Production of Fibronectin Binding Protein A at the Surface of Lactococcus lactis Increases Plasmid Transfer In Vitro and In Vivo

Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL) expressing the Fibronectin-Binding Protein A of Staphylococcus aureus (LL-FnBPA+) showed higher internalization rates in vitro in Caco-2 cells than the native (wt) lactococci and were able to deliver a eukaryotic Green Fluorescent Protein (GFP) expression plasmid in 1% of human Caco-2 cells. Here, using the bovine beta-lactoglobulin (BLG), one of the major cow''s milk allergen, and GFP we characterized the potential of LL-FnBPA+ as an in vivo DNA vaccine delivery vehicle. We first showed that the invasive strain LL-FnBPA+ carrying the plasmid pValac:BLG (LL-FnBPA+ BLG) was more invasive than LL-BLG and showed the same invasivity as LL-FnBPA+. Then we demonstrated that the Caco-2 cells, co-incubated with LL-FnBPA+ BLG produced up to 30 times more BLG than the Caco-2 cells co-incubated with the non invasive LL-BLG. Using two different gene reporters, BLG and GFP, and two different methods of detection, EIA and fluorescence microscopy, we showed in vivo that: i) in order to be effective, LL-FnBPA+ required a pre-coating with Fetal Calf Serum before oral administration; ii) plasmid transfer occurred in enterocytes without regard to the strains used (invasive or not); iii) the use of LL-FnBPA+ increased the number of mice producing BLG, but not the level of BLG produced. We thus confirmed the good potential of invasive recombinant lactic acid bacteria as DNA delivery vector in vivo.     

MiR-27a Functions as a Tumor Suppressor in Acute Leukemia by Regulating 14-3-3θ

MicroRNAs (miRs) play major roles in normal hematopoietic differentiation and hematopoietic malignancies. In this work, we report that miR-27a, and its coordinately expressed cluster (miR-23a∼miR-27a∼miR-24-2), was down-regulated in acute leukemia cell lines and primary samples compared to hematopoietic stem-progenitor cells (HSPCs). Decreased miR-23a cluster expression in some acute leukemia cell lines was mediated by c-MYC. Replacement of miR-27a in acute leukemia cell lines inhibited cell growth due, at least in part, to increased cellular apoptosis. We identified a member of the anti-apoptotic 14-3-3 family of proteins, which support cell survival by interacting with and negatively regulating pro-apoptotic proteins such as Bax and Bad, as a target of miR-27a. Specifically, miR-27a regulated 14-3-3θ at both the mRNA and protein levels. These data indicate that miR-27a contributes a tumor suppressor-like activity in acute leukemia cells via regulation of apoptosis, and that miR-27a and 14-3-3θ may be potential therapeutic targets.     

Sexual Selection of Human Cooperative Behaviour: An Experimental Study in Rural Senegal

Human cooperation in large groups and between non-kin individuals remains a Darwinian puzzle. Investigations into whether and how sexual selection is involved in the evolution of cooperation represent a new and important research direction. Here, 69 groups of four men or four women recruited from a rural population in Senegal played a sequential public-good game in the presence of out-group observers, either of the same sex or of the opposite sex. At the end of the game, participants could donate part of their gain to the village school in the presence of the same observers. Both contributions to the public good and donations to the school, which reflect different components of cooperativeness, were influenced by the sex of the observers. The results suggest that in this non-Western population, sexual selection acts mainly on men’s cooperative behaviour with non-kin, whereas women’s cooperativeness is mainly influenced by nonsexual social selection.     

Genetic Characterisation of Malawian Pneumococci Prior to the Roll-Out of the PCV13 Vaccine Using a High-Throughput Whole Genome Sequencing Approach

Background

Malawi commenced the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into the routine infant immunisation schedule in November 2011. Here we have tested the utility of high throughput whole genome sequencing to provide a high-resolution view of pre-vaccine pneumococcal epidemiology and population evolutionary trends to predict potential future change in population structure post introduction.

Methods

One hundred and twenty seven (127) archived pneumococcal isolates from randomly selected adults and children presenting to the Queen Elizabeth Central Hospital, Blantyre, Malawi underwent whole genome sequencing.

Results

The pneumococcal population was dominated by serotype 1 (20.5% of invasive isolates) prior to vaccine introduction. PCV13 is likely to protect against 62.9% of all circulating invasive pneumococci (78.3% in under-5-year-olds). Several Pneumococcal Molecular Epidemiology Network (PMEN) clones are now in circulation in Malawi which were previously undetected but the pandemic multidrug resistant PMEN1 lineage was not identified. Genome analysis identified a number of novel sequence types and serotype switching.

Conclusions

High throughput genome sequencing is now feasible and has the capacity to simultaneously elucidate serotype, sequence type and as well as detailed genetic information. It enables population level characterization, providing a detailed picture of population structure and genome evolution relevant to disease control. Post-vaccine introduction surveillance supported by genome sequencing is essential to providing a comprehensive picture of the impact of PCV13 on pneumococcal population structure and informing future public health interventions.     

Initial Characterization of the Pf-Int Recombinase from the Malaria Parasite Plasmodium falciparum

Background

Genetic variation is an essential means of evolution and adaptation in many organisms in response to environmental change. Certain DNA alterations can be carried out by site-specific recombinases (SSRs) that fall into two families: the serine and the tyrosine recombinases. SSRs are seldom found in eukaryotes. A gene homologous to a tyrosine site-specific recombinase has been identified in the genome of Plasmodium falciparum. The sequence is highly conserved among five other members of Plasmodia.

Methodology/Principal Findings

The predicted open reading frame encodes for a ∼57 kDa protein containing a C-terminal domain including the putative tyrosine recombinase conserved active site residues R-H-R-(H/W)-Y. The N-terminus has the typical alpha-helical bundle and potentially a mixed alpha-beta domain resembling that of λ-Int. Pf-Int mRNA is expressed differentially during the P. falciparum erythrocytic life stages, peaking in the schizont stage. Recombinant Pf-Int and affinity chromatography of DNA from genomic or synthetic origin were used to identify potential DNA targets after sequencing or micro-array hybridization. Interestingly, the sequences captured also included highly variable subtelomeric genes such as var, rif, and stevor sequences. Electrophoretic mobility shift assays with DNA were carried out to verify Pf-Int/DNA binding. Finally, Pf-Int knock-out parasites were created in order to investigate the biological role of Pf-Int.

Conclusions/Significance

Our data identify for the first time a malaria parasite gene with structural and functional features of recombinases. Pf-Int may bind to and alter DNA, either in a sequence specific or in a non-specific fashion, and may contribute to programmed or random DNA rearrangements. Pf-Int is the first molecular player identified with a potential role in genome plasticity in this pathogen. Finally, Pf-Int knock-out parasite is viable showing no detectable impact on blood stage development, which is compatible with such function.     

Amyloid-β Acts as a Regulator of Neurotransmitter Release Disrupting the Interaction between Synaptophysin and VAMP2

Background

It is becoming increasingly evident that deficits in the cortex and hippocampus at early stages of dementia in Alzheimer’s disease (AD) are associated with synaptic damage caused by oligomers of the toxic amyloid-β peptide (Aβ42). However, the underlying molecular and cellular mechanisms behind these deficits are not fully understood. Here we provide evidence of a mechanism by which Aβ42 affects synaptic transmission regulating neurotransmitter release.

Methodology/Findings

We first showed that application of 50 nM Aβ42 in cultured neurones is followed by its internalisation and translocation to synaptic contacts. Interestingly, our results demonstrate that with time, Aβ42 can be detected at the presynaptic terminals where it interacts with Synaptophysin. Furthermore, data from dissociated hippocampal neurons as well as biochemical data provide evidence that Aβ42 disrupts the complex formed between Synaptophysin and VAMP2 increasing the amount of primed vesicles and exocytosis. Finally, electrophysiology recordings in brain slices confirmed that Aβ42 affects baseline transmission.

Conclusions/Significance

Our observations provide a necessary and timely insight into cellular mechanisms that underlie the initial pathological events that lead to synaptic dysfunction in Alzheimer’s disease. Our results demonstrate a new mechanism by which Aβ42 affects synaptic activity.     

Analysis of the gut microbiota in the old order amish and its relation to the metabolic syndrome

Obesity has been linked to the human gut microbiota; however, the contribution of gut bacterial species to the obese phenotype remains controversial because of conflicting results from studies in different populations. To explore the possible dysbiosis of gut microbiota in obesity and its metabolic complications, we studied men and women over a range of body mass indices from the Old Order Amish sect, a culturally homogeneous Caucasian population of Central European ancestry. We characterized the gut microbiota in 310 subjects by deep pyrosequencing of bar-coded PCR amplicons from the V1-V3 region of the 16S rRNA gene. Three communities of interacting bacteria were identified in the gut microbiota, analogous to previously identified gut enterotypes. Neither BMI nor any metabolic syndrome trait was associated with a particular gut community. Network analysis identified twenty-two bacterial species and four OTUs that were either positively or inversely correlated with metabolic syndrome traits, suggesting that certain members of the gut microbiota may play a role in these metabolic derangements.     

Hepcidin-25 in chronic hemodialysis patients is related to residual kidney function and not to treatment with erythropoiesis stimulating agents

Hepcidin-25, the bioactive form of hepcidin, is a key regulator of iron homeostasis as it induces internalization and degradation of ferroportin, a cellular iron exporter on enterocytes, macrophages and hepatocytes. Hepcidin levels are increased in chronic hemodialysis (HD) patients, but as of yet, limited information on factors associated with hepcidin-25 in these patients is available. In the current cross-sectional study, potential patient-, laboratory- and treatment-related determinants of serum hepcidin-20 and -25, were assessed in a large cohort of stable, prevalent HD patients. Baseline data from 405 patients (62% male; age 63.7 ± 13.9 [mean SD]) enrolled in the CONvective TRAnsport STudy (CONTRAST; NCT00205556) were studied. Predialysis hepcidin concentrations were measured centrally with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Patient-, laboratory- and treatment related characteristics were entered in a backward multivariable linear regression model. Hepcidin-25 levels were independently and positively associated with ferritin (p<0.001), hsCRP (p<0.001) and the presence of diabetes (p = 0.02) and inversely with the estimated glomerular filtration rate (p = 0.01), absolute reticulocyte count (p = 0.02) and soluble transferrin receptor (p<0.001). Men had lower hepcidin-25 levels as compared to women (p = 0.03). Hepcidin-25 was not associated with the maintenance dose of erythropoiesis stimulating agents (ESA) or iron therapy. In conclusion, in the currently studied cohort of chronic HD patients, hepcidin-25 was a marker for iron stores and erythropoiesis and was associated with inflammation. Furthermore, hepcidin-25 levels were influenced by residual kidney function. Hepcidin-25 did not reflect ESA or iron dose in chronic stable HD patients on maintenance therapy. These results suggest that hepcidin is involved in the pathophysiological pathway of renal anemia and iron availability in these patients, but challenges its function as a clinical parameter for ESA resistance.