheep OXT gene. The results of this study suggest that the SNP g.438T > C might be useful to promote association studies with traits/physiological processes controlled by this hormone.

Design, structural and functional characterization of a Temporin-1b analog active against Gram-negative bacteria.

BACKGROUND: Temporins are small antimicrobial peptides secreted by the Rana temporaria showing mainly activity against Gram-positive bacteria. However, different members of the temporin family, such as Temporin B, act in synergy also against Gram-negative bacteria. With the aim to develop a peptide with a wide spectrum of antimicrobial activity we designed and analyzed a series of Temporin B analogs. METHODS: Peptides were initially obtained by Ala scanning on Temporin B sequence; antimicrobial activity tests allowed to identify the TB_G6A sequence, which was further optimized by increasing the peptide positive charge (TB_KKG6A). Interactions of this active peptide with the LPS of E. coli were investigated by CD, fluorescence and NMR. RESULTS: TB_KKG6A is active against Gram-positive and Gram-negative bacteria at low concentrations. The peptide strongly interacts with the LPS of Gram-negative bacteria and folds upon interaction into a kinked helix. CONCLUSION: Our results show that it is possible to widen the activity spectrum of an antimicrobial peptide by subtle changes of the primary structure. TB_KKG6A, having a simple composition, a broad spectrum of antimicrobial activity and a very low hemolytic activity, is a promising candidate for the design of novel antimicrobial peptides. GENERAL SIGNIFICANCE: The activity of antimicrobial peptides is strongly related to the ability of the peptide to interact and break the bacterial membrane. Our studies on TB_KKG6A indicate that efficient interactions with LPS can be achieved when the peptide is not perfectly amphipathic, since this feature seems to help the toroidal pore formation process.

In vivo biological validation of in silico analysis: A novel approach for predicting the effects of TLR4 exon 3 polymorphisms on brucellosis.

The role of the Toll-like receptor 4 (TLR4) is of recognising intracellular and extracellular pathogens and of activating the immune response. This process can be compromised by single nucleotide polymorphisms (SNPs) which might affect the activity of several TLRs. The aim of this study is of ascertaining whether SNPs in the TLR4 of Bubalus bubalis infected by Brucella abortus, compromise the protein functionality. For this purpose, a computational analysis was performed. Next, computational predictions were confirmed by performing genotyping analysis. Finally, NMR-based metabolomics analysis was performed to identify potential biomarkers for brucellosis. The results indicate two SNPs (c. 672 A > C and c. 902 G > C) as risk factor for brucellosis in Bubalus bubalis, and three metabolites (lactate, 3-hydroxybutyrate and acetate) as biological markers for predicting the risk of developing the disease. These metabolites, together with TLR4 structural modifications in the MD2 interaction domain, are a clear signature of the immune system alteration during diverse Gram-negative bacterial infections. This suggests the possibility to extend this study to other pathogens, including Mycobacterium tuberculosis. In conclusion, this study combines multidisciplinary approaches to evaluate the biological and structural effects of SNPs on protein function.

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.

Heterozygosity at the A625C polymorphic site of the MyD88 gene is associated with Mycobacterium bovis infection in cattle.

The study demonstrates that in cattle, animals heterozygous at the MyD88 A625C polymorphic marker have a 5-fold reduced risk for active pulmonary tuberculosis (odds ratio [OR] = 0.19; P = 6 × 10(-12)). The reduced risk, however, does not extend to animals with latent pulmonary tuberculosis (OR = 0.83; P = 0.40). Heterozygosity at the A625C single nucleotide polymorphism is associated with intermediate levels of tumor necrosis factor alpha, gamma interferon, and nitric oxide synthase (NOS). Accordingly, deficiency as well as overexpression of proinflammatory cytokines or NOS favor tuberculosis, while heterozygosity provides the animals with the optimal level of inflammation.

Structures of free and inhibited forms of the L,D-transpeptidase LdtMt1 from Mycobacterium tuberculosis.

The modelling of peptidoglycan is responsible for key cellular processes in Mycobacterium tuberculosis such as cell growth, division and resuscitation from dormancy. The structure of M. tuberculosis peptidoglycan is atypical since it contains a majority of 3,3 cross-links synthesized by L,D-transpeptidases that replace the 4,3 cross-links formed by the D,D-transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate these L,D-transpeptidases and in combination with clavulanic acid are bactericidal against extensively drug-resistant M. tuberculosis. Here, crystal structures of the L,D-transpeptidase LdtMt1 from M. tuberculosis in a ligand-free form and in complex with the carbapenem imipenem are reported. Elucidation of the structural features of LdtMt1 unveils analogies and differences between the two key transpeptidases of M. tuberculosis: LdtMt1 and LdtMt2. In addition, the structure of imipenem-inactivated LdtMt1 provides a detailed structural view of the interactions between a carbapenem drug and LdtMt1. By providing the key interactions in the binding of carbapenem to LdtMt1, this work will facilitate structure-guided discovery of L,D-transpeptidase inhibitors as novel antitubercular agents against drug-resistant M. tuberculosis.