Daytime relapse of the mean radiant temperature based on the six-directional method under unobstructed solar radiation

This study contributes to the knowledge about the capabilities of the popular “six-directional method” describing the radiation fields outdoors. In Taiwan, measurements were carried out with three orthogonally placed net radiometers to determine the mean radiant temperature (T _mrt). The short- and long-wave radiation flux densities from the six perpendicular directions were recorded in the daylight hours of 12 days. During unobstructed direct irradiation, a specific daytime relapse was found in the temporal course of the T _mrt values referring to the reference shapes of a standing man and also of a sphere. This relapse can be related to the short-wave fluxes reaching the body from the lateral directions. Through deeper analysis, an instrumental shortcoming of the six-directional technique was discovered. The pyranometer pairs of the same net radiometer have a 10–15-min long “blind spot” when the sun beams are nearly perpendicular to them. The blind-spot period is supposed to be shorter with steeper solar azimuth curve on the daylight period. This means that the locations with lower geographical latitude, and the summertime measurements, are affected less by this instrumental problem. A methodological shortcoming of the six-directional technique was also demonstrated. Namely, the sum of the short-wave flux densities from the lateral directions is sensitive to the orientation of the radiometers, and therefore by deviating from the original directions, the T _mrt decrease on clear sunny days will occur in different times and will be different in extent.     

Anthocyanin accumulation and PAL activity in a suspension culture of Daucus carota L.

Cells of Daucus carota grown in a liquid medium produced large amounts of cyanidin as the only flavonoid aglycon. After inoculation in fresh medium a maximum activity of phenylalanine ammonia lyase (PAL; EC 4.3.1.5) was observed within 24 h. L--aminooxy--phenylpropionic acid (L-AOPP), thought to be a competitive inhibitor of PAL, inhibited cyanidin accumulation up to 80%. In order to study the regulatory role of PAL, the effects of L-AOPP and t-cinnamic acid, the product of the deamination of phenylalanine, were investigated. Cinnamic acid, applied in vivo (10^-4 M), was not able to compensate for the inhibition of cyanidin production caused by L-AOPP (10^-4 M) in the same sample. Carrot cells treated with L-AOPP exhibited a super-induction of PAL already described for gherkin hypocotyls (Amrhein and Gerhardt 1979). This effect was not influenced by t-cinnamic acid. L-AOPP seems to be a very specific inhibitor since it affected neither growth nor soluble protein content, whereas t-cinnamic acid inhibited both. Investigations on the content of soluble amino acids in L-AOPP-treated cells revealed a specific accumulation of soluble phenylalanine, whereas treatment with t-cinnamic acid led to an increase of amino acids in general, thus indicating that the latter compound has a rather unspecific effect on cellular metabolism. In vitro studies with PAL isolated from Daucus carota revealed that L-AOPP inhibited the enzyme at very low doses (K _I=2.4·10^-9), whereas t-cinnamic acid, by comparison, affected the enzyme at high concentrations (K _I=1.8·10^-4).Abbreviations PAL phenylalanine ammonia lyase - L-AOPP L--aminooxy--phenylpropionic acid     

Phosphorus and nitrogen allocation in Allium ursinum on an alluvial floodplain (Eastern France). Is there an effect of flooding history?

The change in phosphorus and nitrogen content in a common geophyte spring species, Allium ursinum, is studied in alluvial forests in relation to three flooding histories related to river regulation: (1) annually flooded, (2) unflooded for 30 years, and (3) unflooded for 200 years. Flood suppression leads to a reduction of available P soil content as well as decreasing the biomass and the amount of phosphorus in plants, but has no significant effect on N plant content. Plant N:P ratio increases with the suppression of floods and is primarily driven by soil N:P ratios, in turn markedly linked to the total nitrogen in the soil. We highlighted a lower nutrient accumulation in leaves during plant growth in unflooded forests. Overall, our results suggest that P was the main limiting factor in unflooded forests while nitrogen was the main limiting factor in annually flooded forests. Flood suppression strongly affects the morphology and nutrient uptake by Allium ursinum and thus nutrient cycling in riverine forests.     

Comparative analysis of HIV-1-based lentiviral vectors bearing lyssavirus glycoproteins for neuronal gene transfer

Background

The delivery of therapeutic genes to the central nervous system (CNS) using viral vectors represents an appealing strategy for the treatment of nerve injury and disorders of the CNS. Important factors determining CNS targeting include tropism of the viral vectors and retrograde transport of the vector particles. Retrograde transport of equine anemia virus (EIAV)-based lentiviral vectors pseudotyped with the glycoprotein derived from the Rabies virus RabERA strain from peripheral muscle to spinal motor neurons (MNs) was previously reported. Despite therapeutic effects achieved in mouse models of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), the efficiency of this approach needs to be improved for clinical translation. To date there has not been a quantitative assessment of pseudotyped HIV-1-based lentiviral vectors to transduce MNs. Here, we describe quantitative tests to analyze the retrograde transport capacity of HIV-1 vectors pseudotyped with the G glycoprotein derived from Rabies and Rabies-related viruses (Lyssaviruses).

Methods

With a view toward optimizing the retrograde transport properties of HIV-1-based lentiviral vectors, we compared the glycoproteins from different enveloped viruses belonging to the Rhabdoviridae family, genus Lyssavirus, and evaluated their ability to transduce specific cell populations and promote retrograde axonal transport. We first tested the transduction performance of these pseudotypes in vitro in SH-SY5Y neuroblastoma cells, NSC-34 neuroblastoma-spinal cord hybrid cells, and primary mixed spinal cord and pure astrocyte cultures. We then analyzed the uptake and retrograde transport of these pseudotyped vectors in vitro, using Campenot chambers. Finally, intraneural injections were performed to evaluate the in vivo retrograde axonal transport of these pseudotypes.

Results

Both the in vitro and in vivo studies demonstrated that lentiviral vectors pseudotyped with the glycoprotein derived from the Rabies virus PV strain possessed the best performance and neuronal tropism among the vectors tested.

Conclusion

Our results indicate that HIV-1-based lentiviral vectors pseudotyped with the Rabies PV glycoprotein might provide important vehicles for CNS targeting by peripheral injection in the treatment of motor neuron diseases (MND), pain, and neuropathy.     

Genetic and Structural Characterization of L11 Lipooligosaccharide from Neisseria meningitidis Serogroup A Strains

The lipooligosaccharide (LOS) of immunotype L11 is unique within serogroup A meningococci. In order to resolve its molecular structure, we conducted LOS genotyping by PCR analysis of genes responsible for α-chain sugar addition (lgtA, -B, -C, -E, -H, and -F) and inner core substituents (lgtG, lpt-3, and lpt-6). For this study, we selected seven strains belonging to subgroup III, a major clonal complex responsible for meningococcal meningitis epidemics in Africa. In addition, we sequenced the homopolymeric tract regions of three phase-variable genes (lgtA, lgtG, and lot-3) to predict gene functionality. The fine structure of the L11 LOS of each strain was determined using composition and glycosyl linkage analyses, NMR, and mass spectrometry. The masses of the dephosphorylated oligosaccharides were consistent with an oligosaccharide composed of two hexoses, one N-acetyl-hexosamine, two heptoses, and one KDO, as proposed previously. The molar composition of LOS showed two glucose residues to be present, in agreement with lgtH sequence prediction. Despite phosphoethanolaminetransferase genes lpt-3 and lpt-6 being present in all seven Neisseria meningitidis strains, phosphoethanolamine (PEtn) was found at both O-3 and O-6 of HepII among the three ST-5 strains, whereas among the four ST-7 strains, only one PEtn was found and located at O-3 of the HepII. The L11 LOS was found to be O-acetylated, as was indicated by the presence of the lot-3 gene being in-frame in all of the seven N. meningitidis strains. To our knowledge, these studies represent the first full genetic and structural characterization of the L11 LOS of N. meningitidis. These investigations also suggest the presence of further regulatory mechanisms affecting LOS structure microheterogeneity in N. meningitidis related to PEtn decoration of the inner core.     

Use of Logistic Regression for Prediction of the Fate of Staphylococcus aureus in Pasteurized Milk in the Presence of Two Lytic Phages

The use of bacteriophages provides an attractive approach to the fight against food-borne pathogenic bacteria, since they can be found in different environments and are unable to infect humans, both characteristics of which support their use as biocontrol agents. Two lytic bacteriophages, vB_SauS-phiIPLA35 (phiIPLA35) and vB_SauS-phiIPLA88 (phiIPLA88), previously isolated from the dairy environment inhibited the growth of Staphylococcus aureus. To facilitate the successful application of both bacteriophages as biocontrol agents, probabilistic models for predicting S. aureus inactivation by the phages in pasteurized milk were developed. A linear logistic regression procedure was used to describe the survival/death interface of S. aureus after 8 h of storage as a function of the initial phage titer (2 to 8 log₁₀ PFU/ml), initial bacterial contamination (2 to 6 log₁₀ CFU/ml), and temperature (15 to 37°C). Two successive models were built, with the first including only data from the experimental design and a global one in which results derived from the validation experiments were also included. The temperature, interaction temperature-initial level of bacterial contamination, and initial level of bacterial contamination-phage titer contributed significantly to the first model prediction. However, only the phage titer and temperature were significantly involved in the global model prediction. The predictions of both models were fail-safe and highly consistent with the observed S. aureus responses. Nevertheless, the global model, deduced from a higher number of experiments (with a higher degree of freedom), was dependent on a lower number of variables and had an apparent better fit. Therefore, it can be considered a convenient evolution of the first model. Besides, the global model provides the minimum phage concentration (about 2 × 10⁸ PFU/ml) required to inactivate S. aureus in milk at different temperatures, irrespective of the bacterial contamination level.Staphylococcus aureus is one of the pathogenic bacteria considered a threat to food safety. Worldwide, it has a particular relevance to the food-processing industry because of the ability of some strains to produce heat-stable enterotoxins and other virulence factors responsible for staphylococcal food poisoning (14, 18). The ability of S. aureus to grow in different foodstuffs over a wide range of temperatures (10 to 45°C), pHs (4.5 to 9.3), and NaCl concentrations (up to 15%) explains its incidence in foods subjected to manipulation throughout the manufacturing process (1). Milk and dairy products have often been involved in several episodes of staphylococcal food poisoning (6). Food handlers and cattle are usually the main source of dairy product contamination, as humans and animals are the primary reservoirs for staphylococci (28).S. aureus contamination can be avoided by heat treatment of food, but recontamination postpasteurization can occur if the hygienic conditions are inadequate. Alternative approaches to control S. aureus populations in dairy products, such as the use of bacteriocinogenic strains (24) and, more recently, bacteriophages (8), have been tested. Bacteriophages, as well as bacteriocins, are regarded as natural antibacterial agents since they are able to infect and lyse specific undesired target bacteria without disturbing the normal microbiota (19). Among the advantages of using phages as biocontrol strategies in foods, their ubiquity in the environment, their history of safe use, and their high host specificity can be quoted (10). Likewise, their potential use as biopreservatives in several food systems has recently been reviewed (13).Factors affecting S. aureus growth and survival in foods (physicochemical characteristics of the products and conditions associated with food processing, storage, and distribution) have been studied, and mathematical models have been developed to estimate the microorganism''s response (5, 30). However, the effect of lytic phages on S. aureus growth in milk has not been described by predictive models so far. There are several physical parameters which may influence bacterial inhibition by phages in milk. Among them, temperature is the first choice to be studied, as this parameter is always involved in milk processing and can easily be subjected to manipulation without markedly affecting the typical organoleptic characteristics of milk.In this regard, probabilistic models have been widely used to describe the survival/death interface as a function of environmental hurdles (22, 23), and the positions of these limits are of interest in establishing conditions for product stabilization. A procedure of forward or backward stepwise regression with some criteria to include or reject dummy or quantitative explanatory variables, their quadratic terms, or their interactions is included in most of the standard statistical software packages (11, 12).The aim of the present work was to determine the effectiveness of a cocktail of two lytic phages, selected according to its wide host range (9), at reducing S. aureus contamination in pasteurized milk, using different environmental conditions, such as different temperatures, initial bacterial viable counts, and initial phage titers. For this purpose, the survival/death interface was deduced using a logistic regression model.     

Kinetic parameters of hepatic oxidation of cyclic fatty acid monomers formed from linoleic and linolenic acids

Cyclic fatty acid monomers (CFAM) occur from linoleic (CFAM-18:2) or linolenic (CFAM-18:3) acids present in some edible oils as a result of domestic frying or industrial refining. They present adverse effects in pups and weaning rats. In the present work, we studied the importance of hepatic oxidation in the metabolism of CFAM. For this purpose, kinetic parameters of Carnitine Palmitoyl Transferase I (key enzyme of the channeling of the fatty acids into the mitochondrial beta-oxidation pathway) and Acyl CoA Oxidase (key enzyme of the peroxisomal oxidation pathway) towards CFAM-18:2 and CFAM-18:3 were calculated on hepatic sub-cellular fractions of rats. For mitochondrial oxidation of CFAM, we observed a lower oxygen consumption and a lower activity of Carnitine Palmitoyl Transferase compared to 18:2w6 and 16:0. For peroxisomal oxidation, CFAM-18:2 showed the same kinetic parameters (Vm and K(0.5)) as 18:2w6 and 16:0, used for oxidative controls, whereas CFAM-18:3 presented a lower Vm (-50%). This difference should induce a lower catabolism of CFAM-18:3 in liver. This could contribute to their accumulation and probably to their toxic effect.     

Abnormal intracellular lipid processing contributes to fat malabsorption in cystic fibrosis patients

A common feature of cystic fibrosis (CF) is the functional derangement of the exocrine pancreas, which affects output of pancreatic lipase. This condition results in severe dietary malabsorption due to the poor hydrolysis of triacylglycerol (TG) in the lumen of the small intestine. Despite the benefits of pancreatic enzyme supplements, patients with CF present with persistent intestinal fat malabsorption. The aim of the present investigation was to determine whether defects in the intracellular phase of lipid transport occur in this pathophysiology in addition to the known disturbed digestive processes. Our hypothesis was tested by incubating intestinal biopsies from six CF and six healthy subjects with radiolabeled lipid and protein precursors. Lipid esterification and secretion were markedly decreased by 22-31% and 38-42%, respectively, in CF samples, as noted by the low incorporation of [(14)C]palmitic acid into TGs, phospholipids, and cholesteryl esters in patients' duodenal explants and culture media compared with controls (100%). Accordingly, the output of TG-rich lipoproteins was substantially reduced (P < 0.05), and a similar trend was observed for high-density lipoproteins. Because intestinal lipoprotein assembly/secretion shows an absolute requirement for apolipoprotein (apo) B-48, radioactive labeling experiments were performed; these experiments demonstrated a significantly (P < 0.05) diminished synthesis of apoB-48 (40%) and apoA-I (30%). Given the critical role of microsomal triglyceride transfer protein in the formation of apoB-containing lipoproteins, its activity was determined and not found to be altered in CF intestinal tissue. Together, these results suggest that CF malabsorption may also be caused by defects in mucosal mechanisms leading to abnormal lipoprotein delivery into the blood circulation.     

Targeting dsRNA-specific single-chain Fv antibody fragments to different cellular locations in Nicotiana tabacum L

Expression of antibodies or antibody fragments in plants is a useful tool for producing active antibody derivatives for diagnostic or pharmaceutical purposes as well as for immunomodulation. We investigated the effect of cellular expression site on the stability and yield of double-stranded RNA (dsRNA)-specific single-chain Fv-fragments (scFv) in transgenic tobacco. Two antibodies (J2 and P6) belonging to the V23(J558) heavy chain variable gene family but differing in the light chain variable domain were used. scFvs were targeted to the cytoplasm - with or without anchoring them in the plasma membrane -, into the endoplasmic reticulum (ER) and to the apoplast. Although high mRNA concentrations were detected in all cases, scFv proteins accumulated only when scFvs were made ER-resident by appropriate signal sequences. When the ER retention signal was removed to allow scFv-secretion to the apoplast, no scFv-proteins were detected. Despite the strong homology of the VH-sequences of J2 and P6 antibodies, only P6 provided a stable scFv scaffold for intracytoplasmic expression. J2-scFv could not be stabilised either by adding a C-terminal stabilisation signal or by anchoring the protein on the cytoplasmic side of the plasma membrane (PM). It was found that dsRNA-specific J2-scFvs are active in vivo and enhance Potato Virus Y induced symptoms in infected tobacco. This is the first report describing the expression and biological effect of RNA-specific antibodies in plants.