Effect of Growth Conditions and Trehalose Content on Cryotolerance of Bakers' Yeast in Frozen Doughs

The cryotolerance in frozen doughs and in water suspensions of bakers' yeast (Saccharomyces cerevisiae) previously grown under various industrial conditions was evaluated on a laboratory scale. Fed-batch cultures were very superior to batch cultures, and strong aeration enhanced cryoresistance in both cases for freezing rates of 1 to 56°C min⁻¹. Loss of cell viability in frozen dough or water was related to the duration of the dissolved-oxygen deficit during fed-batch growth. Strongly aerobic fed-batch cultures grown at a reduced average specific rate (μ = 0.088 h⁻¹ compared with 0.117 h⁻¹) also showed greater trehalose synthesis and improved frozen-dough stability. Insufficient aeration (dissolved-oxygen deficit) and lower growth temperature (20°C instead of 30°C) decreased both fed-batch-grown yeast cryoresistance and trehalose content. Although trehalose had a cryoprotective effect in S. cerevisiae, its effect was neutralized by even a momentary lack of excess dissolved oxygen in the fed-batch growth medium.     

Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus

Background

A low genetic diversity in Francisella tularensis has been documented. Current DNA based genotyping methods for typing F. tularensis offer a limited and varying degree of subspecies, clade and strain level discrimination power. Whole genome sequencing is the most accurate and reliable method to identify, type and determine phylogenetic relationships among strains of a species. However, lower cost typing schemes are necessary in order to enable typing of hundreds or even thousands of isolates.

Results

We have generated a high-resolution phylogenetic tree from 40 Francisella isolates, including 13 F. tularensis subspecies holarctica (type B) strains, 26 F. tularensis subsp. tularensis (type A) strains and a single F. novicida strain. The tree was generated from global multi-strain single nucleotide polymorphism (SNP) data collected using a set of six Affymetrix GeneChip^® resequencing arrays with the non-repetitive portion of LVS (type B) as the reference sequence complemented with unique sequences of SCHU S4 (type A). Global SNP based phylogenetic clustering was able to resolve all non-related strains. The phylogenetic tree was used to guide the selection of informative SNPs specific to major nodes in the tree for development of a genotyping assay for identification of F. tularensis subspecies and clades. We designed and validated an assay that uses these SNPs to accurately genotype 39 additional F. tularensis strains as type A (A1, A2, A1a or A1b) or type B (B1 or B2).

Conclusion

Whole-genome SNP based clustering was shown to accurately identify SNPs for differentiation of F. tularensis subspecies and clades, emphasizing the potential power and utility of this methodology for selecting SNPs for typing of F. tularensis to the strain level. Additionally, whole genome sequence based SNP information gained from a representative population of strains may be used to perform evolutionary or phylogenetic comparisons of strains, or selection of unique strains for whole-genome sequencing projects.     

Factors Affecting Residual Dosages of Two Formulations of Bacillus thuringiensis subsp. israelensis Tested in the Same Stream During a 3-Year Experiment

Although many field trials have been conducted using Bacillus thuringiensis subsp. israelensis (Bti)-based formulations, most have been in rivers with different biotic and abiotic conditions thus rendering the evaluation of their performance very difficult. Recently, results of a threeyear experiment using a new field procedure brought new insight into the behavior and the performance (carry) of two liquid formulations of Bti, Teknar HP-D and Vectobac 1200L, tested in the same lotic environment and under similar abiotic and biotic conditions. Factors such as discharge, water temperature and the hyporheic zone were identified as elements affecting the downstream loss of activity of both products. However, to better understand the reduction of black fly mortality along a stream (measured by using gutters), data of residual dosages of both products (measured by laboratory assay with mosquito larvae) were compared with reduction of black fly mortality. Bti toxic activity was monitored from water samples taken at different distances downstream from an application point, and from probes driven into the hyporheic zone, to study the effects of abiotic factors on the loss of the toxic crystals. Results showed that the loss of dosage was exponential for both products but more crystals were recovered from Vectobac 1200L along the stream than from Teknar HP-D. However, the latter was more efficient, i.e. less toxins were needed to kill 50% of black fly larvae both in temperate (16°C) and warmer (19.5-22°C) water. Also, a rise in water temperature had a greater effect in the kill induced by Vectobac 1200L compared to Teknar HP-D. For the same residual dosages present at the stations, longer carries of toxin activity (higher mortalities) were obtained in warmer water. Finally, the hyporheic zone was identified as a major source of loss of activity of Bti products. Large stream discharges decreased the effect of the hyporheic zone and that was reflected in longer carry of the products.     

Effect of angiotensin II and norepinephrine on release of prostaglandins E2 and I2 by the perfused rat mesenteric artery

Prostaglandin E₂ (PGE₂) and 6 keto-PGF_1α, the stable metabolite of prostacyclin (PGI₂), have been measured in the effluent of perfused rat mesenteric arteries by the use of a sensitive and specific radioimmunoadday (RIA) method. The PGE₂ and 6-keto-PGF_1α were continuousyl released by the unstimulated mesenteric artery over a period of 145 min. After 100 min of perfusion the release of PGE₂ and 6-keto-PGF_1α was 4.5 ± 8.4 pg/min and 254 ± 75 pg.min respectively, which is in accord with the general belief that PGI₂ is the major PG synthesized by arterial tissue. Angiotensin II (AII) 5 ng/ml) induced an increased of PGE₂ and 6-keto-PGF_1α release without changing the perfusion pressure. The effect of norepinephrine (NE) injections on release of PGs depended on the duration of the stabilization period. The changes of perfusion pressure induced by NE were not related to changes in release of PGs. Thus, it seems that the increase of PG release induced by AII and NE was due to a direct effect of the drugs on the vascular wall. This may represent an important modulating mechanism in the regulation of vascular tone.     

Identification of the domain in the human interleukin-11 receptor that mediates ligand binding

The interleukin-11 receptor (IL-11R) belongs to the hematopoietic receptor superfamily. The functional receptor complex comprises IL-11, IL-11R and the signal-transducing subunit gp130. The extracellular part of the IL-11R consists of three domains: an N-terminal immunoglobulin-like domain, D1, and two fibronectin-type III-like (FNIII) domains and D2 and D3. The two FNIII domains comprise the cytokine receptor-homology region defined by a set of four conserved cysteine residues in the N-terminal domain (D2) and a WSXWS sequence motif in the C-terminal domain (D3). We investigated the structural and functional role of the third extracellular receptor domain of IL-11R. A molecular model of the human IL-11/IL-11R complex allowed the identification of amino acid residues in IL-11R to be involved in ligand binding. Most of them were located in the third extracellular domain, which therefore should be able to bind with high affinity to IL-11. To prove this prediction, domain D3 of the IL-11R was expressed in Escherichia coli, refolded and purified. For structural characterization, circular dichroism, fluorescence and NMR spectroscopy were used. By plasmon resonance experiments, we show that the ligand-binding capacity of this domain is as high as that one for the whole receptor. These results provide a basis for further structural investigations that could be used for the rational design of potential agonists and antagonists essential in human therapy.     

LOLA ZRREGULARIS (CHLOROPHYTA-CLADOPHORACEAE): A NEW SPECIES FROM THE ROSS SEA,ANTARCTICA1

A small, loose lying, unbranched, filamentous alga from the Ross Sea, Antarctica, is described as a new species, Lola irregularis. The genus Lola is closely related to the genera Hormiscia, Rhizoclonium, and Chaetomorpha in the family Cladophoraceae. A key to all known species of Lola is included. This is the first record of a species of Lola south of the Antarctic convergence.     

Multiple mutations in heterogeneous miltefosine-resistant Leishmania major population as determined by whole genome sequencing

Background

Miltefosine (MF) is the first oral compound used in the chemotherapy against leishmaniasis. Since the mechanism of action of this drug and the targets of MF in Leishmania are unclear, we generated in a step-by-step manner Leishmania major promastigote mutants highly resistant to MF. Two of the mutants were submitted to a short-read whole genome sequencing for identifying potential genes associated with MF resistance.

Methods/Principal Findings

Analysis of the genome assemblies revealed several independent point mutations in a P-type ATPase involved in phospholipid translocation. Mutations in two other proteins—pyridoxal kinase and α-adaptin like protein—were also observed in independent mutants. The role of these proteins in the MF resistance was evaluated by gene transfection and gene disruption and both the P-type ATPase and pyridoxal kinase were implicated in MF susceptibility. The study also highlighted that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes.

Conclusions/Significance

Whole genome sequencing was used to pinpoint known and new resistance markers associated with MF resistance in the protozoan parasite Leishmania. The study also demonstrated the polyclonal nature of a resistant population with individual cells with varying susceptibilities and genotypes.     

Mouse natural killer (NK) cells express the nerve growth factor receptor TrkA, which is dynamically regulated

Background

Nerve growth factor (NGF) is a neurotrophin crucial for the development and survival of neurons. It also acts on cells of the immune system which express the NGF receptors TrkA and p75^NTR and can be produced by them. However, mouse NK cells have not yet been studied in this context.

Methodology/Principal Findings

We used cell culture, flow cytometry, confocal microscopy and ELISA assays to investigate the expression of NGF receptors by NK cells and their secretion of NGF. We show that resting NK cells express TrkA and that the expression is different on NK cell subpopulations defined by the relative presence of CD27 and CD11b. Expression of TrkA is dramatically increased in IL-2-activated NK cells. The p75^NTR is expressed only on a very low percentage of NK cells. Functionally, NGF moderately inhibits NK cell degranulation, but does not influence proliferation or cytokine production. NK cells do not produce NGF.

Conclusions/Significance

We demonstrate for the first time that mouse NK cells express the NGF receptor TrkA and that this expression is dynamically regulated.     

Automated analysis of vapor diffusion crystallization drops with an X-ray beam

Crystallogenesis, usually based on the vapor diffusion method, is currently considered one of the most difficult steps in macromolecular X-ray crystallography. Due to the increasing number of crystallization assays performed by protein crystallographers, several automated analysis methods are under development. Most of these methods are based on microscope images and shape recognition. We propose an alternative method of identifying protein crystals: by directly exposing the crystallization drops to an X-ray beam. The resulting diffraction provides far more information than classical microscope images. Not only is the presence of diffracting crystals revealed, but also a first estimation of the space group, cell parameters, and mosaicity is obtained. In certain cases, it is also possible to collect enough data to verify the presence of a specific substrate or a heavy atom. All these steps are performed without the sometimes tedious necessity of removing crystals from their crystallization drop.     

Selfish male‐determining element favors the transition from hermaphroditism to androdioecy

According to the current, widely accepted paradigm, the evolutionary transition from hermaphroditism toward separate sexes occurs in two successive steps: an initial, intermediate step in which unisexual individuals, male or female, sterility mutants coexist with hermaphrodites and a final step that definitively establishes dioecy. Two nonexclusive processes can drive this transition: inbreeding avoidance and reallocation of resources from one sexual function to the other. Here, we report results of controlled crosses between males and hermaphrodites in Phillyrea angustifolia, an androdioecious species with two mutually intercompatible, but intraincompatible groups of hermaphrodites. We observed different segregation patterns that can be explained by: (1) epistatic interactions between two unlinked diallelic loci, determining sex and mating compatibility, and (2) a mutation with pleiotropic effects: female sterility, full compatibility of males with both hermaphrodite incompatibility groups, and complete male‐biased sex‐ratio distortion in one of the two groups. Modeling shows that these mechanisms can explain the high frequency of males in populations of P. angustifolia and can promote the maintenance of androdioecy without requiring inbreeding depression or resource reallocation. We thus argue that segregation distortion establishes the right conditions for the evolution of cryptic dioecy and potentially initiates the evolution toward separate sexes.