Unleashing the full power of LHCb to probe stealth new physics.

In this paper, we describe the potential of the LHCb experiment to detect stealth physics. This refers to dynamics beyond the standard model that would elude searches that focus on energetic objects or precision measurements of known processes. Stealth signatures include long-lived particles and light resonances that are produced very rarely or together with overwhelming backgrounds. We will discuss why LHCb is equipped to discover this kind of physics at the Large Hadron Collider and provide examples of well-motivated theoretical models that can be probed with great detail at the experiment.

Root Growth Adaptation to Climate Change in Crops.

Climate change is threatening crop productivity worldwide and new solutions to adapt crops to these environmental changes are urgently needed. Elevated temperatures driven by climate change affect developmental and physiological plant processes that, ultimately, impact on crop yield and quality. Plant roots are responsible for water and nutrients uptake, but changes in soil temperatures alters this process limiting crop growth. With the predicted variable climatic forecast, the development of an efficient root system better adapted to changing soil and environmental conditions is crucial for enhancing crop productivity. Root traits associated with improved adaptation to rising temperatures are increasingly being analyzed to obtain more suitable crop varieties. In this review, we will summarize the current knowledge about the effect of increasing temperatures on root growth and their impact on crop yield. First, we will describe the main alterations in root architecture that different crops undergo in response to warmer soils. Then, we will outline the main coordinated physiological and metabolic changes taking place in roots and aerial parts that modulate the global response of the plant to increased temperatures. We will discuss on some of the main regulatory mechanisms controlling root adaptation to warmer soils, including the activation of heat and oxidative pathways to prevent damage of root cells and disruption of root growth; the interplay between hormonal regulatory pathways and the global changes on gene expression and protein homeostasis. We will also consider that in the field, increasing temperatures are usually associated with other abiotic and biotic stresses such as drought, salinity, nutrient deficiencies, and pathogen infections. We will present recent advances on how the root system is able to integrate and respond to complex and different stimuli in order to adapt to an increasingly changing environment. Finally, we will discuss the new prospects and challenges in this field as well as the more promising pathways for future research.

Association of a single nucleotide polymorphism in the ubxn6 gene with long-term non-progression phenotype in HIV-positive individuals.

OBJECTIVES: The long-term non-progressors (LTNPs) are a heterogeneous group of HIV-positive individuals characterized by their ability to maintain high CD4+ T-cell counts and partially control viral replication for years in the absence of antiretroviral therapy. The present study aims to identify host single nucleotide polymorphisms (SNPs) associated with non-progression in a cohort of 352 individuals. METHODS: DNA microarrays and exome sequencing were used for genotyping about 240 000 functional polymorphisms throughout more than 20 000 human genes. The allele frequencies of 85 LTNPs were compared with a control population. SNPs associated with LTNPs were confirmed in a population of typical progressors. Functional analyses in the affected gene were carried out through knockdown experiments in HeLa-P4, macrophages and dendritic cells. RESULTS: Several SNPs located within the major histocompatibility complex region previously related to LTNPs were confirmed in this new cohort. The SNP rs1127888 (UBXN6) surpassed the statistical significance of these markers after Bonferroni correction (q = 2.11 × 10-6). An uncommon allelic frequency of rs1127888 among LTNPs was confirmed by comparison with typical progressors and other publicly available populations. UBXN6 knockdown experiments caused an increase in CAV1 expression and its accumulation in the plasma membrane. In vitro infection of different cell types with HIV-1 replication-competent recombinant viruses caused a reduction of the viral replication capacity compared with their corresponding wild-type cells expressing UBXN6. CONCLUSIONS: A higher prevalence of Ala31Thr in UBXN6 was found among LTNPs within its N-terminal region, which is crucial for UBXN6/VCP protein complex formation. UBXN6 knockdown affected CAV1 turnover and HIV-1 replication capacity.

Purification, immobilization, and stabilization of a lipase from Bacillus thermocatenulatus by interfacial adsorption on hydrophobic supports.

A lipase from Bacillus thermocatenulatus (BTL2) cloned in E. coli has been purified using a very simple method: interfacial activation on a hydrophobic support followed by desorption with Triton. Only one band was detected by SDS-PAGE. The pure enzyme was immobilized using different methodologies. BTL2 adsorbed on a hydrophobic support (octadecyl-Sepabeads) exhibited a hyperactivation with respect to the soluble enzyme, whereas the other immobilized preparations suffered a slight decrease in the expressed activity. The soluble enzyme was very stable, but all immobilized preparations were much more stable than the soluble enzyme, the octadecyl-Sepabeads-BTL2 preparation being the most stable one in all conditions (high temperature or in the presence of organic cosolvents), maintaining 100% of the activity at 65 degrees C or 30% of dioxane and 45 degrees C after several days of incubation. The glyoxyl preparation, the second more stable, retained 80% of the initial activity after 2 days, respectively. The adsorption of this thermophilic lipase on octadecyl-Sepabeads permitted an increase in the optimal temperature of the enzyme of 10 degrees C.

Crystallization and preliminary X-ray diffraction studies of two different crystal forms of the lipase 2 isoform from the yeast Candida rugosa.

The yeast Candida rugosa produces several closely related lipases which show a high degree of sequence identity (between 77 and 88% for pairs of proteins). Despite this high sequence identity, they exhibit markedly different substrate specificities, indicating that subtle structural differences may produce significant functional changes. Isoform 2 (lip2) has been crystallized using the hanging-drop vapour-diffusion method at 291 K. Diffraction-quality crystals have been obtained from two different experimental conditions (designated A and B, respectively). Type A crystals belong to space group P1 and have unit-cell parameters a = 62.15, b = 91.14, c = 108.46 A, alpha = 90.78, beta = 106.31, gamma = 86.91 degrees; type B crystals are monoclinic with a nearly hexagonal topology, with unit-cell parameters a = 116.11, b = 225.55, c = 116.06 A, beta = 119.89 degrees, and belong to space group P2(1). Diffraction data were collected to a resolution of 1.97 A at a synchrotron facility from type A crystals and to 2.65 A on an in-house rotating-anode generator from type B crystals. Whereas the triclinic crystal reveals monomeric lip2, the monoclinic crystal contains dimeric lip2.

Influence of the conformational flexibility on the kinetics and dimerisation process of two Candida rugosa lipase isoenzymes.

We have investigated the interfacial activation process of two isoenzymes from Candida rugosa (Lip1 and Lip3) using triacetin as substrate. Kinetics were coupled to inhibition experiments in order to analyse the transition between the open and closed conformers. This process was slow, particularly for Lip1, in the absence of an interface provided by the substrate or a detergent. Dimers of Lip3 were also purified and their catalytic action was closer to that of a typical esterase. In spite of the high sequence homology between Lip1 and Lip3, small changes enhance hydrophobicity in the binding pocket of Lip3 and increase the flexibility of its flap. We postulated that these factors account for the higher tendency of Lip3 to dimerise fixing its open conformation.

Purification and characterization of Lip2 and Lip3 isoenzymes from a Candida rugosa pilot-plant scale fed-batch fermentation.

Previous purification of a crude extracellular enzyme preparation from Candida rugosa ATCC 14830 pilot-plant fed-batch fermentations showed the presence of two lipase isoenzymes, Lip2 and Lip3, differing in their molecular masses (58 and 62 kDa, respectively). These enzymes were purified but the lipases were forming active aggregates with a molecular mass higher than 200 kDa. In this work we developed a purification method following three steps: ammonium sulfate precipitation, sodium cholate treatment and ethanol/ether precipitation, and anion exchange chromatography which allowed the sequential disaggregation of the isoenzymes. Pure and monomeric Lip2 and Lip3 were characterized according to pI, glycosylation and activity for p-nitrophenol esters and triacylglycerols of varying acyl chain. Lip3 was the best catalyst for the hydrolysis of the simple esters and triacylglycerols with short and medium acyl chains.

Lipid-transfer proteins as potential plant panallergens: cross-reactivity among proteins of Artemisia pollen, Castanea nut and Rosaceae fruits, with different IgE-binding capacities.

BACKGROUND: Lipid-transfer proteins (LTPs), but not Bet v 1 homologues, have been identified as major allergens of apple and peach in the Rosaceae fruit-allergic population in the Mediterranean area. Many of these patients show cosensitization to mugwort pollen. LTPs have an ubiquitous distribution in tissues of many plant species, and have been proposed as a novel type of plant panallergens. OBJECTIVE: We sought to isolate LTPs from Artemisia pollen and from a plant food not belonging to the Rosaceae family, such as chestnut nut, and to compare their amino acid sequences and IgE-binding capacities with those of apple and peach LTPs. METHODS: Allergens (LTPs) were isolated by different chromatographic methods (gel-filtration, ion exchange and/or reverse-phase HPLC), and characterized by N-terminal amino acid sequencing and MALDI analysis. Specific IgE-quantification and immunodetection, as well as immunoblot and ELISA inhibition assays, were carried out using sera from patients allergic to both apple and peach. RESULTS: Purified LTPs from Artemisia pollen and from chestnut seed showed molecular masses about 9 700d, and 43-50% sequence identity with the equivalent allergens of apple and peach in the first 30 N-terminal residues, which comprise about one third of the total amino acid sequence. A similar degree of sequence identity (50%) was found between the Artemisia and chestnut proteins. Both isolated LTPs bound specific IgE of sera from Rosaceae fruits allergic patients. However, substantially lower values of specific IgE-binding and maximum ELISA inhibition percentages were obtained for Artemisia and chestnut LTPs when compared to those from apple and peach. CONCLUSION: LTPs from Artemisia pollen and chestnut crossreact with allergens (LTPs) of Rosaceae fruits, but significant differences in specific IgE-binding capacities were observed among members of the plant LTP family. Thus, further studies are needed to evaluate the clinical significance of the observed cross-reactivities of plant LTPs.

Der p 1 and Der f 1, the highly related and major allergens from house dust mites, are differentially affected by a plant cystatin.

BACKGROUND: Der p 1 and Der f 1 are highly related allergens from the two main house dust mite species, Dermatophagoides pteronyssinus and Dermatophagoides farinae, respectively. A link between the cysteine proteinase activity of Der p 1 and its allergenicity has recently been demonstrated. OBJECTIVE: To test the effects of several proteinase inhibitors, mainly a chestnut cystatin (CsC), on the enzymatic activity of Der p 1 and Der f 1, as well as to study the potential acaricide properties of the inhibitors. METHODS: Inhibition tests were performed using mite extracts, as well as isolated Der p 1 and Der f 1 as targets. Immunodetection after SDS-PAGE and N-terminal amino acid sequencing were used to demonstrate the inactivation of chestnut cystatin by Der p 1. Bioassays including different inhibitors in a semisynthetic diet were performed to evaluate a potential effect on larval survival. RESULTS: CsC inhibited the crude digestive proteinase activity of D. farinae, but it was not active towards the equivalent enzyme from D. pteronyssinus. This differential behaviour was fully explained by testing CsC against the two purified allergens, Der f 1 and Der p 1; the former was highly susceptible, whereas the latter was not affected. In contrast, other cysteine proteinase inhibitors, such as egg white cystatin and E-64, inhibited the proteinase activity of both mite allergens. Der p 1 inactivated CsC by a specific proteolytic cleavage between Gly 6 and Val 7, thus given rise to a noninhibitory processed protein. Besides the in vitro activity, CsC also showed an in vivo effect on D. farinae, drastically increasing the larval mortality when added to a semisynthetic diet. CONCLUSION: Der p 1 and Der f 1 behave very differently towards a plant cystatin, thus indicating substantial differences in their proteolytic activity. This fact could be significant with regards to the immunogenic properties of both allergens.

Biotic and abiotic stress can induce cystatin expression in chestnut.

A cysteine proteinase inhibitor (cystatin) from chestnut (Castanea sativa) seeds, designated CsC, has been previously characterized. Its antifungal, acaricide and inhibitory activities have allowed to involve CsC in defence mechanisms. The CsC transcription levels decreased during seed maturation and increased throughout germination, an opposite behavior to that shown by most phytocystatins. No inhibition of endogenous proteinase activity by purified CsC was found during the seed maturation or germination processes. CsC message accumulation was induced in chestnut leaves after fungal infection, as well as by wounding and jasmonic acid treatment. Induction in roots was also observed by the last two treatments. Furthermore, CsC transcript levels strongly raised, both in roots and leaves, when chestnut plantlets were subjected to cold- and saline-shocks, and also in roots by heat stress. All together, these data suggest that chestnut cystatin is not only involved in defence responses to pests and pathogen invasion, but also in those related to abiotic stress.

A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests.

Cystatin CsC, a cysteine proteinase inhibitor from chestnut (Castanea sativa) seeds, has been purified and characterized. Its full-length cDNA clone was isolated from an immature chestnut cotyledon library. The inhibitor was expressed in Escherichia coli and purified from bacterial extracts. Identity of both seed and recombinant cystatin was confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis, two-dimensional electrophoresis and N-terminal sequencing. CsC has a molecular mass of 11,275 Da and pI of 6.9. Its amino acid sequence includes all three motifs that are thought to be essential for inhibitory activity, and shows significant identity to other phytocystatins, especially that of cowpea (70%). Recombinant CsC inhibited papain (Ki 29 nM), ficin (Ki 65 nM), chymopapain (Ki 366 nM), and cathepsin B (Ki 473 nM). By contrast with most cystatins, it was also effective towards trypsin (Ki 3489 nM). CsC is active against digestive proteinases from the insect Tribolium castaneum and the mite Dermatophagoides farinae, two important agricultural pests. Its effects on the cysteine proteinase activity of two closely related mite species revealed the high specificity of the chestnut cystatin.

Specific depletion of the house dust mite allergen Der p 1 cereal flour prolamins.

BACKGROUND: Quantitation of Der 1 and Der 2 in dust samples by specific monoclonal antibodies is a method used increasingly to evaluate mite allergen exposure. The level of Der 1 has been proposed as a risk factor for sensitization. AIM: We report a drastic decrease in the Der 1/Der 2 ratio when dust samples are collected in bakeries. METHODS: Wheat flour and purified mites were extracted simultaneously; levels of Der p 1 and Der p 2 and cysteine protease activity were determined by ELISA and inhibition experiments. RESULTS: High titers of Der 2, but only trace amounts of Der p 1, were detected in dust collected from bakeries. Both the level and proteolytic activity of Der p 1 appeared greatly decreased when mites and wheat flour were coextracted. CONCLUSION: Group 1 protein was found to be masked by flour components, resulting in an underestimation of the mite content in bakery dust. This problem was not found for group 2 allergen.

Linkage mapping of '25-kDa globulin' genes on homoeologous group-1 chromosomes of bread and durum wheat.

Acid polyacrylamide-gel electrophoresis (A-PAGE) of ethanol-soluble proteins from the endosperm of bread and durum wheats reveals some bands encoded by genes on the homoeologous group-1 chromosomes with higher mobility than the α-gliadins. The isolation of these proteins showed that they were the previously described '25-kDa globulins' encoded by genes at the Glo-A1, Glo-B1, and Glo-D1 loci. The variability found among a collection of 51 bread and 81 durum wheats was very low: two allelic variants at Glo-A1 and no variants at Glo-B1 in each of the two species, and two allelic variants at Glo-D1 in bread wheat. Inheritance studies of '25-kDa globulin' genes on group-1 chromosomes of bread and durum wheat were carried out on the F2 progeny from four crosses, two in bread wheat and two in durum wheat. The linkage mapping of the 1A '25-kDa globulin' genes of bread wheat was done based on four prolamin loci: Glu-A1, Glu-A3, Gli-A1 and Gli -A3. The percentages of recombination and the distances found allowed a re-evaluation of the linkage map of endosperm protein loci on this chromosome. The Glo-A1 locus was found to be located at the distal end of the short arm of 1A chromosome, at a distance of 5.23±1.99 cM from Gli-A1, 6.85±2.22 cM from Glu-A3, 22.64±3.62 cM from Gli-A1, and at a recombination percentage of 49.30±4.40 from Glu-A1. A similar distance between Gli-A1 and Glo-A1 (4.82±1.75 and 6.66±2.26 cM) was found in durum wheat. The distance between Gli-D1 and Glo-D1 on chromosome 1D was 2.86±1.39 cM.

Sequence of non-structural regions 3 and 5 of hepatitis C virus genomes from Spanish patients: existence of a predominant variant related to type 1b.

Several hepatitis C viruses (HCV) have been described. In this study, the prevalence of HCV subtypes 1a, 1b, 2a and 2b has been studied by means of specific PCR in 93 serum samples of Spanish patients. Among these, the HCV-1b subtype was the most frequently detected (62%). Complementary DNA fragments from non-structural region 3 (NS3) and 5 (NS5), obtained from serum samples of three Spanish patients, were amplified by PCR and the products were cloned and sequenced. Comparison of the sequence obtained with those previously published shows the highest homology (91.7% in NS3 and 91.8% in NS5) with the HCV-1b subtype. The incidence of the local variant was analysed among the HCV-1b-infected patients. In order to distinguish between the local and HCV-1b prototype subtype, a new specific PCR assay was designed using primers from NS5. In the majority of the 76 HCV-1b-infected patients, the local variant was the only subtype detected (53%). These findings support the existence of a local variant, belonging to the HCV-1b subtype.

Evidence of subtype-specific antibodies to antigenic epitopes in the NS5 region of hepatitis C virus in the circulation of patients with chronic hepatitis C.

The antigenicity of the NS5 region of type 1 hepatitis C virus (HCV) was investigated by epitope mapping of the region spanning amino acids 2530 to 2723 of the HCV polyprotein. Six antigenic regions were recognized by anti-HCV positive sera from individuals with chronic hepatitis C in a modified enzyme-linked immunosorbent assay with synthetic oligopeptides. Five of these regions demonstrated intra-type 1 sequence variations defining subtype 1a and 1b HCV sequences. The region between amino acids 2623 and 2634 allowed testing of subtype-specific anti-NS5 antibodies; serological reactivity to subtypes 1a and 1b was observed in 27 and 61%, respectively, of 150 cases with chronic hepatitis C. Simultaneous reactivity to subtypes 1a and 1b was found in 23% of the patients. Detection of subtype-specific anti-NS5 antibody correlated in more than 80% of the cases with the HCV genotype (subtypes 1a and 1b) analyzed by PCR amplification of the NS5 sequence. These data provide evidence of the existence of a subtype-specific anti-NS5 response in the circulation of patients with chronic hepatitis C.

[The effect of clindamycin on intraphagocytic Staphylococcus aureus in leukocytes from patients with chronic osteomyelitis]. / Efecto de clindamicina sobre Staphylococcus aureus intrafacogíticos en leucocitos de pacientes con osteomielitis crónica.

BACKGROUND: Osteomyelitis are infections difficult to be completely cured, and its optimal therapy had not clearly been established. METHODS: We study the bactericidal activity of polymorphonuclear leukocytes and also of serum from 13 patients with Staphylococcus aureus chronic osteomyelitis. We also studied the activity of clindamycin against intraphagocytic S. aureus. The study was done in vitro using control S. aureus strain ATCC 25923 and also microorganisms recovered from infected bone. RESULTS: S. aureus two hours survival rates in polymorphonuclear leukocytes were 13.5 +/- 4.4 x 10(6), 10.5 +/- 4.0 x 10(6), 11.0 +/- 4.0 x 10(6), using polymorphonuclear leukocytes from controls and patients, with control and autologous sera respectively (p greater than 0.05). We have observed a bactericidal activity defect in polymorphonuclear leukocytes from one patient and in the sera of 7 other patients. The incubation with clindamycin (10 MIC) reduces the number of cfu/ml to 2.1 +/- 0.9 x 10(6), 1.1 +/- 0.7 x 10(6) y 1.9 +/- 1.1 x 10(6), and we also detected and additional inhibition of 81.7 +/- 7%, 70.7 +/- 17% and 79.0 +/- 4% respectively. CONCLUSION: The results of our study confirm that clindamycin has an intracellular action against intraphagocytic S. aureus and also showed the ability of this antimicrobial agent to cover defects in defensive mechanism of the host. Both statements give support the potential usefulness of clindamycin as therapy for bone infections due to S. aureus.