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The Middle Member of the Llopis Fm in the Sierra de Bedmar-Jódar Unit of the Prebetic Zone of Jaén (southern Spain) was deposited on a shallow-marine platform of the Southern Iberian Continental Margin during the earliest Aptian. Detailed field logging of nine stratigraphic sections and facies mapping have allowed seven lithofacies associations (L1–L6) to be distinguished, one siliciclastic (L1) and five carbonate facies (L2–6). The succession is composed of eight consecutive elemental sequences of lithofacies associations L1–L6. Each elemental sequence is interpreted as representing one episode of shallowing-upwards carbonate deposition in a very shallow platform-lagoon that was bounded shoreward by clastic/ooid bars and passed seaward either to stromatoporoid bioconstructions (bioherms and biostromes) or rudist biostromes. The successive elemental sequences show north-eastward progradational geometries. Three phases of platform development are identified: (1) installation of the shallow platform; (2) development of a lagoon bounded by a stromatoporoid barrier and (3) development of an Urgonian-type platform dominated by rudists. During the early Aptian, the Bedmar-Jódar platform was partially isolated from the rest of the Prebetic platform and showed overall progradation towards the NE, in contrast to the general south-eastward progradational trend of the Prebetic platform. Sedimentation was controlled by rift-generated extensional tectonics that resulted in tilting of the platform block, causing the deviation of progradation from the general trends of the Prebetic Platform. In addition, climatic influence is inferred from the presence of siliciclastic sediments derived from weathering of the hinterland, which restricted the carbonate factory.

Objective Pharmacological interventions remain the cornerstone of chronic pain treatment; however, nearly 40% of the prescription medicines are not taken as prescribed. The present study aims at understanding and describing non-adherence from the perspective of chronic pain patients during a 1-year follow-up study. Methods A cohort of 950 consecutive patients referred to a first consultation in Multidisciplinary Chronic Pain Clinics was followed with a standardized protocol for 1 year. This included assessment of pain characteristics; prescribed medication; therapeutic adherence; effectiveness of treatment, non-adherence and its perceived reasons; clinical outcomes and quality of life. We used a mixed methods approach, including qualitative and quantitative analyses. Results Forty-nine percent of the 562 patients who responded to all assessments during follow-up were adherent after 1 year of chronic pain treatment. The core associations between each "non-adherence reason" and Anatomical Therapeutic Chemical Code (ATC) group were perceived side effects (p=0.019) and delayed start (p=0.022) for narcotic analgesics (opioids); perceived non-efficacy (p=0.017) and delayed start (p=0.004) for antiepileptics and anticonvulsants; perceived low necessity (p=0.041) and delayed start (p=0.036) for analgesics antipyretics; change in prescriptions because of a new clinical condition for antidepressants (p=0.024); high concerns (p=0.045) and change in prescriptions because of a new clinical condition (p

Vaccine hesitation is a topic of utmost importance, with the COVID-19 pandemic serving as a clear reminder of its timeliness. Besides evaluating COVID-19 vaccine acceptance in a sample of Portuguese people, this study aims at understanding cognitive and emotional representations related to vaccination, and their influence on vaccination hesitation. A cross-sectional online survey was conducted between 27 December 2020 and 27 January 2021. It assessed cognitive and emotional COVID-19 representations; vaccination status; cognitive and emotional representations of vaccination and perceived necessity and concerns about vaccines. Of 31 × 58 participants, 91% accepted taking a COVID-19 vaccine. Among several other significant findings, women (71.3%) more often considered that the pandemic affected their lives (p < 0.001) and were more often concerned with being infected (p < 0.001). Likewise, there were significantly more female participants concerned about taking a COVID-19 vaccine and its possible effects, when compared to the number of male participants (p < 0.001). The number of participants with a higher education level that were more worried about becoming infected was greater (p = 0.001), when compared with those less educated. Regarding age groups, people aged 18 to 24 had fewer concerned participants (9.6%), while the number of individuals aged 55 to 64 had the most (p < 0.001). Somewhat surprisingly, perceiving oneself as extremely informed about COVID-19 was not associated with greater vaccine acceptance (OR = 1.534 [1.160–2.029]; (p = 0.003)). Moreover, people aged 25 to 64 years old and with lower education level were more likely not to accept vaccination (OR = 2.799 [1.085–7.221]; (p = 0.033)). Finally, being more concerned about taking a vaccine lowers its acceptance (OR = 4.001 [2.518–6.356]; (p < 0.001)). Cognitive and emotional representations have a great impact and are reliable predictors of vaccine acceptance. Thus, it is of extreme importance that public health messages be adapted to the different characteristics of the population.

Background The assessment of pain as the fifth vital sign (P5VS) is of paramount importance since it leads to the management of undertreated pain, consequently reducing suffering, readmissions and emergency department visits after hospital discharge. Objective To evaluate the implementation of P5VS in public and private hospitals. Methods Data analysis on validated questionnaires was sent to all 171 Portuguese hospitals via an official letter. Results When compared to private hospitals, public hospitals presented a higher adherence to the process related to the P5VS. It has demonstrated superiority in the charts properly placed to record P5VS, in the number of emergency departments recording P5VS, in the regularity of audits, and in the existence of guidelines and staff training on pain assessment and management. Conclusion The standardization of both evaluation and recording of pain intensity constitutes a measure of good clinical practice. Public hospitals demonstrated better commitment to these procedures that should be properly carried out in all health care institutions.

This study consisted of a palynological, microbiological, and physicochemical characterization of fifteen samples of Spanish honey sold under quality brands with different botanical and geographical origins from two consecutive harvest years (2010 and 2011). Eight of the fifteen honey samples were classified as monofloral honey from botanical origins Persea americana, Castanea sativa, Rosmarinus officinalis, Eucalyptus sp., and Thymus sp. With regard to microbiological analyses, mold, and yeast counting, Staphylococcus aureus, Salmonella, sulfite-reducing clostridia, and Escherichia coli were not detected in any of the samples. Aerobic mesophilic microorganisms were detected only in some samples and the counts in these cases were low. Despite the great variability between samples, the results obtained in the physicochemical analysis were consistent with the limits set by the Council Directive 2001/100. Honey samples showed high variability between two consecutive harvests, since, even if they had similar geographical origins they showed different nectar floral origins. Mieles españolas con marca de calidad: un enfoque multivariado de parámetros fisicoquímicos, calidad microbiológica y origen floral Este estudio consistió en la caracterización botánica, microbiológica y fisicoquímica de quince muestras de miel españolas acogidas a marcas de calidad diferenciada de diferentes orígenes botánicos y geográficos y procedentes de dos cosechas consecutivas (años 2010 y 2011). Ocho de las quince muestras de miel estudiadas fueron clasificadas como mieles monoflorales de Persea americana, Castanea sativa, Rosmarinus officinalis, Eucalyptus sp. y Thymus sp. En relación a los análisis microbiológicos los recuentos de mohos y levaduras, Staphylococcus aureus, Salmonella, clostridios sulfito reductores y Escherichia coli no fueron detectados en ninguna de las muestras. Se detectaron microorganismos aerobios mesófilos solo en algunas muestras y en este caso los recuentos fueron bajos. Aunque se detectaron coliformes, estos podrían estar asociados a un origen ambiental. A pesar de la gran variabilidad entre las muestras, los resultados obtenidos en el análisis fisicoquímico se encontraron dentro de los límites establecidos por la Directiva del Consejo 2001/100. Las muestras de miel mostraron una gran variabilidad entre cosechas porque aunque tuvieron un mismo origen geográfico, hubo diferencias en su origen floral. Este estudio consistió en la caracterización botánica, microbiológica y fisicoquímica de quince muestras de miel españolas acogidas a marcas de calidad diferenciada de diferentes orígenes botánicos y geográficos y procedentes de dos cosechas consecutivas (años 2010 y 2011). Ocho de las quince muestras de miel estudiadas fueron clasificadas como mieles monoflorales de Persea americana, Castanea sativa, Rosmarinus officinalis, Eucalyptus sp. y Thymus sp. En relación a los análisis microbiológicos los recuentos de mohos y levaduras, Staphylococcus aureus, Salmonella, clostridios sulfito reductores y Escherichia coli no fueron detectados en ninguna de las muestras. Se detectaron microorganismos aerobios mesófilos solo en algunas muestras y en este caso los recuentos fueron bajos. Aunque se detectaron coliformes, estos podrían estar asociados a un origen ambiental. A pesar de la gran variabilidad entre las muestras, los resultados obtenidos en el análisis fisicoquímico se encontraron dentro de los l ımites establecidos por la Directiva del Consejo 2001/100. Las muestras de miel mostraron una gran variabilidad entre cosechas porque aunque tuvieron un mismo origen geográfico, hubo diferencias en su origen floral. This work was supported by the Consejería de Sanidad. Junta de Castilla y León [GRS 551/A/10]; Patricia Combarros- Fuertes PhD Research was funded by Consejería de Educación. Junta de Castilla y León and Fondo Social Europeo. info:eu-repo/semantics/publishedVersion

Background. High rates of opioid use for chronic noncancer pain (CNCP) have been reported worldwide, despite its association with adverse events, inappropriate use, and limited analgesic effect. Opioid-induced constipation (OIC) is the most prevalent and disabling adverse effect associated with opioid therapy. Our aim was to assess the incidence, health related quality of life (HRQOL), and disability in OIC patients. Methods. A prospective cohort study was performed, with 6 months of follow-up, of adult CNCP patients consecutively admitted in 4 multidisciplinary pain clinics (MPC). Demographic and clinical data have been collected. Brief Pain Inventory (BPI) and Short version of Treatment Outcomes in Pain Survey (S-TOPS) were used to measure functional outcomes and HRQOL. OIC was assessed using Bowel Function Index (BFI). Results. 694 patients were recruited. OIC prevalence at baseline was 25.8%. At 6 months, OIC incidence was 24.8%. Female gender (OR = 1.65, p=0.039), opioid therapy (OR 1.65, p=0.026), and interference pain score on BPI (OR 1.10, p=0.009) were identified as OIC independent predictors. OIC patients presented higher disability and pain interference and severity scores. OIC patients reported less satisfaction with outcome (p=0.038). Discussion. Constipation is a common adverse event among opioid users with major functional and quality of life impairment. These findings emphasise the need of OIC adequate assessment and management.

This study assessed 16 different honey samples in order to select the best one for therapeutic purposes. First, a study of honey's main bioactive compounds was carried out. Then phenolic profiles were determined and specific compounds quantified using a HPLC system coupled to a mass spectrometer. Then, antioxidant activity, by three in vitro methods, and antibacterial activity against reference strains and clinical isolates were evaluated. Great variability among samples was observed regarding ascorbic acid (between 0.34 ± 0.00 and 75.8 ± 0.41 mg/100 g honey; p < 0.001), total phenolic compounds (between 23.1 ± 0.83 and 158 ± 5.37 mg/100 g honey; p < 0.001), and total flavonoid contents (between 1.65 ± 0.11 and 5.93 ± 0.21 mg/100 g honey; p < 0.001). Forty-nine different phenolic compounds were detected, but only 46 of them were quantified by HPLC. The concentration of phenolic compounds and the phenolic profiles varied widely among samples (between 1.06 ± 0.04 and 18.6 ± 0.73 mg/100 g honey; p < 0.001). Antioxidant activity also varied significantly among the samples. All honey varieties exhibited antibacterial activity against both reference and clinical strains (effective concentrations ranged between 0.05 and 0.40 g/mL depending on the honey sample and bacteria tested). Overall, samples with better combinations of bioactive properties were avocado and chestnut honeys. This work was supported by the ConsejeriaFundinǵ de Sanidad of Junta de Castilla y Leoń (Spain); under grant GRS 551/A/10. P.C.-F. was funded by the Consejeriá de Educacioń of Junta de Castilla y Leoń and European Social Fund. Notes The authors declare no competing financial interest. info:eu-repo/semantics/publishedVersion

AIM The aim of present study was to perform the translation, cultural adaptation, and validation of the Medication Adherence Report Scale (MARS-P9) for the European Portuguese language in a sample of chronic pain patients. METHODS A Portuguese version of the nine-item MARS (©Professor Rob Horne) scale (MARS-P9) was constructed through a process of translation, back translation, and experts' panel evaluation. A total of 141 chronic pain patients were subsequently evaluated at four time assessments during a 1-year pain medication treatment. The protocol interview included the assessment of pain intensity and interference (BPI), clinical outcomes and quality of life (S-TOPS), and MARS-P9. RESULTS The internal consistency coefficient was acceptable for the total scale (α = 0.84). Exploratory factor analysis revealed a two-factor structure (intentional and unintentional nonadherence) that explained 61% of the variance. Convergent and discriminant validity were demonstrated by correlations between MARS scores and pain interference (r = 0.180, P ≤ 0.01) and S-TOPS (r = 0.242, P ≤ 0.05). CONCLUSION MARS-P9 has been shown to be an adequate instrument for Portuguese researchers and clinicians to assess the pattern of adherence during the management of chronic pain.

Four batches of Armada semi-hard goat cheese were elaborated from pasteurized milk inoculated with a natural starter culture, constituted of two lactococci strains, combined with a Geotrichum candidum strain. The four G. candidum strains tested as co-starter were selected for their proteolytic and lipolytic activities. The effects of an autochthonous starter on physico-chemical, microbiological and sensorial characteristics during the ripening of this cheese were evaluated. The depth of proteolysis was very low, which confirms the presence of low-level aminopeptidase activity. Fungal population was involved in lipolysis. The cheeses elaborated with G. candidum strains developed a desirable flavour characteristic of goat cheese and a creamy texture. Cheeses from batch I (that included the strain of G. candidum with high lipolytic activity and low proteolytic activity) presented a hardness profile that differed from the others, as it was the batch with the highest scores at thirty days of ripening and was even the best evaluated at the end of ripening because of their odour, fresh balanced pleasant taste and creamy smooth texture. Key words: Cheese, starter culture, Geotrichum candidum, lactic acid bacteria.

Supplemental Digital Content is Available in the Text., Introduction: The burden of untreated postoperative pain is high. Objective: This study assessed feasibility of using quality improvement (QI) tools to improve management of perioperative pain in hospitals in multiple developing countries. Methods: The International Pain Registry and Developing Countries working groups, from the International Association for the Study of Pain (IASP), sponsored the project and PAIN OUT, a QI and research network, coordinated it, and provided the research tools. The IASP published a call about the project on its website. Principal investigators (PIs) were responsible for implementing a preintervention and postintervention study in 1 to 2 surgical wards in their hospitals, and they were free to choose the QI intervention. Trained surveyors used standardized and validated web-based tools for collecting findings about perioperative pain management and patient reported outcomes (PROs). Four processes and PROs, independent of surgery type, assessed effectiveness of the interventions. Results: Forty-three providers responded to the call; 13 applications were selected; and PIs from 8 hospitals, in 14 wards, in 7 countries, completed the study. Interventions focused on teaching providers about pain management. Processes improved in 35% and PROs in 37.5% of wards. Conclusions: The project proved useful on multiple levels. It offered PIs a framework and tools to perform QI work and findings to present to colleagues and administration. Management practices and PROs improved on some wards. Interpretation of change proved complex, site-dependent, and related to multiple factors. PAIN OUT gained experience coordinating a multicentre, international QI project. The IASP promoted research, education, and QI work.

Traditional cheeses are an important reservoir of microbial diversity that can have important biotechnological applications, especially with a view to improving the characteristics unique to each type of cheese, and in this respect, starter cultures consisting of autochthonous lactic acid bacteria strains are of particular interest. In the present study, we investigated lactic acid bacteria population diversity in San Simon da Costa cheese (PDO, Galicia, Spain) and found a predominance of the genus Lactobacillus, which by the end of ripening accounted for 78% of the strains isolated in Rogosa agar, around 40% of those in M17 agar and about 10% of those in MSE agar. The main species of lactic acid bacteria identified were Lactococcus lactis subsp. lactis, Lactobacillus casei subsp. casei, Lb. paracasei subsp. paracasei, Leuconostoc mesenteroides and Enterococcus faecalis. Virtually all the strains studied from a technological point of view yielded more than or equal to 0.24 g 100 mL-1 lactic acid. Lactococcus lactis subsp. lactis (SS 194), Lactobacillus paracasei (SS 1695 and SS 1689) and Enterococcus faecalis (SS 1378 and SS 1449) strains exhibited the greatest proteolytic capacity. Based on the overall technological aptitude of the tested strains, we can propose starter cultures and co-cultures that include different combinations of previous strains with a view to manufacturing San Simon da Costa cheese from pasteurised milk.

This review describes some aspects related to the technological barriers encountered in the development and stability of probiotic cheeses. Aspects concerning the viability of probiotic cultures in this matrix are discussed and the potential of cheese as a biofunctional food carrier is analyzed, outlying some points related to health and safety. In general, the manufacture of probiotic cheese should have little change when compared with the elaboration of cheese in the traditional way. The physicochemical and technological parameters influencing the quality of these products have also to be measured so as to obtain a process optimization.

Partial sequencing of 16S ribosomal RNA (rRNA) gene is commonly used for the identification of lactic acid bacteria (LAB). The present study evaluated the performance in the identification of dairy LAB of two universal (515FPL-13B and 91E-13) and one Lactobacillus group-specific (S-D-Bact-0011-a-S-17-S-G-Lab-0677-a-A-17) primer pairs. After amplification, the 16S rRNA gene portions were sequenced, and sequences were analysed using the Ribosomal Database Project (RDP-II) and Basic Length Alignment Search Tool (BLAST) programs. The primers were used with three reference strains and eight isolates from Oaxaca cheese. The 515FLP-13B primer misled the identification of one reference strain and two isolates. Five among the eight isolates were identified as Lactococcus lactis by at least two of the primers, and the specific primer showed higher RDP-II identity scores than the universal primers. The specific primer was successfully used for the identification of other 24 cheese isolates. The joint use of the two ...

The aim of this work was to identify and characterize lactobacilli strains from Mexican Oaxaca cheese. Twenty-seven lactobacilli isolated from Oaxaca cheese were identified at species level by 16S rRNA sequencing. Selected isolates were further characterized by ribotyping. Isolates were screened, among others, by acidifying capacity, antibiotic resistance, and activity against pathogens. Lactobacillus plantarum was predominant in Oaxaca cheese. The intraspecies variability of Lb. plantarum isolates was great. Multiple antibiotic resistances were observed. Eight isolates showed antimicrobial activity against the pathogenic species tested. Four Lb. plantarum strains showing low antibiotic resistance index, antimicrobial activity against enterotoxigenic Staphylococcus aureus and Listeria innocua stains, amine-negative decarboxylase activity, and resistance to NaCl and bile salt solutions, could be preselected to complete studies focused on designing a culture for use in pasteurized-milk Oaxaca cheese manufacturing.

Opioids use for Chronic Non cancer Pain (CNCP) treatment has been increasing worldwide. Despite their important role for moderate to severe pain treatment, their use in CNCP has been subject of great controversy. Opioid long-term use is associated with adverse effects development such as hypogonadism, osteoporosis, immunosuppression, cognitive disorders, opioid induced hyperalgesia (OIH) and opioid addiction. Moreover, there is uncertainty about their long-term effectiveness and safety. It is important to raise awareness among physicians of opioid long-term effects, as well as, strategies for their prevention and treatment.

The aim of this work was to study the antimicrobial activity of 395 strains of lactic acid bacteria isolated from Genestoso cheese throughout manufacture and ripening. These were: Lactobacillus (137 strains), Lactococcus (125 strains), Leuconostoc (58 strains) and Enterococcus (75 strains). After the extract had been neutralized and treated with catalase to exclude the action of hydrogen peroxide, 13 lactococci, three lactobacilli, three leuconostocs and five enterococci continued to show antimicrobial activity against one or more of the reference varieties used as indicator strains (Enterococcus faecalis CECT 481, Staphylococcus aureus CECT 240, Lactobacillus plantarum CECT 748, Clostridium tyrobutyricum CECT 4011 and Listeria monocytogenes CECT 4031). The 24 strains of lactic acid bacteria showing antimicrobial activity after excluding the action of hydrogen peroxide were identified at species level by genetic methods. The majority of these strains were isolated from the batch ripened in autumn (37.5% of strains) and the batch ripened in spring (29.2% of 15 strains) manufactured by the same cheesemaker and mainly during manufacture and the first days of ripening.

Six batches of Armada cheese were produced, one from raw milk with no added starter, another from pasteurized milk with a commercial mesophilic starter and four from pasteurized milk with experimental starters. These starters included: Lactococcus lactis subsp. lactis and L. lactis subsp. lactis biovar. diacetylactis; or the same strains plus either Enterococcus raffinosus, Leuconostoc mesenteroides subsp. dextranicum or Lactobacillus plantarum, all isolated from Armada cheese made from raw milk. The highest counts of aerobic mesophilic bacteria and populations of lactic acid bacteria for all batches were reached in the first week of ripening. These counts declined later throughout the ripening, although not at identical rates in every batch. Counts of lactobacilli were significantly higher in cheeses made from raw milk and those inoculated with the lactobacilli strain than in the other batches, over the whole ripening period. Added native starters minimized growth of Enterobacteriaceae. Cheeses made with the starter containing the Enterococcus strain had the most favourable sensory attributes throughout ripening.

Thirty-one lactic acid bacteria isolated from Armada cheese and previously selected in accordance with their technological properties, were screened for antimicrobial activity one against another. Four strains showed inhibitory activity against 14 strains when tested by well diffusion assay after the effects of organic acid and hydrogen peroxide were eliminated. Extracts of the strains did not show inhibitory activity after treatment with proteinase K, trypsin, α-chymotrypsin or pepsin. None of the strains showed antimicrobial activity against several pathogenic and spoilage reference strains. Eighteen representative strains were tested for their antibiotic resistance. None of the strains were totally susceptible to all antibiotics tested and multiple resistance was observed. Most of the tested strains were resistant to cefotoxin, oxacillin, vancomycin, teicoplanin, nitrofurantoin and trimethoprim.

The aim of this work was the genetic characterization at the strain level of 39 presumed Geotrichum candidum isolates isolated throughout the artisanal manufacturing and ripening of Armada cheese and tentatively identified at genus and/or species level by phenotypic characteristics. The molecular identification of the strains included among others the amplification and sequencing of the D1/D2 domains of the 26S rRNA gene. A restriction fragment length polymorphism (RFLP) analysis with the ITS1-5.8S-ITS2 PCR amplicons and a randomly amplified polymorphic DNA (RAPD) analysis with five different primers were carried out. The bands pattern profile obtained through RFLP by enzymatic restriction with HinfI was the same for all the strains studied, which confirmed the classification of the strains at species level. A RAPD-PCR analysis with three different primers was applied to assess the intraspecific diversity, in this way 16 band profiles were obtained for the 39 strains studied by the combined use of primers Ari1 and Omt1. This study contributes to know the occurrence and genotypic biodiversity of G.candidum in Armada cheese. © 2013., The authors would like to thank to the “Consejería de Educación of the Junta de Castilla y León” (the Regional Government) for the grant to the author N. Sacristán.

7 páginas, 2 figuras, 1 tabla.-Received: 13 December 2012 / Accepted: 18 January 2013 / Published online: 6 February 2013, DNA primers were designed from the 18S rRNA sequence from the relevant digenean trematode Dicrocoelium dendriticum to evaluate a polymerase chain reaction-based diagnostic method of this parasite from its eggs in faeces of naturally and experimentally infected sheep. In order to get DNA from D. dendriticum eggs, several hatching mechanisms were studied. Successful results were obtained when the eggs were frozen to -80 A degrees C and/or in liquid nitrogen and then defrosted. This method allowed the opening of the egg operculum and the liberation of the miracidium. DNA from D. dendriticum adults and from hatching egg miracidia was obtained and an amplification single band of 1.95 kb was observed using primers designed for the total 18S rRNA sequence in both cases as well as when the template DNA was from adults of the closely related parasite Fasciola hepatica; in addition, a single and specific 0.8-kb band was obtained when primers based on an internal partial 18S rRNA sequence were used. The method showed to be useful not only in samples coming from adults, but in eggs from gall bladder and faeces as well. F. hepatica internal 18S rRNA primers were also designed and used as a negative control to prove that the eggs in faeces came from D. dendriticum and not from F. hepatica. A molecular tool able to detect a minimum of about 40 D. dendriticum eggs in one of the definitive host faeces has been developed for the first time and could provide a useful molecular tool to improve the conventional coprological diagnosis for detecting D. dendriticum eggs., This work was supported by the Spanish CICYT (Project No. AGF96-0416) and Castile and León Autonomy (Proyect No. CSI5/98). H. Sandoval was supported by a Scientist Research Contract from the CSIC.

Directed integration of heterologous DNA into the insertion sequence IS13869 of Brevibacterium lactofermentum has been achieved by using integrative vectors that contain a 600 bp fragment from IS13869 and a kanamycin resistance cartridge with unique PacI, SwaI and PmeI sites. The incorporation of these restriction sites for rare-cutting enzymes, which recognize octanucleotide sequences, allowed rapid mapping of the location of plasmid insertion into the chromosome. Mapping by pulsed-field gel electrophoresis and hybridization with an IS13869 probe showed four types of transformants. In three of these types, integration of the vector occurred at each of three different sites in the chromosome. These vectors have the advantage of increasing the number of integration events due to the presence of multiple targets in the genome.

Pain is a prominent feature of osteoarthritis (OA). To further understand the primary mechanisms of nociception in OA, we studied the expression of the phenotype markers calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), and neurofilament 200 (NF200) in sensory neurons innervating the OA knee joint in rats.OA was induced in rats by intraarticular injection of 2 mg of mono-iodoacetate (MIA) into the knee. Neurons innervating the joint were identified by retrograde labeling with fluorogold in dorsal root ganglia (DRG) and colocalized with neurochemical markers by immunofluorescence. The total number of DRG cells was determined by stereologic methods in Nissl-stained sections.A 37% decrease in the number of fluorogold-backlabeled cells was observed in rats with OA when compared with control rats, even though no decrease in the total number of cells was observed. However, an increase in the number of medium/large cell bodies and a decrease in the number of the smallest cells were observed, suggesting the occurrence of perikarya hypertrophy. The percentage of CGRP-positive cells increased significantly, predominantly in medium/large cells, suggesting the occurrence of a phenotypic switch. Colocalization of CGRP and NF200 revealed no significant changes in the percentage of double-labeled cells, but an increase in the number of medium/large double-labeled cells was observed. No differences in the expression of either IB4 or NF200 were observed in fluorogold-backlabeled cells.These results indicate that MIA-induced OA causes an up-regulation of CGRP in different subpopulations of primary afferent neurons in DRG due to a phenotypic switch and/or cell hypertrophy which may be functionally relevant in terms of the onset of pain in this pathologic condition.

2 figures, 2 tables.-- PMID: 11269322 [PubMed]., Genetic variability of adult specimens of Dicrocoelium dendriticum has been studied using random amplified polymorphic DNA (RAPD). The worms were collected from the infected livers of different sheep from several localities in León province (NW Spain). DNA fragments were amplified by means of decamer primer oligonucleotides of arbitrary sequence. Some primers produce complex and highly variable patterns of amplified DNA in D. dendriticum. Intra- and inter-population genetic variability of adult parasites were analyzed, scoring polymorphic and monomorphic reproducible bands by means of the Jaccard similarity, and dendrograms showing genetic relationships between individuals were obtained using the FITCH method. Genetic variability seems to be high in this parasite and genetic similarity within a population (worms infecting a single animal) is similar to the average similarity between worms from different sheep. These results suggest that each sheep is infected by numerous genetically different parasites from one or more populations of infected ants., This study was supported by the Spanish CICYT, Projects number AGF92-0588 and AGF96-0416.

Summary: Five rRNA operons (rrn) were found by hybridization in the genome of ‘Brevibacterium lactofermentum’ ATCC 13869 and Corynebacterium glutamicum ATCC 13032. ‘B. lactofermentum’ DSM 20412 differed from the other corynebacteria tested in showing six hybridizing BamHI bands. Two of the rrn operons (rrnD and rrnE) were located in a single cosmid. Sequencing of the rrnD operon showed that it contains a complete 16S rRNA-23S RNA-55 rRNA gene cluster. Phylogenetic studies using the complete 16S rRNA sequence showed that ‘B. lactofermentum’ is closely related to several species of the genus Corynebacterium but only distantly related to the type species Brevibacterium linens and the authors suggest that it should be reclassified as Corynebacterium lactofermentum. The 5′ end of mature 16S rRNA was identified by primer extension. Sequence elements similar to those of mycobacteria implicated in transcription antitermination (Boxes A, B, C) and in processing of the pre-rRNA to 16S rRNA were identified. An open reading frame encoding an rpoD-like sigma factor (named SigC) different from the previously reported SigA and SigB proteins was found upstream of rrnD in the opposite orientation. Both rpoD and sigC seem to be expressed from a bidirectional promoter region.

In an effort to relate structural, kinetic, and thermodynamic features in a model macromolecular recognition process, the amino acid residues in the reactive surface of bovine pancreatic trypsin inhibitor (BPTI) and surrounding residues were substituted individually by alanine, and the effects of the point-mutations on the kinetics and thermodynamics of inhibition by BPTI toward trypsin and chymotrypsin were investigated. Fifteen alanine mutants were produced. The majority of the BPTI mutants exhibited a binding affinity similar to that of the wild-type protein. The exceptions were the primary specificity site (PI) mutant and those mutants that seem to have nonlocal perturbations of structure, as revealed by circular dichroism and thermostability measurements. The mutation at the P1 site caused a reduction in the binding free energy of 10 and 1.8 kcal mol-1 for trypsin and chymotrypsin, respectively. The losses in binding affinity were determined almost exclusively by an increase in the dissociation rate constant. However, the rate of association of the P1 mutant, Lys-15-Ala, with trypsin was also drastically reduced (200-fold). Calorimetric measurements of the heats of binding for the association of chymotrypsin with the wild-type inhibitor and its alanine mutants allowed determination of the relative contributions of the changes in enthalpy and entropy to the free energy of binding. Compensatory changes in the two parameters were observed in several cases, which were attributed to desolvation effects at the binding interface.