The enzymatic properties of Arabidopsis thaliana DNA polymerase λ suggest a role in base excision repair.

Base excision repair (BER) generates gapped DNA intermediates containing a 5'-terminal 2-deoxyribose-5-phosphate (5'-dRP) group. In mammalian cells, gap filling and dRP removal are catalyzed by Pol ß, which belongs to the X family of DNA polymerases. In higher plants, the only member of the X family of DNA polymerases is Pol λ. Although it is generally believed that plant Pol λ participates in BER, there is limited experimental evidence for this hypothesis. Here we have characterized the biochemical properties of Arabidopsis thaliana Pol λ (AtPol λ) in a BER context, using a variety of DNA repair intermediates. We have found that AtPol λ performs gap filling inserting the correct nucleotide, and that the rate of nucleotide incorporation is higher in substrates containing a C in the template strand. Gap filling catalyzed by AtPol λ is most efficient with a phosphate at the 5'-end of the gap and is not inhibited by the presence of a 5'-dRP mimic. We also show that AtPol λ possesses an intrinsic dRP lyase activity that is reduced by mutations at two lysine residues in its 8-kDa domain, one of which is present in Pol λ exclusively and not in any Pol ß homolog. Importantly, we also found that the dRP lyase activity of AtPol λ allows efficient completion of uracil repair in a reconstituted short-patch BER reaction. These results suggest that AtPol λ plays an important role in plant BER.

Co-culture of chondrons and mesenchymal stromal cells reduces the loss of collagen VI and improves extracellular matrix production.

Adult articular chondrocytes are surrounded by a pericellular matrix (PCM) to form a chondron. The PCM is rich in hyaluronan, proteoglycans, and collagen II, and it is the exclusive location of collagen VI in articular cartilage. Collagen VI anchors the chondrocyte to the PCM. It has been suggested that co-culture of chondrons with mesenchymal stromal cells (MSCs) might enhance extracellular matrix (ECM) production. This co-culture study investigates whether MSCs help to preserve the PCM and increase ECM production. Primary bovine chondrons or chondrocytes or rat MSCs were cultured alone to establish a baseline level for ECM production. A xenogeneic co-culture monolayer model using rat MSCs (20, 50, and 80%) was established. PCM maintenance and ECM production were assessed by biochemical assays, immunofluorescence, and histological staining. Co-culture of MSCs with chondrons enhanced ECM matrix production, as compared to chondrocyte or chondron only cultures. The ratio 50:50 co-culture of MSCs and chondrons resulted in the highest increase in GAG production (18.5 ± 0.54 pg/cell at day 1 and 11 ± 0.38 pg/cell at day 7 in 50:50 co-culture versus 16.8 ± 0.61 pg/cell at day 1 and 10 ± 0.45 pg/cell at day 7 in chondron monoculture). The co-culture of MSCs with chondrons appeared to decelerate the loss of the PCM as determined by collagen VI expression, whilst the expression of high-temperature requirement serine protease A1 (HtrA1) demonstrated an inverse relationship to that of the collagen VI. Together, this implies that MSCs directly or indirectly inhibited HtrA1 activity and the co-culture of MSCs with chondrons enhanced ECM synthesis and the preservation of the PCM.

Development of a novel lateral flow assay for detection of African swine fever in blood.

BACKGROUND: African swine fever (ASF) is a viral infectious disease of domestic and wild suids of all breeds and ages, causing a wide range of hemorrhagic syndromes and frequently characterized by high mortality. The disease is endemic in Sub-Saharan Africa and Sardinia. Since 2007, it has also been present in different countries of Eastern Europe, where control measures have not been effective so far. The continued spread poses a serious threat to the swine industry worldwide. In the absence of vaccine, early detection of infected animals is of paramount importance for control of the outbreak, to prevent the transmission of the virus to healthy animals and subsequent spreading of the disease. Current laboratory diagnosis is mainly based on virological methods (antigen and genome detection) and serodiagnosis. RESULTS: In the present work, a Lateral Flow Assay (LFA) for antigen detection has been developed and evaluated. The test is based on the use of a MAb against VP72 protein of ASFV, the major viral capsid protein and highly immunogenic. First experiments using VP72 viral and recombinant protein or inactivated culture virus showed promising results with a sensitivity similar to that of a commercially available Antigen-ELISA. Moreover, these strips were tested with blood from experimentally infected pigs and field animals and the results compared with those of PCR and Antigen-ELISA. For the experimentally infected samples, there was an excellent correlation between the LFA and the ELISA, while the PCR always showed to be more sensitive (38 % positive samples by PCR versus 27 % by LFA). The LFA was demonstrated to be positive for animals with circulating virus levels exceeding 10(4) HAU. With the field samples, once again, the PCR detected more positives than either the Antigen-ELISA or LFA, although here the number of positive samples scored by the LFA exceeded the values obtained with the Antigen-ELISA, showing 60 % positivity vs 48 % for the ELISA. For the two groups of sera, the specificity was close to 100 % indicating that hardly any false positive samples were found. CONCLUSIONS: The newly developed LFA allows rapid and reliable detection of ASFV, at field and laboratory level, providing a new useful tool for control programs and in situations where laboratory support and skilled personnel are limited.

Use of Phosphite Salts in Laboratory and Semicommercial Tests to Control Citrus Postharvest Decay.

Potassium phosphite (KP) concentrations that inhibited the germination of 50% of Penicillium digitatum conidia were 229, 334, 360, 469, 498, or 580 mg/liter at pH 3, 4, 5, 6, 7, or 8, respectively. Increasing phosphate content in media reduced phosphite toxicity. To control green or blue mold, fruit were inoculated with P. digitatum or P. italicum, then immersed 24 h later in KP, calcium phosphite (CaP), sodium carbonate, sodium bicarbonate, or potassium sorbate for 1 min at 20 g/liter for each at 25 or 50°C. Mold incidence was lowest after potassium sorbate, CaP, or KP treatments at 50°C. CaP was often more effective than KP but left a white residue on fruit. KP was significantly more effective when fruit were stored at 10 or 15°C after treatment compared with 20°C. Acceptable levels of control were achieved only when KP was used in heated solutions or with fungicides. KP was compatible with imazalil (IMZ) and other fungicides and improved their effectiveness. KP increased thiabendazole or IMZ residues slightly. Phosphite residues did not change during storage for 3 weeks, except they declined when KP was applied with IMZ. KP caused no visible injuries or alteration in the rate of color change of citrus fruit in air or ethylene at 5 µl/liter.

Experimental study of vertical stress profiles of a confined granular bed under static and dynamic conditions.

In a wet agglomeration process inside a low shear mixer, the blade function is to induce i) homogenization of the liquid sprayed on the powder surface and ii) a stress field able to transfer the mechanical energy at the particle scale. In this work we study the mechanical state of a confined powder bed through the analysis of stress distributions (by force measurements) in a rectangular cell in two cases: for a classical model powder (i.e. glass beads) and a complex powder (i.e. wheat semolina). Two types of vertical stress profiles are obtained according to the type of measurements carried out in the powder bed, either locally (at different positions in the cell) or globally (at the entire base). The global vertical stress profile follows Janssen's model and the local vertical stress profile highlights a critical length, identified as the percolation threshold of the force network, and a shielding length near the bottom, which is similar to an influence length of the side walls. In the context of wet agglomeration, the results allow to consider the role of the characteristic lengths in the mixing bowl under vertical mechanical solicitation.

The C-terminal domain of Arabidopsis ROS1 DNA demethylase interacts with histone H3 and is required for DNA binding and catalytic activity.

Active DNA demethylation plays an important role in controlling methylation patterns in eukaryotes. In plants, the DEMETER-LIKE (DML) family of 5-methylcytosine DNA glycosylases initiates DNA demethylation through a base excision repair pathway. However, it is poorly understood how these DNA demethylases are recruited to their target loci and the role that histone marks play in this process. Arabidopsis REPRESSOR OF SILENCING 1 (ROS1) is a representative enzyme of the DML family, whose members are uniquely characterized by a basic amino-terminal domain mediating nonspecific binding to DNA, a discontinuous catalytic domain, and a conserved carboxy-terminal domain of unknown function. Here, we show that ROS1 interacts with the N-terminal tail of H3 through its C-terminal domain. Importantly, phosphorylation at H3 Ser28, but not Ser10, abrogates ROS1 interaction with H3. Conserved residues at the C-terminal domain are not only required for H3 interaction, but also for efficient DNA binding and catalytic activity. Our findings suggest that the C-terminal domain of ROS1 may function as a histone reader module involved in recruitment of the DNA demethylase activity to specific genomic regions.

The extended signal peptide of the trimeric autotransporter EmaA of Aggregatibacter actinomycetemcomitans modulates secretion.

The extracellular matrix protein adhesin A (EmaA) of the Gram-negative bacterium Aggregatibacter actinomycetemcomitans is a fibrillar collagen adhesin belonging to the family of trimeric autotransporters. The protein forms antenna-like structures on the bacterial surface required for collagen adhesion. The 202-kDa protein monomers are proposed to be targeted and translocated across the inner membrane by a long signal peptide composed of 56 amino acids. The predicted signal peptide was functionally active in Escherichia coli and A. actinomycetemcomitans using truncated PhoA and Aae chimeric proteins, respectively. Mutations in the signal peptide were generated and characterized for PhoA activity in E. coli. A. actinomycetemcomitans strains expressing EmaA with the identical mutant signal peptides were assessed for cellular localization, surface expression, and collagen binding activity. All of the mutants impaired some aspect of EmaA structure or function. A signal peptide mutant that promoted alkaline phosphatase secretion did not allow any cell surface presentation of EmaA. A second mutant allowed for cell surface exposure but abolished protein function. A third mutant allowed for the normal localization and function of EmaA at 37°C but impaired localization at elevated temperatures. Likewise, replacement of the long EmaA signal peptide with a typical signal peptide also impaired localization above 37°C. The data suggest that the residues of the EmaA signal peptide are required for protein folding or assembly of this collagen adhesin.

[Validation of the Scale of Behavior Indicators of Pain (ESCID) in critically ill, non-communicative patients under mechanical ventilation: results of the ESCID scale]. / Validación de la Escala de Conductas Indicadoras de Dolor para valorar el dolor en pacientes críticos, no comunicativos y sometidos a ventilación mecánica: resultados del proyecto ESCID.

OBJECTIVE: To determine the reliability and validity of the "Scale of Behavior Indicators of Pain" (Escala de Conductas Indicadoras de Dolor: ESCID) as a tool to assess pain in the critically ill, non-communicative patients with mechanical ventilation. METHODS: An observational study of development and validation of this scale as an instrument for pain measurement in ICU patients over 18 years of age, who are uncommunicative and under mechanical ventilation. Their pain was assessed with the Behavioral Pain Scale (BPS) and the ESCID simultaneously, by two independent observers, when the painful maneuvers (PM), secretion aspiration and mobilization, were applied. Measurements were obtained before, during and after the PM. A descriptive analysis of the general characteristics of the population was carried out. The reliability of the ESCID was measured through the internal consistency of each item using Cronbach's alpha. Intraobserver and interobserver concordance was measured with the repeated measurements analysis of variance test. The components of the two pain scales were compared to obtain the change between the results obtained based on time, observer and procedure. The correlation between the scales was measured with the Pearson's correlation. RESULTS: A total of 480 observations were obtained in 42 patients, 62% were males; age 57.33 ± 16.35 years. The most frequent ICU admission was due to infectious disease (36%) and neurological disease (35%). Glasgow Coma Scale 8.45±1.2 and Richmond Agitation-Sedation Scale -2.55±1.5. Arterial blood pressure, heart rate and respiratory rate remained stable. Cronbach's s Alpha Coefficient for ESCID ranged from 0.70-0.80. There is a good correlation between the ESCID and BPS in the three measurement points in time: Pearson's correlation: before 0.97, during 0.94 and after 0.95. CONCLUSIONS: ESCID is a reliable and valid tool to assess pain in critically ill, non-communicative patients under mechanical ventilation.

The structure of eukaryotic and prokaryotic complex I.

The structures of the NADH dehydrogenases from Bos taurus and Aquifex aeolicus have been determined by 3D electron microscopy, and have been analyzed in comparison with the previously determined structure of Complex I from Yarrowia lipolytica. The results show a clearly preserved domain structure in the peripheral arm of complex I, which is similar in the bacterial and eukaryotic complex. The membrane arms of both eukaryotic complexes show a similar shape but also significant differences in distinctive domains. One of the major protuberances observed in Y. lipolytica complex I appears missing in the bovine complex, while a protuberance not found in Y. lipolytica connects in bovine complex I a domain of the peripheral arm to the membrane arm. The structural similarities of the peripheral arm agree with the common functional principle of all complex Is. The differences seen in the membrane arm may indicate differences in the regulatory mechanism of the enzyme in different species.

Prevalence and factors linked to atopic eczema in 10- and 11-year-old schoolchildren. Isaac 2 in Almeria, Spain.

BACKGROUND: Atopic eczema affects 5-10% of the Spanish paediatric population, and has increased in frequency over the last few decades, probably due to changes in the environment and lifestyle. Phase II of the ISAAC (International Study of Asthma and Allergies in Childhood) uses a standardised methodology to establish the prevalence of allergic disorders and factors linked to them in each centre. OBJECTIVES: To assess the prevalence and severity of atopic eczema, and to establish factors linked to atopic eczema in 10-11 year-old school children in the city of Almeria (South-East coast of Spain). MATERIAL AND METHODS: An ecological study was carried out as part of ISAAC II, using homologated questionnaires and allergic tests in 1143 schoolchildren. Statistic association was assessed by means of chi(2) test, and then logistic regression analysis was performed with the most significant variables from the univariant analysis. RESULTS: The prevalence of atopic eczema was 11.4%. The risk factors found in the multiple logistic regression analysis were: personal antecedents of severe asthma (OR 19 CI 95% 1.35-266) and severe rhinitis (OR 7.7 CI 95% 1.79-33), fungi in bedroom during the first year of life (OR 4.2 CI 95% 1.17-15.1) and atopic eczema in one parent (OR 5.2 CI 95% 2.69-10.1). CONCLUSIONS: The prevalence of atopic eczema is similar to that found in other studies within ISAAC Phase I. The most important risk factors for atopic eczema are family and personal history of other atopic diseases and the presence of fungi in the home.

Prevalence and factors linked to allergic rhinitis in 10 and 11-year-old children in Almería. Isaac Phase II, Spain.

BACKGROUND: Allergic rhinitis affects 10-30% of children in developed countries and has increased in frequency over the last few decades, probably due to changes in the environment and life style. AIM: To assess the prevalence, severity, and factors linked to rhinitis in 10 and 11-year-old children from Almeria (Spain). METHODS: As part of ISAAC II, a cross-sectional survey was conducted among a representative sample of 1143 schoolchildren in spring and autumn of 2001, using homologated questionnaires and skin-prick testing. RESULTS: The overall prevalence of rhinitis and rhinoconjunctivitis were 38.9% and 24.8%, respectively, 17.9% had medically diagnosed rhinitis. During the previous year symptoms disturbed daily activities and school attendance in some measure in 40% and 26% of children with rhinitis, respectively. The risk factors found in the multiple logistic regression analysis were atopy (OR 2.57; 95% CI 1.92-3.42); cat contact at home during first year of life (OR 2.4 95% CI 1.13-5.12); prior medical diagnosis of asthma (OR 2.2; 95% CI 1.22-4.02); nocturnal cough in absence of colds (OR 1.9; 95% CI 1.25-2.97); diagnosis of rhinitis in one of the parents (OR 1.8; 95% CI 1.31-2.59); wheezing at any time (OR 1.6; 95% CI 1.18-2.28); and nursery school attendance (OR 1.5; 95% CI 1.21-2.5). CONCLUSIONS: The prevalence of rhinitis found is superior to that of other centres participating in the ISAAC Phases I and II, and coexists with asthma and eczema in many children. The independent risk factors associated to rhinitis are in accordance with previous reports.

In vivo, villin is required for Ca(2+)-dependent F-actin disruption in intestinal brush borders.

Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca(2+)-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca(2+) differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca(2+), whereas Ca(2+) had no effect in villin-null isolates. Moreover, increase in intracellular Ca(2+) by serosal carbachol or mucosal Ca(2+) ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 +/- 9.6%, compared with wild-type mice, 70 +/- 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury.

Interleukin-12 gene polymorphisim in patients with giant cell arteritis, polymyalgia rheumatica and elderly-onset rheumatoid arthritis.

OBJECTIVE: The cytokine profile suggests that giant cell arteritis (GCA) is a Th1-driven disease, in which local IFN-gamma plays a critical role in the development of a systemic arteritis. IL-12 is a potent inducer of IFN-gamma and is critically involved in biasing an immune response towards a Th1 pathway. The purpose of this study was to investigate whether there was an association between an IL-12 gene polymorphism (-1188 A/C 3UTR) and disease susceptibility for GCA and two other age-related inflammatory conditions, such as polymyalgia rheumatica (PMR) and elderly-onset rheumatoid arthritis (EORA). Furthermore, we attempted to correlate such polymorphism with in vitro IL-12 production. MATERIALS AND METHODS: We analyzed genotypes at -1188 in the 3UTR of the IL-12 promoter by PCR-RFLP in 68 GCA, 138 PMR, and 72 EORA patients as well as in 465 healthy controls (HC). IL-12p70 levels in culture supernatants after stimulation with PMA+Ionomycin was assessed by ELISA. RESULTS: All groups were in Hardy-Weinberg equilibrium. Allelic and gen-omic distribution was not significantly different among the study groups. None of the genetic variants was associated with disease severity. Although the differences were not statistically significant, HC genotypes were associated with distinct IL-12 p70 production. CONCLUSIONS: The IL-12 (-1188 A/C 3UTR) gene polymorphism is not associated with disease susceptibility or severity in three age-related chronic inflammatory syndromes. The production of IL-12 p70 is dependent on the genetic background in HC, although in patients such association may be biased by other unknown factors.

Malignant colonic adenomas. Therapeutic criteria. Long-term results of therapy in a series of 42 patients in our healthcare area.

OBJECTIVE: a) to evaluate the appropriateness of histological criteria as proposed by Morson as indicators for surgery; and b) to compare the adequacy of Morson s criteria with Haggitt s levels as indicators for surgery in the case of malignant sessile lesions. MATERIAL AND METHODS: we carried out a prospective, protocolized study of 42 patients with polyps with invasive carcinoma (IC) who underwent colonoscopic polypectomy from 1979 through 2008. We applied the histological criteria proposed by Morson to all the patients included in the series. RESULTS: we treated 24 polyps with IC and favorable histological criteria (FC) and 18 polyps showing unfavorable histological criteria (UC). All polyps with FC were treated by means of colonoscopic polypectomy. None of the patients showed signs of disease after a mean follow-up period o f 9.67 yrs. Patients with polyps with UC were recommended to undergo surgery. The presence of unfavorable histological criteria in the polypectomy sample was clearly associated with an unfavorable patient outcome and showed a 100% sensitivity level, a negative prognostic value (NPV) of 100%, and a false negative (FN) percentage of 0% for the prognosis of the disease. We found 12 sessile polyps (Haggitt s level 4). Colonoscopic polypectomy was the treatment employed in 9 out of 12 cases. All patients are free from disease (mean follow-up 7.3 yrs). If Haggitt s level criteria had been applied, all 12 patients would have undergone surgery. This means 58% more patients than following Morson s criteria. CONCLUSIONS: Morson s criteria are considered an adequate diagnostic tool for the indication of surgery in patients with malignant adenomas. Haggitt s invasion levels do not accurately discriminate the necessity for surgery in case of malignant sessile lesions.

Twin product/process approach for pellet preparation by extrusion/spheronisation. Part I: hydro-textural aspects.

The extrusion/spheronisation technique has made a notable contribution to the existing range of pharmaceutical forms especially in the area of modified-release products. The twin product/process approach adopted in this work is based on the on-line monitoring of the hydro-textural characteristics of the product up to its final form. The objective is to balance the influence of the operating parameters for each successive stage against the influence of product characteristics. A coherent "representational framework" is proposed for insoluble substances through a diagram locating intergranular porosity value depending on water content. The wetting/kneading operation brings the material to a state in which porosity is linked to water content. The extrusion operation densifies the material to saturation point, while spheronisation is only a shaping process which maintains hydro-textural state. The drying operation finalises the textural characteristics of the product by densifying the medium through induced shrinkage.