Celiac disease‐associated Neisseria flavescens decreases mitochondrial respiration in CaCo‐2 epithelial cells: Impact of Lactobacillus paracasei CBA L74 on bacterial‐induced cellular imbalance

We previously identified a Neisseria flavescens strain in the duodenum of celiac disease (CD) patients that induced immune inflammation in ex vivo duodenal mucosal explants and in CaCo‐2 cells. We also found that vesicular trafficking was delayed after the CD‐immunogenic P31‐43 gliadin peptide‐entered CaCo‐2 cells and that Lactobacillus paracasei CBA L74 (L. paracasei‐CBA) supernatant reduced peptide entry. In this study, we evaluated if metabolism and trafficking was altered in CD‐N. flavescens‐infected CaCo‐2 cells and if any alteration could be mitigated by pretreating cells with L. paracasei‐CBA supernatant, despite the presence of P31‐43. We measured CaCo‐2 bioenergetics by an extracellular flux analyser, N. flavescens and P31‐43 intracellular trafficking by immunofluorescence, cellular stress by TBARS assay, and ATP by bioluminescence. We found that CD‐N. flavescens colocalised more than control N. flavescens with early endocytic vesicles and more escaped autophagy thereby surviving longer in infected cells. P31‐43 increased colocalisation of N. flavescens with early vesicles. Mitochondrial respiration was lower (P < .05) in CD‐N. flavescens‐infected cells versus not‐treated CaCo‐2 cells, whereas pretreatment with L. paracasei‐CBA reduced CD‐N. flavescens viability and improved cell bioenergetics and trafficking. In conclusion, CD‐N. flavescens induces metabolic imbalance in CaCo‐2 cells, and the L. paracasei‐CBA probiotic could be used to correct CD‐associated dysbiosis.     

An update on magnesium and bone health

In 2009 EFSA Panel concludes that a cause and effect relationship has been established between the dietary intake of magnesium (Mg) and maintenance of normal bone. After 2009, numerous studies have been published, but no reviews have made an update on this topic. So, the aim of this narrative review was to consider the state of the art since 2009 on relationship between Mg blood levels, Mg dietary intake and Mg dietary supplementation (alone or with other micronutrients; this last topic has been considered since 1990, because it is not included in the EFSA claims) and bone health in humans. This review included 28 eligible studies: nine studies concern Mg blood, 12 studies concern Mg intake and seven studies concern Mg supplementation, alone or in combination with other nutrients. From the various studies carried out on the serum concentration of Mg and its relationship with the bone, it has been shown that lower values are related to the presence of osteoporosis, and that about 30–40% of the subjects analyzed (mainly menopausal women) have hypomagnesaemia. Various dietetic investigations have shown that many people (about 20%) constantly consume lower quantities of Mg than recommended; moreover, in this category, a lower bone mineral density and a higher fracturing risk have been found. Considering the intervention studies published to date on supplementation with Mg, most have used this mineral in the form of citrate, carbonate or oxide, with a dosage varying between 250 and 1800 mg. In all studies there was a benefit both in terms of bone mineral density and fracture risk.

     

Interleukin‐6 neutralization ameliorates symptoms in prematurely aged mice

Hutchinson–Gilford progeria syndrome (HGPS) causes premature aging in children, with adipose tissue, skin and bone deterioration, and cardiovascular impairment. In HGPS cells and mouse models, high levels of interleukin‐6, an inflammatory cytokine linked to aging processes, have been detected. Here, we show that inhibition of interleukin‐6 activity by tocilizumab, a neutralizing antibody raised against interleukin‐6 receptors, counteracts progeroid features in both HGPS fibroblasts and Lmna^G609G/G609G progeroid mice. Tocilizumab treatment limits the accumulation of progerin, the toxic protein produced in HGPS cells, rescues nuclear envelope and chromatin abnormalities, and attenuates the hyperactivated DNA damage response. In vivo administration of tocilizumab reduces aortic lesions and adipose tissue dystrophy, delays the onset of lipodystrophy and kyphosis, avoids motor impairment, and preserves a good quality of life in progeroid mice. This work identifies tocilizumab as a valuable tool in HGPS therapy and, speculatively, in the treatment of a variety of aging‐related disorders.     

Association between Recruitment Methods and Attrition in Internet-Based Studies

Internet-based systems for epidemiological studies have advantages over traditional approaches as they can potentially recruit and monitor a wider range of individuals in a relatively inexpensive fashion. We studied the association between communication strategies used for recruitment (offline, online, face-to-face) and follow-up participation in nine Internet-based cohorts: the Influenzanet network of platforms for influenza surveillance which includes seven cohorts in seven different European countries, the Italian birth cohort Ninfea and the New Zealand birth cohort ELF. Follow-up participation varied from 43% to 89% depending on the cohort. Although there were heterogeneities among studies, participants who became aware of the study through an online communication campaign compared with those through traditional offline media seemed to have a lower follow-up participation in 8 out of 9 cohorts. There were no clear differences in participation between participants enrolled face-to-face and those enrolled through other offline strategies. An Internet-based campaign for Internet-based epidemiological studies seems to be less effective than an offline one in enrolling volunteers who keep participating in follow-up questionnaires. This suggests that even for Internet-based epidemiological studies an offline enrollment campaign would be helpful in order to achieve a higher participation proportion and limit the cohort attrition.     

Thiol oxidation is crucial in the desensitization of the mitochondrial F1FO-ATPase to oligomycin and other macrolide antibiotics

Background

The macrolide antibiotics oligomycin, venturicidin and bafilomycin, sharing the polyketide ring and differing in the deoxysugar moiety, are known to block the transmembrane ion channel of ion-pumping ATPases; oligomycins are selective inhibitors of mitochondrial ATP synthases.

Methods

The inhibition mechanism of macrolides was explored on swine heart mitochondrial F₁F_O-ATPase by kinetic analyses. The amphiphilic membrane toxicant tributyltin (TBT) and the thiol reducing agent dithioerythritol (DTE) were used to elucidate the nature of the macrolide–enzyme interaction.

Results

When individually tested, the macrolide antibiotics acted as uncompetitive inhibitors of the ATPase activity. Binary mixtures of macrolide inhibitors I₁ and I₂ pointed out a non-exclusive mechanism, indicating that each macrolide binds to its binding site on the enzyme. When co-present, the two macrolides acted synergistically in the formed quaternary complex (ESI₁I₂), thus mutually strengthening the enzyme inhibition. The enzyme inhibition by macrolides displaying a shared mechanism was dose-dependently reduced by TBT ≥ 1 μM. The TBT-driven enzyme desensitization was reversed by DTE.

Conclusions

The macrolides tested share uncompetitive inhibition mechanism by binding to a specific site in a common macrolide-binding region of F_O. The oxidation of highly conserved thiols in the ATP synthase c-ring of F_O weakens the interaction between the enzyme and the macrolides. The native macrolide-inhibited enzyme conformation can be restored by reducing crucial thiols oxidized by TBT.

General significance

The findings, by elucidating the macrolide inhibitory mechanism on F_O, indirectly cast light on the F₁F_O torque generation involving crucial amino acid residues and may address drug design and antimicrobial therapy.     

Molecular control of seasonal flowering in rice,arabidopsis and temperate cereals

Background

Rice (Oryza sativa) and Arabidopsis thaliana have been widely used as model systems to understand how plants control flowering time in response to photoperiod and cold exposure. Extensive research has resulted in the isolation of several regulatory genes involved in flowering and for them to be organized into a molecular network responsive to environmental cues. When plants are exposed to favourable conditions, the network activates expression of florigenic proteins that are transported to the shoot apical meristem where they drive developmental reprogramming of a population of meristematic cells. Several regulatory factors are evolutionarily conserved between rice and arabidopsis. However, other pathways have evolved independently and confer specific characteristics to flowering responses.

Scope

This review summarizes recent knowledge on the molecular mechanisms regulating daylength perception and flowering time control in arabidopsis and rice. Similarities and differences are discussed between the regulatory networks of the two species and they are compared with the regulatory networks of temperate cereals, which are evolutionarily more similar to rice but have evolved in regions where exposure to low temperatures is crucial to confer competence to flower. Finally, the role of flowering time genes in expansion of rice cultivation to Northern latitudes is discussed.

Conclusions

Understanding the mechanisms involved in photoperiodic flowering and comparing the regulatory networks of dicots and monocots has revealed how plants respond to environmental cues and adapt to seasonal changes. The molecular architecture of such regulation shows striking similarities across diverse species. However, integration of specific pathways on a basal scheme is essential for adaptation to different environments. Artificial manipulation of flowering time by means of natural genetic resources is essential for expanding the cultivation of cereals across different environments.     

Molecular Cloning and Functional Characterization of Components of the Capsule Biosynthesis Complex of Neisseria meningitidis Serogroup A: TOWARD IN VITRO VACCINE PRODUCTION*

The human pathogen Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis globally. A major virulence factor of Nm is the capsular polysaccharide (CPS), which in Nm serogroup A consists of N-acetyl-mannosamine-1-phosphate units linked together by phosphodiester linkages [→6)-α-d-ManNAc-(1→OPO₃⁻→]_n. Acetylation in O-3 (to a minor extent in O-4) position results in immunologically active polymer. In the capsule gene cluster (cps) of Nm, region A contains the genetic information for CPSA biosynthesis. Thereby the open reading frames csaA, -B, and -C are thought to encode the UDP-N-acetyl-d-glucosamine-2-epimerase, poly-ManNAc-1-phosphate-transferase, and O-acetyltransferase, respectively. With the aim to use a minimal number of recombinant enzymes to produce immunologically active CPSA, we cloned the genes csaA, csaB, and csaC and functionally characterized the purified recombinant proteins. If recombinant CsaA and CsaB were combined in one reaction tube, priming CPSA-oligosaccharides were efficiently elongated with UDP-GlcNAc as the donor substrate, confirming that CsaA is the functional UDP-N-acetyl-d-glucosamine-2-epimerase and CsaB the functional poly-ManNAc-1-phosphate-transferase. Subsequently, CsaB was shown to transfer ManNAc-1P onto O-6 of the non-reducing end sugar of priming oligosaccharides, to prefer non-O-acetylated over O-acetylated primers, and to efficiently elongate the dimer of ManNAc-1-phosphate. The in vitro synthesized CPSA was purified, O-acetylated with recombinant CsaC, and proven to be identical to the natural CPSA by ¹H NMR, ³¹P NMR, and immunoblotting. If all three enzymes and their substrates were combined in a one-pot reaction, nature identical CPSA was obtained. These data provide the basis for the development of novel vaccine production protocols.     

Acidic pH Strongly Enhances In Vitro Biofilm Formation by a Subset of Hypervirulent ST-17 Streptococcus agalactiae Strains

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30 to 76% of the cases of neonatal meningitis. In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low-, and non-biofilm-forming strains, and to facilitate interpretation of data. This protocol was used to screen the biofilm-forming abilities of 366 GBS clinical isolates from pregnant women and from neonatal infections of different serotypes in relation to medium composition and pH. The results identified a subset of isolates of serotypes III and V that formed strong biofilms under acidic conditions. Importantly, the best biofilm formers belonged to serotype III hypervirulent clone ST-17. Moreover, the abilities of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm initiation and contribute to biofilm structural stability.     

Hitching a Ride on Vesicles: Cauliflower Mosaic Virus Movement Protein Trafficking in the Endomembrane System

The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata. Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance. Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis (Arabidopsis thaliana) μA-adaptin subunit. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide. The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection. In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation. Altogether, these results demonstrate that CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes.Membrane trafficking is essential in eukaryotic cells. Cellular membranes serve as a delivery system for newly synthesized proteins such as transporters and receptors exiting the endoplasmic reticulum after proper folding. They then transit through the Golgi complex, reaching the plasma membrane (PM) or the tonoplast via intermediate endomembrane compartments. Receptors and transporters returning from the PM are either recycled or targeted to the vacuole for degradation. Delivery and recycling sorting pathways overlap in the trans-Golgi network (TGN)/early endosome (EE), an intermediate compartment for both exocytosis and endocytosis (Reyes et al., 2011). In plant systems, the endoplasmic reticulum and PM provide membrane continuity between cells through the connections made by plasmodesmata (PD), cytoplasmic channels that regulate traffic in the symplasm (Maule et al., 2011).The selective transport of macromolecules between different compartments of the endomembrane system is mediated by coat proteins promoting the generation of small cargo-trafficking coated vesicles (Spang, 2008). The recognition and recruitment of cargo proteins are mediated by so-called adaptor complexes (AP complexes [AP-1–AP-4]; Robinson, 2004) one of which, AP-1, is localized on the TGN/EE and endosomes, whereas AP-2 is in the PM. The μ-subunit of AP complexes is devoted to cargo protein selection via a specific and well-characterized interaction with a Tyr-sorting signal, YXXΦ, where Φ is a bulky hydrophobic residue and X is any amino acid (Bonifacino and Dell’Angelica, 1999). YXXΦ motifs are present in the cytoplasmic tail of many proteins integral to the PM and TGN/EE and have been found in the movement proteins (MPs) of some viruses (Laporte et al., 2003; Haupt et al., 2005). Plant viruses are obligate parasites that exploit host components to move within the cell and from cell to cell into the vascular system for systemic invasion of the host. Virus movement, which requires the passage of macromolecules through PD connections, is mediated by one or more virus-encoded MPs with the help of the host cytoskeleton and/or endomembranes (Harries et al., 2010). While most MPs act to increase the size exclusion limit of PD to facilitate the passage of the viral nucleoprotein complex, other MPs are assembled in tubules that pass inside highly modified PD and transport encapsidated particles through their lumen.Here, we focus on this second group of tubule-forming MPs and examine the intracellular trafficking of cauliflower mosaic virus (CaMV) MP. The MP encoded by CaMV forms tubules guiding encapsidated virus particle cell-to-cell transport via an indirect MP-virion interaction (Stavolone et al., 2005; Sánchez-Navarro et al., 2010). However, how CaMV MP (and the other tubule-forming MPs) targets the PM and forms tubules remains to be elucidated. Tubule-forming MPs do not require an intact cytoskeleton for PM targeting (Huang et al., 2000; Pouwels et al., 2002) and/or tubule formation (Laporte et al., 2003). However, suppression of tubule formation upon treatment with brefeldin A (BFA), a specific inhibitor of secretion or endocytosis, suggests the involvement of the endomembrane system in correct functioning of some tubule-forming MPs (Huang et al., 2000; Laporte et al., 2003). In this study, we examined the three Tyr-sorting motifs in CaMV MP and show that each of the three domains interacts directly with subunit μ of an Arabidopsis (Arabidopsis thaliana) AP complex. Mutations in these domains revert in the viral context to maintain CaMV viability. MP is found in endosomal compartments labeled by AtRAB-F2b (ARA7) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64). The presence of at least one functional YXXΦ domain is essential for the localization of MP to endosomes and for tubule assembly but is not required for MP targeting to the PM. We provide several lines of evidence to show CaMV MP trafficking in the endocytic pathway. Our findings are discussed in the light of the recent demonstration that the TGN/EE functions as a major hub controlling secretory and endocytic pathways in plants.