The role of aminoglycoside--modifying enzymes in resistance of Gram-negative rods

The aim of the study was to evaluate the aminoglycoside resistance of Gram-negative bacilli isolated from patients. To the examination 35 strains of Enterobacteriaceae and 18 of non-fermentative bacteria were included. Resistance to aminoglycosides (gentamicin (G), netilmicin (Nt), tobramycin (T), amikacin (A), kanamycin (K), neomycin (N)) was established by disk diffusion method. Interpretation of enzymatic mechanisms was performed by Livermore. The most common enzymes AAC(6')I were found in Enterobacteriaceae group (mostly in E. cloaceae and P. mirabilis) and AAC(3') and in non-fermentative bacteria: AAC(6')I in P. aeruginosa and APH(3')VI and AAC(3')I in A. baumanii. The most frequent phenotype was resistance to six antibiotics (G, Nt, T, A, K, N) Resistance rates were high for gentamicin (>70 %) in both groups and amikacin (88,89 %) in non-fermentatives.     

Proposed Classification of Auriculotemporal Nerve,Based on the Root System

The topography of the auriculotemporal nerve (ATN) root system is the main criterion of this nerve classification. Previous publications indicate that ATN may have between one and five roots. Most common is a one- or two-root variant of the nerve structure. The problem of many publications is the inconsistency of nomenclature which concerns the terms “roots”, “connecting branches”, or “branches” that are used to identify the same structures. This study was performed on 80 specimens (40 adults and 40 fetuses) to propose a classification based on: (i) the number of roots, (ii) way of root division, and (iii) configuration of interradicular fibers that form the ATN trunk. This new classification is a remedy for inconsistency of nomenclature of ATN in the infratemporal fossa. This classification system has proven beneficial when organizing all ATN variants described in previous studies and could become a helpful tool for surgeons and dentists. Examination of ATN from the infratemporal fossa of fetuses (the youngest was at 18 weeks gestational age) showed that, at that stage, the nerve is fully developed.     

Colonization behaviour of Pseudomonas fluorescens and Sinorhizobium meliloti in the alfalfa (Medicago sativa) rhizosphere

The colonization ability of Pseudomonas fluorescens F113rif in alfalfa rhizosphere and its interactions with the alfalfa microsymbiont Sinorhizobium meliloti EFB1 has been analyzed. Both strains efficiently colonize the alfalfa rhizosphere in gnotobiotic systems and soil microcosms. Colonization dynamics of F113rif on alfalfa were similar to other plant systems previously studied but it is displaced by S. meliloti EFB1, lowering its population by one order of magnitude in co-inoculation experiments. GFP tagged strains used to study the colonization patterns by both strains indicated that P. fluorescens F113rif did not colonize root hairs while S. meliloti EFB1 extensively colonized this niche. Inoculation of F113rif had a deleterious effect on plants grown in gnotobiotic systems, possibly because of the production of HCN and the high populations reached in these systems. This effect was reversed by co-inoculation. Pseudomonas fluorescens F113 derivatives with biocontrol and bioremediation abilities have been developed in recent years. The results obtained support the possibility of using this bacterium in conjunction with alfalfa for biocontrol or rhizoremediation technologies.     

Exome sequences and multi‐environment field trials elucidate the genetic basis of adaptation in barley

Broadening the genetic base of crops is crucial for developing varieties to respond to global agricultural challenges such as climate change. Here, we analysed a diverse panel of 371 domesticated lines of the model crop barley to explore the genetics of crop adaptation. We first collected exome sequence data and phenotypes of key life history traits from contrasting multi‐environment common garden trials. Then we applied refined statistical methods, including some based on exomic haplotype states, for genotype‐by‐environment (G×E) modelling. Sub‐populations defined from exomic profiles were coincident with barley's biology, geography and history, and explained a high proportion of trial phenotypic variance. Clear G×E interactions indicated adaptation profiles that varied for landraces and cultivars. Exploration of circadian clock‐related genes, associated with the environmentally adaptive days to heading trait (crucial for the crop's spread from the Fertile Crescent), illustrated complexities in G×E effect directions, and the importance of latitudinally based genic context in the expression of large‐effect alleles. Our analysis supports a gene‐level scientific understanding of crop adaption and leads to practical opportunities for crop improvement, allowing the prioritisation of genomic regions and particular sets of lines for breeding efforts seeking to cope with climate change and other stresses.     

18F-EF5 PET Is Predictive of Response to Fractionated Radiotherapy in Preclinical Tumor Models

We evaluated the relationship between pre-treatment positron emission tomography (PET) using the hypoxic tracer ¹⁸F-[2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3- pentafluoropropyl) acetamide] (¹⁸F-EF5) and the response of preclinical tumor models to a range of fractionated radiotherapies. Subcutaneous HT29, A549 and RKO tumors grown in nude mice were imaged using ¹⁸F-EF5 positron emission tomography (PET) in order to characterize the extent and heterogeneity of hypoxia in these systems. Based on these results, 80 A549 tumors were subsequently grown and imaged using ¹⁸F-EF5 PET, and then treated with one, two, or four fraction radiation treatments to a total dose of 10–40 Gy. Response was monitored by serial caliper measurements of tumor volume. Longitudinal post-treatment ¹⁸F-EF5 PET imaging was performed on a subset of tumors. Terminal histologic analysis was performed to validate ¹⁸F-EF5 PET measures of hypoxia. EF5-positive tumors responded more poorly to low dose single fraction irradiation relative to EF5-negative tumors, however both groups responded similarly to larger single fraction doses. Irradiated tumors exhibited reduced ¹⁸F-EF5 uptake one month after treatment compared to control tumors. These findings indicate that pre- treatment ¹⁸F-EF5 PET can predict the response of tumors to single fraction radiation treatment. However, increasing the number of fractions delivered abrogates the difference in response between tumors with high and low EF5 uptake pre-treatment, in agreement with traditional radiobiology.     

Involvement of the pyrophosphate and the 2'-phosphate binding regions of ferredoxin-NADP+ reductase in coenzyme specificity

Previous studies indicated that the determinants of coenzyme specificity in ferredoxin-NADP+ reductase (FNR) from Anabaena are situated in the 2'-phosphate (2'-P) NADP+ binding region, and also suggested that other regions must undergo structural rearrangements of the protein backbone during coenzyme binding. Among the residues involved in such specificity could be those located in regions where interaction with the pyrophosphate group of the coenzyme takes place, namely loops 155-160 and 261-268 in Anabaena FNR. In order to learn more about the coenzyme specificity determinants, and to better define the structural basis of coenzyme binding, mutations in the pyrophosphate and 2'-P binding regions of FNR have been introduced. Modification of the pyrophosphate binding region, involving residues Thr-155, Ala-160, and Leu-263, indicates that this region is involved in determining coenzyme specificity and that selected alterations of these positions produce FNR enzymes that are able to bind NAD+. Thus, our results suggest that slightly different structural rearrangements of the backbone chain in the pyrophosphate binding region might determine FNR specificity for the coenzyme. Combined mutations at the 2'-P binding region, involving residues Ser-223, Arg-224, Arg-233, and Tyr-235, in combination with the residues mentioned above in the pyrophosphate binding region have also been carried out in an attempt to increase the FNR affinity for NAD+/H. However, in most cases the analyzed mutants lost the ability for NADP+/H binding and electron transfer, and no major improvements were observed with regard to the efficiency of the reactions with NAD+/H. Therefore, our results confirm that determinants for coenzyme specificity in FNR are also situated in the pyrophosphate binding region and not only in the 2'-P binding region. Such observations also suggest that other regions of the protein, yet to be identified, might also be involved in this process.     

Modification by glucose of the flocculent phenotype of a <Emphasis Type="Italic">Kloeckera apiculata</Emphasis> wine strain

We have evaluated the induction of the flocculent phenotype of Kloeckera apiculata by glucose mc1 and propose a pathway involved in carbohydrate flocculation induction. Pulses of glucose were given to cells growing in glucose-poor medium (2 g l(-1)) and the flocculation percentage was measured. To elucidate the mechanism involved in flocculation induction, cycloheximide was injected into the cultures 120 min before the glucose pulse. 2,4-Dinitrophenol or cAMP was added to the media instead, or simultaneously with glucose, while a protein kinase A (PKA) inhibitor was added 30 min before the glucose pulse. With 20 and 50 g l(-1) glucose pulse, the yeast flocculation percentage arises to 55 and 65%, respectively. The quantity of proteins and the reflocculating capacity of a lectinic protein extract from the yeast cell wall increase as the concentration of glucose pulse was higher. Cycloheximide prevented the glucose-induced flocculation, while cAMP or 2,4-dinitrophenol increased it 4- and 5-fold, respectively. PKA inhibitor completely prevented the glucose induction flocculation. The flocculent phenotype of K. apiculata mc1 was induced by glucose and the mechanism seems to imply de novo protein (lectin) synthesis via the PKA transduction pathway. This work contributes to the elucidation of the mechanism involved in flocculation induction by glucose of a non-Saccharomyces wine yeast, K. apiculata, which has not been reported. The induction of flocculation by glucose could be a biotechnological tool for the early removal of the indigenous microorganisms from the grape must before the inoculation of a selected starter strain to conduct the alcohol fermentation.     

Present and future extension of the Iberian submediterranean territories as determined from the distribution of marcescent oaks

The present work proposes new boundaries for the current submediterranean territories of the Iberian Peninsula, defining them at the smallest scale attempted to date. The boundaries proposed are not sharp divisions but somewhat ‘gradual’, reflecting the transitional nature of the territories they encompass. Climate change predictions were used to estimate how the distribution of these submediterranean regions might change in the near future. The maps constructed are based on the distribution of marcescent Quercus species—trees that characterise the submediterranean plant landscape where they form the main forest communities. To determine their climatic range, the distribution of different types of Iberian oak forest was represented in ‘climate diagrams’ (ordination diagrams derived from principal components analysis), both in terms of individual species and groups of species based on leaf ecophysiological type, i.e. marcescent (Submediterranean), sclerophyllous (Mediterranean), semideciduous (Mediterranean) and deciduous (Eurosiberian). The climate range of each type of forest was determined, and the means of representative climate variables are analysed by one way ANOVA. The variables differentiating the forest groups were also examined by discriminant analysis. The range of the climate variables found to be associated with the majority of marcescent forests was used to determine the distribution of territories throughout the Peninsula with the same conditions (i.e. whether marcescent forests were present or not), thus providing a map of the Iberian submediterranean territories. Predictions of climate change were used to investigate possible climate-induced modifications in the boundaries of these territories in the near future. The patterns obtained show dramatic reductions in the extension of the Iberian submediterranean environment. Submediterranean conditions will probably disappear from the areas where they currently reign, and it seems unlikely that any new, large submediterranean areas will form by displacement towards higher altitudes. The outlook for the unique submediterranean vegetation of the Iberian Peninsula is gloomy.

The G428A Nonsense Mutation in FUT2 Provides Strong but Not Absolute Protection against Symptomatic GII.4 Norovirus Infection

In November 2004, 116 individuals in an elderly nursing home in El Grao de Castellón, Spain were symptomatically infected with genogroup II.4 (GII.4) norovirus. The global attack rate was 54.2%. Genotyping of 34 symptomatic individuals regarding the FUT2 gene revealed that one patient was, surprisingly, a non-secretor, hence indicating secretor-independent infection. Lewis genotyping revealed that Lewis-positive and negative individuals were susceptible to symptomatic norovirus infection indicating that Lewis status did not predict susceptibility. Saliva based ELISA assays were used to determine binding of the outbreak virus to saliva samples. Saliva from a secretor-negative individual bound the authentic outbreak GII.4 Valencia/2004/Es virus, but did not in contrast to secretor-positive saliva bind VLP of other strains including the GII.4 Dijon strain. Amino acid comparison of antigenic A and B sites located on the external loops of the P2 domain revealed distinct differences between the Valencia/2004/Es and Dijon strains. All three aa in each antigenic site as well as 10/11 recently identified evolutionary hot spots, were unique in the Valencia/2004/Es strain compared to the Dijon strain. To the best of our knowledge, this is the first example of symptomatic GII.4 norovirus infection of a Le^a+b− individual homozygous for the G428A nonsense mutation in FUT2. Taken together, our study provides new insights into the host genetic susceptibility to norovirus infections and evolution of the globally dominating GII.4 viruses.