Design of novel urogenital pharmabiotic formulations containing lactobacilli,salivaricin CRL 1328 and non-microbial compounds with different functionalities

Context: The administration of pharmabiotics is a promising alternative to antimicrobial drugs for the treatment and/or prevention of female urogenital infections.

Objective: To design pharmabiotic formulations including bioactive ingredients of microbial origin combined with non-microbial substances and then to evaluate the stability of the combinations during freeze-drying and storage.

Materials and methods: Different formulations including Lactobacillus gasseri CRL 1263, Lactobacillus salivarius CRL 1328, salivaricin CRL 1328 (a bacteriocin) and non-microbial compounds (lactose, inulin and ascorbic acid) were assayed, and the ingredients were freeze-dried together or separately. The formulations were stored in gelatin capsules at 4?°C for 360?d.

Results: The viability of lactobacilli was affected to different extents depending on the strains and on the formulations assayed. L. salivarius and ascorbic acid were successfully combined only after the freeze-drying process. Salivaricin activity was not detected in formulations containing L. gasseri. However, when combined with ascorbic acid, lactose, inulin or L. salivarius, the bacteriocin maintained its activity for 360?d. The selected microorganisms proved to be compatible for their inclusion in multi-strain formulations together with lactose, inulin and ascorbic acid. Salivaricin could be included only in a L. salivarius CRL 1328 single-strain formulation together with non-microbial substances.

Conclusions: This study provides new insights into the design of urogenital pharmabiotics combining beneficial lactobacilli, salivaricin CRL 1328 and compounds with different functionalities.     

Synthesis of Long-Chain β-Lactones and Their Antibacterial Activities against Pathogenic Mycobacteria

In the quest for new antibacterial agents, a series of novel long- and medium-chain mono- and disubstituted β-lactones was developed. Their activity against three pathogenic mycobacteria—M. abscessus, M. marinum, and M. tuberculosis—was assessed by the resazurin microtiter assay (REMA). Among the 16 β-lactones synthesized, only 3-hexadecyloxetan-2-one (VM005) exhibited promising activity against M. abscessus, whereas most of the β-lactones showed interesting activities against M. marinum, similar to that of the classical antibiotic, isoniazid. Regarding M. tuberculosis, six compounds were found to be active against this mycobacterium, with β-lactone VM008 [trans-(Z)-3-(hexadec-7-en-1-yl)-4-propyloxetan-2-one] being the best growth inhibitor. The promising antibacterial activities of the best compounds in this series suggest that these molecules may serve as leads for the development of much more efficient antimycobacterial agents.     

Alteration of the Route to Menaquinone towards Isochorismate-Derived Metabolites

Chorismate and isochorismate constitute branch-point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α-ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double-variant Asn117Arg–Leu478Thr preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHD), the hydrolysis product of isochorismate, with a >70-fold higher ratio than that for the wild type. The single-variant Arg107Ile uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHA) as substrate with >6-fold conversion compared to wild-type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L⁻¹). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild-type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.     

Effect of processing on the microbiological quality and safety of chicken carcasses at slaughterhouse

Microbiological contamination of chicken meat depends on the conditions under which the animals are reared, slaughtered and processed. The aim of this study was to determine the influence of farm origin and processing stages at slaughterhouse on the microbial safety and quality of chicken. Samples of chicken carcasses from three different farms were taken from a slaughterhouse. Mesophiles, Escherichia coli, coagulase positive Staphylococcci counts, presence of Listeria monocytogenes,Campylobacter and Salmonella were determined at five sampling points: after defeathering, after evisceration, after washing, after chilling and after cutting. Chilling reduced log numbers of mesophiles, coagulase positive Staphylococci and E. coli by 0.85, 1.52 and 2.2 log units, respectively. Salmonella was not detected after chilling. High prevalence of Campylobacter spp was observed at all the stages ranging between 84% and 100%. L. monocytogenes was not detected in chicken carcasses after defeathering. However, it was detected after evisceration and after washing and chilling. The most critical stage for L. monocytogenes contamination was the portioning operation, the prevalence in breast and legs being 88% and 84%, respectively.     

Salivary Proteome Changes in Response to Acute Psychological Stress Due to an Oral Exam Simulation in University Students: Effect of an Olfactory Stimulus

The autonomic nervous system (ANS) plays a crucial role both in acute and chronic psychological stress eliciting changes in many local and systemic physiological and biochemical processes. Salivary secretion is also regulated by ANS. In this study, we explored salivary proteome changes produced in thirty-eight University students by a test stress, which simulated an oral exam. Students underwent a relaxation phase followed by the stress test during which an electrocardiogram was recorded. To evaluate the effect of an olfactory stimulus, half of the students were exposed to a pleasant odor diffused in the room throughout the whole session. Saliva samples were collected after the relaxation phase (T0) and the stress test (T1). State anxiety was also evaluated at T0 and T1. Salivary proteins were separated by two-dimensional electrophoresis, and patterns at different times were compared. Spots differentially expressed were trypsin digested and identified by mass spectrometry. Western blot analysis was used to validate proteomic results. Anxiety scores and heart rate changes indicated that the fake exam induced anxiety. Significant changes of α-amylase, polymeric immunoglobulin receptor (PIGR), and immunoglobulin α chain (IGHA) secretion were observed after the stress test was performed in the two conditions. Moreover, the presence of pleasant odor reduced the acute social stress affecting salivary proteome changes. Therefore, saliva proteomic analysis was a useful approach to evaluate the rapid responses associated to an acute stress test also highlighting known biomarkers.     

Structural Cues for Understanding eEF1A2 Moonlighting

Spontaneous mutations in the EEF1A2 gene cause epilepsy and severe neurological disabilities in children. The crystal structure of eEF1A2 protein purified from rabbit skeletal muscle reveals a post-translationally modified dimer that provides information about the sites of interaction with numerous binding partners, including itself, and maps these mutations onto the dimer and tetramer interfaces. The spatial locations of the side chain carboxylates of Glu301 and Glu374, to which phosphatidylethanolamine is uniquely attached via an amide bond, define the anchoring points of eEF1A2 to cellular membranes and interorganellar membrane contact sites. Additional bioinformatic and molecular modeling results provide novel structural insight into the demonstrated binding of eEF1A2 to SH3 domains, the common MAPK docking groove, filamentous actin, and phosphatidylinositol-4 kinase IIIβ. In this new light, the role of eEF1A2 as an ancient, multifaceted, and articulated G protein at the crossroads of autophagy, oncogenesis and viral replication appears very distant from the “canonical” one of delivering aminoacyl-tRNAs to the ribosome that has dominated the scene and much of the thinking for many decades.