Design and expression of peptides with antimicrobial activity against Salmonella typhimurium

We showed previously that insertion of Synechocystis Δ¹²‐desaturase in salmonella's membrane alters membrane physical state (MPS), followed by the expression of stress genes causing inability to survive within murine macrophages (MΦ). Recently, we showed that expression of one membrane lipid domain (MLD) of Δ¹²‐desaturase (ORF200) interferes with salmonella MPS, causing loss of virulence in mice and immunoprotection. Here, we postulate that an α‐antimicrobial peptide (α‐AMP) intercalates within membrane lipids, and depending on its amino acid sequence, it does so within specific key sensors of MLD. In this study, we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg²⁺ concentration. We synthesised a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in salmonella typhimurium. We showed that the pattern of gene expression controlled by PhoP/PhoQ highlights dysregulation of pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens. RNA‐Seq of strain expressing ORF200 showed that the pattern of those genes is also altered here. Accumulation of NUF conferred temporary immunoprotection. This represents a powerful procedure to address synthetic α‐AMPs to a specific MLD generating live non‐virulent bacterial strains.     

Accumulation of Polyphosphate in Lactobacillus spp. and Its Involvement in Stress Resistance

Polyphosphate (poly-P) is a polymer of phosphate residues synthesized and in some cases accumulated by microorganisms, where it plays crucial physiological roles such as the participation in the response to nutritional stringencies and environmental stresses. Poly-P metabolism has received little attention in Lactobacillus, a genus of lactic acid bacteria of relevance for food production and health of humans and animals. We show that among 34 strains of Lactobacillus, 18 of them accumulated intracellular poly-P granules, as revealed by specific staining and electron microscopy. Poly-P accumulation was generally dependent on the presence of elevated phosphate concentrations in the culture medium, and it correlated with the presence of polyphosphate kinase (ppk) genes in the genomes. The ppk gene from Lactobacillus displayed a genetic arrangement in which it was flanked by two genes encoding exopolyphosphatases of the Ppx-GppA family. The ppk functionality was corroborated by its disruption (LCABL_27820 gene) in Lactobacillus casei BL23 strain. The constructed ppk mutant showed a lack of intracellular poly-P granules and a drastic reduction in poly-P synthesis. Resistance to several stresses was tested in the ppk-disrupted strain, showing that it presented a diminished growth under high-salt or low-pH conditions and an increased sensitivity to oxidative stress. These results show that poly-P accumulation is a characteristic of some strains of lactobacilli and may thus play important roles in the physiology of these microorganisms.     

A small unstructured nucleic acid disrupts a trinucleotide repeat hairpin

A variety of neurodegenerative disorders are associated with the expansion of trinucleotide repeat (TNR) sequences. These repetitive sequences are prone to adopting non-canonical structures, such as intrastrand stem-loop hairpins. Indeed, the formation and persistence of these hairpins during DNA replication and/or repair have been proposed as factors that facilitate TNR expansion. Given this proposed contribution of TNR hairpins to the expansion mechanism, disruption of such structures via strand invasion offers a potential means to negate the disease-initiating expansion. In this work, we investigated the strand invading abilities of a (CTG)₃ unstructured nucleic acid on a (CAG)₁₀ TNR hairpin. Using fluorescence, optical, and electrophoretic methods, instantaneous disruption of the (CAG)₁₀ hairpin by (CTG)₃ was observed at low temperatures. Additionally, we have identified three distinct duplex-like species that form between (CAG)₁₀ and (CTG)₃; these include 1, 2, or 3 (CTG)₃ sequences hybridized to (CAG)₁₀. The results presented here showcase (CTG)₃ as an invader of a TNR hairpin and suggest that unstructured nucleic acids could serve as a scaffold to design agents to prevent TNR expansion.     

Quantitative trait loci and underlying candidate genes controlling agronomical and fruit quality traits in octoploid strawberry <Emphasis Type="Italic">(Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis>)

Breeding for fruit quality traits in strawberry (Fragaria × ananassa, 2n = 8x = 56) is complex due to the polygenic nature of these traits and the octoploid constitution of this species. In order to improve the efficiency of genotype selection, the identification of quantitative trait loci (QTL) and associated molecular markers will constitute a valuable tool for breeding programs. However, the implementation of these markers in breeding programs depends upon the complexity and stability of QTLs across different environments. In this work, the genetic control of 17 agronomical and fruit quality traits was investigated in strawberry using a F₁ population derived from an intraspecific cross between two contrasting selection lines, ‘232’ and ‘1392’. QTL analyses were performed over three successive years based on the separate parental linkage maps and a pseudo-testcross strategy. The integrated strawberry genetic map consists of 338 molecular markers covering 37 linkage groups, thus exceeding the 28 chromosomes. 33 QTLs were identified for 14 of the 17 studied traits and approximately 37% of them were stable over time. For each trait, 1–5 QTLs were identified with individual effects ranging between 9.2 and 30.5% of the phenotypic variation, indicating that all analysed traits are complex and quantitatively inherited. Many QTLs controlling correlated traits were co-located in homoeology group V, indicating linkage or pleiotropic effects of loci. Candidate genes for several QTLs controlling yield, anthocyanins, firmness and l-ascorbic acid are proposed based on both their co-localization and predicted function. We also report conserved QTLs among strawberry and other Rosaceae based on their syntenic location.     

Influence of environmental and genetic factors linked to celiac disease risk on infant gut colonization by Bacteroides species

Celiac disease (CD) is an immune-mediated enteropathy involving genetic and environmental factors whose interaction might influence disease risk. The aim of this study was to determine the effects of milk-feeding practices and the HLA-DQ genotype on intestinal colonization of Bacteroides species in infants at risk of CD development. This study included 75 full-term newborns with at least one first-degree relative suffering from CD. Infants were classified according to milk-feeding practice (breast-feeding or formula feeding) and HLA-DQ genotype (high or low genetic risk). Stools were analyzed at 7 days, 1 month, and 4 months by PCR and denaturing gradient gel electrophoresis (DGGE). The Bacteroides species diversity index was higher in formula-fed infants than in breast-fed infants. Breast-fed infants showed a higher prevalence of Bacteroides uniformis at 1 and 4 months of age, while formula-fed infants had a higher prevalence of B. intestinalis at all sampling times, of B. caccae at 7 days and 4 months, and of B. plebeius at 4 months. Infants with high genetic risk showed a higher prevalence of B. vulgatus, while those with low genetic risk showed a higher prevalence of B. ovatus, B. plebeius, and B. uniformis. Among breast-fed infants, the prevalence of B. uniformis was higher in those with low genetic risk than in those with high genetic risk. Among formula-fed infants, the prevalence of B. ovatus and B. plebeius was increased in those with low genetic risk, while the prevalence of B. vulgatus was higher in those with high genetic risk. The results indicate that both the type of milk feeding and the HLA-DQ genotype influence the colonization process of Bacteroides species, and possibly the disease risk.     

Metabolic alterations in K562 cells exposed to taxol and tyrphostin AG957: 1H NMR and biochemical studies

K562 cells exposed for 3 h to taxol or taxol plus tyrphostin AG957 exhibited a significant variation in the concentration of the water-soluble metabolites glutathione, myo-inositol and phosphorylcholine, as evaluated by (1)H NMR up to 72 h incubation in drug-free medium. Cells treated with both drugs showed an increase of glutathione and glutathione reductase at 24 h and a sharp decrease of myo-inositol between 8 and 24 h. Phosphorylcholine increased at 8 h both in taxol and taxol plus AG957-treated cells, which was then abruptly inverted to a significantly lower concentration at 24 h, subsequently increasing again to values higher than those found in taxol-treated and control cells. All the above reported effects were lacking in cells exposed to AG957 alone. These modifications, despite the enhancement of the overall apoptotic cascade in taxol plus AG957-treated cells, can be related to the activation of cellular detoxification mechanisms, to the correct osmolarity maintenance, and to alterations of phospholipid metabolism.     

Serum Levels of Vasoactive Intestinal Peptide as a Prognostic Marker in Early Arthritis

Objective

Suitable biomarkers are essential for the design of therapeutic strategies in personalized medicine. Vasoactive intestinal peptide (VIP) has demonstrated immunomodulatory properties in autoimmune murine and ex vivo human models. Our aim was to study serum levels of VIP during the follow-up of an early arthritis (EA) cohort and to analyze its value as a biomarker predicting severity and therapeutic requirements.

Methods

Data from 91 patients on an EA register were analyzed (76% rheumatoid arthritis (RA), 24% undifferentiated arthritis, 73% women, and median age 54 years; median disease duration at entry, 5.4 months). We collected per protocol sociodemographic, clinical, and therapeutic data. VIP levels were determined by enzyme immunoassay in sera harvested from the 91 patients (353 visits; 3.9 visit/patient) and from 100 healthy controls. VIP values below the 25^th percentile of those assessed in healthy population were considered low. To determine the effect of independent variables on VIP levels, we performed a longitudinal multivariate analysis nested by patient and visit. A multivariate ordered logistic regression was modeled to determine the effect of low VIP serum levels on disease activity at the end of follow-up.

Results

VIP concentrations varied considerably across EA patients. Those fulfilling the criteria for RA had the lowest values in the whole sample, although no significant differences were observed compared with healthy donors. Disease activity, which was assessed using DAS28, inversely correlated with VIP levels. After a two-year follow-up, those patients with low baseline levels of VIP displayed higher disease activity and received more intensive treatment.

Conclusion

Patients who are unable to up-regulate VIP seem to have a worse clinical course despite receiving more intense treatment. Therefore, measurement of VIP levels may be suitable as a prognostic biomarker.     

Change in Body Size and Mortality: Results from the Melbourne Collaborative Cohort Study

Background

The association between change in weight or body mass index, and mortality is widely reported, however, both measures fail to account for fat distribution. Change in waist circumference, a measure of central adiposity, in relation to mortality has not been studied extensively.

Methods

We investigated the association between mortality and changes in directly measured waist circumference, hips circumference and weight from baseline (1990–1994) to wave 2 (2003–2007) in a prospective cohort study of people aged 40–69 years at baseline. Cox regression, with age as the time metric and follow-up starting at wave 2, adjusted for confounding variables, was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for change in body size in relation to mortality from all causes, cardiovascular disease and cancer.

Results

There were 1465 deaths (109 cancer, 242 cardiovascular disease) identified during an average 7.7 years of follow-up from 21 298 participants. Compared to minimal increase in body size, loss of waist circumference (HR: 1.26; 95% CI: 1.09–1.47), weight (1.80; 1.54–2.11), or hips circumference (1.35; 1.15–1.57) were associated with an increased risk of all-cause mortality, particularly for older adults. Weight loss was associated with cardiovascular disease mortality (2.40; 1.57–3.65) but change in body size was not associated with obesity-related cancer mortality.

Conclusion

This study confirms the association between weight loss and increased mortality from all-causes for older adults. Based on evidence from observational cohort studies, weight stability may be the recommended option for most adults, especially older adults.