Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-Suisse Cheeses under In Vitro Gastrointestinal Conditions by Propidium Monoazide - qPCR

Species-specific Quantitative Real Time PCR (qPCR) alone and combined with the use of propidium monoazide (PMA) were used along with the plate count method to evaluate the survival of the probiotic strains Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12, and the bacteriocinogenic and potentially probiotic strain Lactobacillus sakei subsp. sakei 2a in synbiotic (F1) and probiotic (F2) petit-suisse cheeses exposed throughout shelf-life to in vitro simulated gastrointestinal tract conditions. The three strains studied showed a reduction in their viability after the 6 h assay. Bb-12 displayed the highest survival capacity, above 72.6 and 74.6% of the initial populations, respectively, by plate count and PMA-qPCR, maintaining population levels in the range or above 6 log CFU/g. The prebiotic mix of inulin and FOS did not offer any additional protection for the strains against the simulated gastrointestinal environment. The microorganisms'' populations were comparable among the three methods at the initial time of the assay, confirming the presence of mainly viable and culturable cells. However, with the intensification of the stress induced throughout the various stages of the in vitro test, the differences among the methods increased. The qPCR was not a reliable enumeration method for the quantification of intact bacterial populations, mixed with large numbers of injured and dead bacteria, as confirmed by the scanning electron microscopy results. Furthermore, bacteria plate counts were much lower (P<0.05) than with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms unable to form colonies. The use of PMA overcame the qPCR inability to differentiate between dead and alive cells. The combination of PMA and species-specific qPCR in this study allowed a quick and unequivocal way of enumeration of viable closely related species incorporated into probiotic and synbiotic petit-suisse cheeses and under stress conditions.     

The calcium-binding C-terminal domain of Escherichia coli alpha-hemolysin is a major determinant in the surface-active properties of the protein

alpha-Hemolysin (HlyA) from Escherichia coli is a protein toxin (1024 amino acids) that targets eukaryotic cell membranes, causing loss of the permeability barrier. HlyA consists of two main regions, an N-terminal domain rich in amphipathic helices, and a C-terminal Ca(2+)-binding domain containing a Gly- and Asp-rich nonapeptide repeated in tandem 11-17 times. The latter is called the RTX domain and gives its name to the RTX protein family. It had been commonly assumed that membrane interaction occurred mainly if not exclusively through the amphipathic helix domain. However, we have cloned and expressed the C-terminal region of HlyA, containing the RTX domain plus a few stabilizing sequences, and found that it is a potent surface-active molecule. The isolated domain binds Ca(2+) with about the same affinity (apparent K(0.5) approximately 150 microM) as the parent protein HlyA, and Ca(2+) binding induces in turn a more compact folding with an increased proportion of beta-sheet structure. Both with and without Ca(2+) the C-terminal region of HlyA can interact with lipid monolayers spread at an air-water interface. However, the C-terminal domain by itself is devoid of membrane lytic properties. The present results can be interpreted in the light of our previous studies that involved in receptor binding a peptide in the C-terminal region of HlyA. We had also shown experimentally the distinction between reversible membrane adsorption and irreversible lytic insertion of the toxin. In this context, the present data allow us to propose that both major domains of HlyA are directly involved in membrane-toxin interaction, the nonapeptide repeat, calcium-binding RTX domain being responsible for the early stages of HlyA docking to the target membrane.     

Event-driven time-optimal control for a class of discontinuous bioreactors

Discontinuous bioreactors may be further optimized for processing inhibitory substrates using a convenient fed-batch mode. To do so the filling rate must be controlled in such a way as to push the reaction rate to its maximum value, by increasing the substrate concentration just up to the point where inhibition begins. However, an exact optimal controller requires measuring several variables (e.g., substrate concentrations in the feed and in the tank) and also good model knowledge (e.g., yield and kinetic parameters), requirements rarely satisfied in real applications. An environmentally important case, that exemplifies all these handicaps, is toxicant wastewater treatment. There the lack of online practical pollutant sensors may allow unforeseen high shock loads to be fed to the bioreactor, causing biomass inhibition that slows down the treatment process and, in extreme cases, even renders the biological process useless. In this work an event-driven time-optimal control (ED-TOC) is proposed to circumvent these limitations. We show how to detect a "there is inhibition" event by using some computable function of the available measurements. This event drives the ED-TOC to stop the filling. Later, by detecting the symmetric event, "there is no inhibition," the ED-TOC may restart the filling. A fill-react cycling then maintains the process safely hovering near its maximum reaction rate, allowing a robust and practically time-optimal operation of the bioreactor. An experimental study case of a wastewater treatment process application is presented. There the dissolved oxygen concentration was used to detect the events needed to drive the controller.     

The biofilm formation defect of a Bacillus subtilis flotillin‐defective mutant involves the protease FtsH

Biofilm formation in Bacillus subtilis requires the differentiation of a subpopulation of cells responsible for the production of the extracellular matrix that structures the biofilm. Differentiation of matrix‐producing cells depends, among other factors, on the FloT and YqfA proteins. These proteins are present exclusively in functional membrane microdomains of B. subtilis and are homologous to the eukaryotic lipid raft‐specific flotillin proteins. In the absence of FloT and YqfA, diverse proteins normally localized to the membrane microdomains of B. subtilis are not functional. Here we show that the absence of FloT and YqfA reduces the level of the septal‐localized protease FtsH. The flotillin homologues FloT and YqfA are occasionally present at the midcell in exponentially growing cells and the absence of FloT and YqfA negatively affects FtsH concentration. Biochemical experiments indicate a direct interaction between FloT/YqfA and FtsH. Moreover, FtsH is essential for the differentiation of matrix producers and hence, biofilm formation. This molecular trigger of biofilm formation may therefore be used as a target for the design of new biofilm inhibitors. Accordingly, we show that the small protein SpoVM, known to bind to and inhibit FtsH activity, inhibits biofilm formation in B. subtilis and other distantly related bacteria.     

Structures of Human DPP7 Reveal the Molecular Basis of Specific Inhibition and the Architectural Diversity of Proline-Specific Peptidases

Proline-specific dipeptidyl peptidases (DPPs) are emerging targets for drug development. DPP4 inhibitors are approved in many countries, and other dipeptidyl peptidases are often referred to as DPP4 activity- and/or structure-homologues (DASH). Members of the DASH family have overlapping substrate specificities, and, even though they share low sequence identity, therapeutic or clinical cross-reactivity is a concern. Here, we report the structure of human DPP7 and its complex with a selective inhibitor Dab-Pip (L-2,4-diaminobutyryl-piperidinamide) and compare it with that of DPP4. Both enzymes share a common catalytic domain (α/β-hydrolase). The catalytic pocket is located in the interior of DPP7, deep inside the cleft between the two domains. Substrates might access the active site via a narrow tunnel. The DPP7 catalytic triad is completely conserved and comprises Ser162, Asp418 and His443 (corresponding to Ser630, Asp708 and His740 in DPP4), while other residues lining the catalytic pockets differ considerably. The "specificity domains" are structurally also completely different exhibiting a β-propeller fold in DPP4 compared to a rare, completely helical fold in DPP7. Comparing the structures of DPP7 and DPP4 allows the design of specific inhibitors and thus the development of less cross-reactive drugs. Furthermore, the reported DPP7 structures shed some light onto the evolutionary relationship of prolyl-specific peptidases through the analysis of the architectural organization of their domains.     

Genetic and functional analyses of SHANK2 mutations suggest a multiple hit model of autism spectrum disorders

Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders with a complex inheritance pattern. While many rare variants in synaptic proteins have been identified in patients with ASD, little is known about their effects at the synapse and their interactions with other genetic variations. Here, following the discovery of two de novo SHANK2 deletions by the Autism Genome Project, we identified a novel 421 kb de novo SHANK2 deletion in a patient with autism. We then sequenced SHANK2 in 455 patients with ASD and 431 controls and integrated these results with those reported by Berkel et al. 2010 (n = 396 patients and n = 659 controls). We observed a significant enrichment of variants affecting conserved amino acids in 29 of 851 (3.4%) patients and in 16 of 1,090 (1.5%) controls (P = 0.004, OR = 2.37, 95% CI = 1.23–4.70). In neuronal cell cultures, the variants identified in patients were associated with a reduced synaptic density at dendrites compared to the variants only detected in controls (P = 0.0013). Interestingly, the three patients with de novo SHANK2 deletions also carried inherited CNVs at 15q11–q13 previously associated with neuropsychiatric disorders. In two cases, the nicotinic receptor CHRNA7 was duplicated and in one case the synaptic translation repressor CYFIP1 was deleted. These results strengthen the role of synaptic gene dysfunction in ASD but also highlight the presence of putative modifier genes, which is in keeping with the “multiple hit model” for ASD. A better knowledge of these genetic interactions will be necessary to understand the complex inheritance pattern of ASD.     

Repeated Administration of the Neurotensin Receptor Antagonist SR 48692 Differentially Regulates Mesocortical and Mesolimbic Dopaminergic Systems

Abstract: The purpose of the present study was to investigate the effects of repeated administration of the neurotensin receptor antagonist, SR 48692, on the activity of the mesocortical and mesolimbic dopaminergic (DA) systems. We showed that daily administration of SR 48692 for 15 days (1 mg/kg i.p.) to Wistar rats increased the expression of tyrosine hydroxylase mRNA and protein in the ventral mesencephalon. Simultaneous in vivo microdialysis in the shell part of the nucleus accumbens (AcbSh) and the medial prefrontal cortex (mPFC) revealed that blockade of neurotensin receptors for 15 days decreased basal extracellular levels of DA (∼50%) and its metabolites in the AcbSh, whereas no modification in DA levels was observed in the mPFC. In animals submitted to a forced swimming stress, which preferentially enhanced extracellular DA levels in the mPFC, treatment with SR 48692 failed to affect the stress-induced increase in DA. Moreover, given that glucocorticoids can modulate the activity of mesencephalic DA neurons, we examined the effect of the same SR 48692 treatment on corticosterone levels in dialysates from the AcbSh. We found that repeated SR 48692 did not affect the basal levels of free corticosterone, but significantly reduced the increase induced by forced swimming stress. The present results demonstrate that repeated treatment with SR 48692 modulates selectively the DA mesolimbic system when compared with the mesocortical pathway. These findings suggest that long-term treatment with selective neurotensin receptor antagonists could have potential clinical utility in the treatment of neuropsychiatric disorders associated with hyperactivity of the mesolimbic DA systems or the hypothalamic-pituitary-adrenal axis.