Heterologous protein production and delivery systems for Lactococcus lactis

Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as live vehicles for the production and delivery of heterologous proteins of vaccinal, medical or technological interest has therefore been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium). A promising application of L. lactis is its use as an antigen delivery vehicle, for the development of live mucosal vaccines. The expression of heterologous proteins and antigens as well as the various delivery systems developed in L. lactis, and its use as an oral vaccine carrier are discussed.     

Short term hypothyroidism affects ovarian function in the cycling rat

Background  

Rats made hypothyroid with propilthyouracil start showing abnormal cycling on the second cycle after the start of the treatment, with a high proportion of spontaneous pseudopregnancies and reduced fertility.     

An Appended Domain Results in an Unusual Architecture for Malaria Parasite Tryptophanyl-tRNA Synthetase

Specific activation of amino acids by aminoacyl-tRNA synthetases (aaRSs) is essential for maintaining fidelity during protein translation. Here, we present crystal structure of malaria parasite Plasmodium falciparum tryptophanyl-tRNA synthetase (Pf-WRS) catalytic domain (AAD) at 2.6 Å resolution in complex with L-tryptophan. Confocal microscopy-based localization data suggest cytoplasmic residency of this protein. Pf-WRS has an unusual N-terminal extension of AlaX-like domain (AXD) along with linker regions which together seem vital for enzymatic activity and tRNA binding. Pf-WRS is not proteolytically processed in the parasites and therefore AXD likely provides tRNA binding capability rather than editing activity. The N-terminal domain containing AXD and linker region is monomeric and would result in an unusual overall architecture for Pf-WRS where the dimeric catalytic domains have monomeric AXDs on either side. Our PDB-wide comparative analyses of 47 WRS crystal structures also provide new mechanistic insights into this enzyme family in context conserved KMSKS loop conformations.     

Conjugation of a new two-photon fluorophore to poly(ethylenimine) for gene delivery imaging

We report herein the molecular engineering of an efficient two-photon absorbing (TPA) chromophore based on a donor-donor bis-stilbenyl entity to allow conjugation with biologically relevant molecules. The dye has been functionalized using an isothiocyanate moiety to conjugate it with the amine functions of poly(ethylenimine) (PEI), which is a cationic polymer commonly used for nonviral gene delivery. Upon conjugation, the basic architecture and photophysical properties of the active TPA chromophore remain unchanged. At the usual N/P ratio (ratio of the PEI positive charges to the DNA negative charges) of 10 used for transfection, the transfection efficiency and cytotoxicity of the labeled PEI/DNA complexes were found to be comparable to those of the unlabeled PEI/DNA complexes. Moreover, when used in combination with unlabeled PEI (at a ratio of 1 labeled PEI to 3 unlabeled PEI), the labeled PEI does not affect the size of the complexes with DNA. The labeled PEI was successfully used in two-photon fluorescence correlation spectroscopy measurements, showing that at N/P = 10 most PEI molecules are free and the diffusion coefficient of the complexes is consistent with the 360 nm size measured by quasielastic light scattering. Finally, two-photon images of the labeled PEI/DNA complexes confirmed that the complexes enter into the cytoplasm of HeLa cells by endocytosis and hardly escape from the endosomes. As a consequence, the functionalized TPA chromophore appears to be an adequate tool to label the numerous polyamines used in nonviral gene delivery and characterize their complexes with DNA in two-photon applications.     

Climate change, humans, and the extinction of the woolly mammoth

Woolly mammoths inhabited Eurasia and North America from late Middle Pleistocene (300 ky BP [300,000 years before present]), surviving through different climatic cycles until they vanished in the Holocene (3.6 ky BP). The debate about why the Late Quaternary extinctions occurred has centred upon environmental and human-induced effects, or a combination of both. However, testing these two hypotheses—climatic and anthropogenic—has been hampered by the difficulty of generating quantitative estimates of the relationship between the contraction of the mammoth's geographical range and each of the two hypotheses. We combined climate envelope models and a population model with explicit treatment of woolly mammoth–human interactions to measure the extent to which a combination of climate changes and increased human pressures might have led to the extinction of the species in Eurasia. Climate conditions for woolly mammoths were measured across different time periods: 126 ky BP, 42 ky BP, 30 ky BP, 21 ky BP, and 6 ky BP. We show that suitable climate conditions for the mammoth reduced drastically between the Late Pleistocene and the Holocene, and 90% of its geographical range disappeared between 42 ky BP and 6 ky BP, with the remaining suitable areas in the mid-Holocene being mainly restricted to Arctic Siberia, which is where the latest records of woolly mammoths in continental Asia have been found. Results of the population models also show that the collapse of the climatic niche of the mammoth caused a significant drop in their population size, making woolly mammoths more vulnerable to the increasing hunting pressure from human populations. The coincidence of the disappearance of climatically suitable areas for woolly mammoths and the increase in anthropogenic impacts in the Holocene, the coup de grâce, likely set the place and time for the extinction of the woolly mammoth.     

Dihydroxynonene mercapturic acid, a urinary metabolite of 4-hydroxynonenal, as a biomarker of lipid peroxidation

The objective of our study was to compare the information obtained through the use of three different urinary biomarkers of lipoperoxidation during the time course of a bromotrichloromethane (BrCCl3) induced oxidative stress in rats. These biomarkers were malondialdehyde (MDA) measured by LC/MS after derivatization, the isoprostane 8-iso-PGF2alpha measured by enzyme immunoassay and 1,4-dihydroxynonene mercapturic acid (DHN-MA), the major 4-hydroxynonenal urinary metabolite [1], measured by LC-MS. Male Wistar rats received a single dose of 100 microL/kg BrCCl3 per os and lipid peroxidation was estimated every day for a 4-day-period after treatment. MDA, 8-iso-PGF2alpha and DHN-MA significantly increased in response to BrCCl3 treatment for this period of time, and DHN-MA showed the main increase during the 24-48 h period after treatment.     

Enzymatic synthesis of poly-l-lactide and poly-l-lactide-co-glycolide in an ionic liquid

The syntheses of poly-l-lactide (PLLA) and poly-l-lactide-co-glycolide (PLLGA) is reported in the ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate [HMIM][PF₆] mediated by the enzyme lipase B from Candida antarctica (Novozyme 435). The highest PLLA yield (63%) was attained at 90 °C with a molecular weight (M _n ) of 37.8 × 10³ g/mol determined by size exclusion chromatography. This procedure produced relatively high crystalline polymers (up to 85% PLLA) as determined by DSC. In experiments at 90 °C product synthesis also occurred without biocatalyst, however, PLLA synthesis in [HMIM][PF₆] at 65 °C followed only the enzymatic mechanism as ring opening was not observed without the enzyme. In addition, the enzymatic synthesis of PLLGA is first reported here using Novozyme 435 biocatalyst with up to 19% of lactyl units in the resulting copolymer as determined by NMR. Materials were also characterized by TGA, MALDI-TOF–MS, X-ray diffraction, polarimetry and rheology.     

Cryptosporidium tyzzeri and Cryptosporidium muris originated from wild West-European house mice (Mus musculus domesticus) and East-European house mice (Mus musculus musculus) are non-infectious for pigs

Three and 8 week old pigs were inoculated with Cryptosporidium muris HZ206 (Mus musculus musculus isolate), Cryptosporidium tyzerri CR2090 (M. m. musculus isolate) or C. tyzzeri CR4293 (isolate from a hybrid between Mus musculus domesticus and M. m. musculus) at a dose of 1 × 10(7) oocysts per animal. Inoculated pigs showed no detectable infection and no clinical symptoms of cryptosporidiosis during 30 days post infection (DPI), and no macroscopic changes were detected in the digestive tract following necropsy. Developmental stages were not detected in gastrointestinal tract tissue by histology or PCR throughout the duration of the experiment. The infectivity of isolates was verified on SCID mice, in which oocysts shedding started from 4 to 8 DPI. Based on our findings, it can be concluded that pigs are not susceptible to C. muris or C. tyzzeri infection.     

Is hyper-hardness more chemically relevant than expected?

In this work, the third derivative of the energy with respect to the number of electrons, the so-called hyper-hardness, is investigated to assess whether this quantity has a chemical meaning. To achieve this goal a new working expression for hyper-hardness is developed and analyzed. It transpired from this analysis that hyper-hardness, just like hardness, can measure the reactivity or the stability of electron systems. Interestingly, positive values of hyper-hardness point to quite stable species such as noble gases and molecules. On the other hand, radicals almost always display large negative values of hyper-hardness.     

DNA replication stress-induced loss of reproductive capacity in S. cerevisiae and its inhibition by caloric restriction

In many organisms, attenuation of growth signaling by caloric restriction or mutational inactivation of growth signaling pathways extends lifespan and protects against cancer and other age-related diseases. The focus of many efforts to understand these effects has been on the induction of oxidative stress defenses that inhibit cellular senescence and cell death. Here we show that in the model organism S. cerevisiae, growth signaling induces entry of cells in stationary phase into S phase in parallel with loss of reproductive capacity, which is enhanced by elevated concentrations of glucose. Overexpression of RNR1 encoding a ribonucleotide reductase subunit required for the synthesis of deoxynucleotide triphosphates and DNA replication suppresses the accelerated loss of reproductive capacity of cells cultured in high glucose. The reduced reproductive capacity of these cells is also suppressed by excess threonine, which buffers dNTP pools when ribonucleotide reductase activity is limiting. Caloric restriction or inactivation of the AKT homolog Sch9p inhibits senescence and death in stationary phase cells caused by the DNA replication inhibitor hydroxyurea or by inactivation of the DNA replication and repair proteins Sgs1p or Rad27p. Inhibition of DNA replication stress represents a novel mechanism by which caloric restriction promotes longevity in S. cerevisiae. A similar mechanism may promote longevity and inhibit cancer and other age-related diseases in humans.