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MRI and swelling experiments are used to probe the state of water and infer the microstructure of chitosan hydrogels. SEM reveals a porous open scaffold‐type structure for hydrogels that were equilibrated at 2 °C before freezing as compared to those equilibrated at 37 °C. ADC MRI measurements reveal an anisotropy in the microstructure of these gels. T1relaxation MRI values were larger as the pH increased from 7.6 to 12.0, the result of a lower rate of exchange between protons of the hydration sphere of the polymer and bulk water. The thermosensitive and pH‐sensitive properties of these hydrogels can be utilized in the development of innovative materials for biotechnological and biomedical applications, including criobiocatalysis and bioremediation as well as in programmed drug delivery.

ABSTRACTHigh-resolution melt (HRM) analysis can identify sequence polymorphisms by comparing the melting curves of amplicons generated by real-time PCR amplification. We describe the application of this technique to identify Mycobacterium aviumsubspecies paratuberculosistypes I, II, and III. The HRM approach was based on type-specific nucleotide sequences in MAP1506, a member of the PPE (proline-proline-glutamic acid) gene family.

Chitosan is a natural polymer composed of randomly distributed -(1-4)-linked D-glucosamine (deacetylated unit) and Nacetyl- D-glucosamine (acetylated unit). It has been described as a non-toxic, biodegradable and biocompatible polymer with very interesting biological properties, such as permeation-enhancing and mucoadhesive properties, anticoagulant and antimicrobial activity and so on. Chitosan has been used in several areas such as biomedical, pharmaceutical and biotechnological fields as well as in the food industry. Recently, there has been a growing interest in the modification of chitosan to improve its solubility in physiological conditions, to introduce new applications or to improve chitosan biological properties. Research and development on a variety of amphiphilic copolymers containing hydrophobic and hydrophilic segments, have been very active due to their spontaneous self-assembly behaviour in aqueous media These smart transitions often lead to diverse functional compartment structures like micelles, vesicles and gels, which represent promising applications in the field of biotechnology and pharmaceutics. The aim of the present paper is to review the latest advances in the synthesis of chitosan amphiphilic derivatives with a special emphasis in their applications.

ABSTRACTInsertion sequence IS900is used as a target for the identification of Mycobacterium aviumsubsp. paratuberculosis.Previous reports have revealed single nucleotide polymorphisms within IS900. This study, which analyzed the IS900sequences of a panel of isolates representing M. aviumsubsp. paratuberculosisstrain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes.

Chitin and its deacetylated derivative chitosan are natural polymers composed of randomly distributed -(1-4)- linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitin is insoluble in aqueous media while chitosan is soluble in acidic conditions due to the free protonable amino groups present in the D-glucosamine units. Due to their natural origin, both chitin and chitosan can not be defined as a unique chemical structure but as a family of polymers which present a high variability in their chemical and physical properties. This variability is related not only to the origin of the samples but also to their method of preparation. Chitin and chitosan are used in fields as different as food, biomedicine and agriculture, among others. The success of chitin and chitosan in each of these specific applications is directly related to deep research into their physicochemical properties. In recent years, several reviews covering different aspects of the applications of chitin and chitosan have been published. However, these reviews have not taken into account the key role of the physicochemical properties of chitin and chitosan in their possible applications. The aim of this review is to highlight the relationship between the physicochemical properties of the polymers and their behaviour. A functional characterization of chitin and chitosan regarding some biological properties and some specific applications (drug delivery, tissue engineering, functional food, food preservative, biocatalyst immobilization, wastewater treatment, molecular imprinting and metal nanocomposites) is presented. The molecular mechanism of the biological properties such as biocompatibility, mucoadhesion, permeation enhancing effect, anticholesterolemic, and antimicrobial has been updated.

Las guías de práctica clínica deben estar adaptadas a la población en la que se van a aplicar. Esto trae consigo la necesidad de someterlas a un proceso de validación de su calidad. El instrumento AGREE es una herramienta genérica diseñada principalmente para ayudar a productores y usuarios de guías de práctica clínica, tanto nuevas como las ya existentes, en la evaluación de la calidad metodológica de éstas.

Ceramic materials are osteoconductive matrices extensively used in bone tissue engineering approaches. The performance of these types of biomaterials can be greatly enhanced by the incorporation of bioactive agents and materials. It is previously reported that chitosan is a biocompatible, biodegradable material that enhances bone formation. In the other hand, bone morphogenetic protein-2 (BMP-2) is a well-known osteoinductive factor. In this work we coated porous β-tricalcium phosphate (β-TCP) scaffolds with recombinant human BMP-2 (rhBMP-2) carrier chitosan films and studied how they could modify the ceramic physicochemical properties, cellular response, and in vivobone generation. Initial β-TCP disks with an average diameter of 5.78 mm, 2.9 mm thickness, and 53 porosity were coated with a chitosan film. These coating properties were studied by X-ray diffraction, Fourier transform-infrared analysis, transmission electron microscopy, scanning electron microscopy, and energy dispersive X-ray analysis (EDX). Treatment modified the scaffold porous distribution and increased the average hardness. The biocompatibility did not seem to be altered. In addition, adhered C2C12 cells expressed alkaline phosphatase activity, related to cell differentiation toward osteogenic lineage, due to the incorporation of rhBMP-2. On the other hand, in vivoobservations showed new bone formation 3 weeks after surgery, a much shorter time than control β-TCP ceramics. These results suggest that developed coating improved porous β-TCP scaffold for bone tissue applications and added osteoinductive properties.

ABSTRACTIsolation of Mycobacterium aviumcomplex (MAC) organisms from clinical samples may occur in patients without clinical disease, making the interpretation of results difficult. The clinical relevance of MAC isolates from different types of clinical samples (n= 47) from 39 patients in different sections of a hospital was assessed by comparison with environmental isolates (n= 17) from the hospital. Various methods for identification and typing (commercial probes, phenotypic characteristics, PCR for detection of IS1245and IS901, sequencing of the hsp65gene, and pulsed-field gel electrophoresis) were evaluated. The same strain was found in all the environmental isolates, 21 out of 23 (91.3%) of the isolates cultured from urine samples, and 5 out of 19 (26.3%) isolates from respiratory specimens. This strain did not cause disease in the patients. Testing best characterized the strain as M. aviumsubsp. hominissuis, with the unusual feature that 81.4% of these isolates lacked the IS1245element. Contamination of certain clinical samples with an environmental strain was the most likely event; therefore, characterization of the environmental mycobacteria present in health care facilities should be performed to discard false-positive isolations in nonsterile samples, mainly urine samples. Molecular techniques applied in this study demonstrated their usefulness for this purpose.

The analysis of the gyrA and gyrB genes of a panel of Mycobacterium avium subsp. paratuberculosis isolates from types I, II, and III detected type-specific single nucleotide polymorphisms. Based on these results, we developed a PCR and restriction enzyme analysis to discriminate type I and III isolates. The application of this technique would be the unique strategy to characterize these strains when there is not enough bacterial growth to perform pulsed-field gel electrophoresis and IS900 restriction fragment length polymorphism.

ABSTRACTThe analysis of the gyrAand gyrBgenes of a panel of Mycobacterium aviumsubsp. paratuberculosisisolates from types I, II, and III detected type-specific single nucleotide polymorphisms. Based on these results, we developed a PCR and restriction enzyme analysis to discriminate type I and III isolates. The application of this technique would be the unique strategy to characterize these strains when there is not enough bacterial growth to perform pulsed-field gel electrophoresis and IS900restriction fragment length polymorphism.

ABSTRACTPCR-based characterization techniques have been adopted in most laboratories for Mycobacterium bovistyping. We report a molecular characterization of human multidrug-resistant M. bovisisolates and three bovine isolates that share the spoligotyping profile. The analysis of the direct repeat region showed that both groups differed in the presence of spacers not included in the current membrane. They were also distinguished by two out of the nine mycobacterial interspersed repetitive unit variable-number tandem repeat loci tested, indicating that the human infection was not acquired from the cattle from which isolates were obtained. These results highlight that a combination of techniques is required for appropriate discrimination, even for those spoligotypes that have a low frequency.

The intradermal tuberculin (IDTB) test and the interferon-gamma (IFN-γ) assay are used worldwide for detection of bovine tuberculosis in cattle, but little is known about the effect of co-infecting agents on the performance of these diagnostic tests. This report describes a field trial conducted in a cattle herd with dual infection (bovine tuberculosis and paratuberculosis) during 3.5 years. It has been based on a strategic approach encompassing serial parallel testing (comparative IDTB test, the IFN-γ assay and serology of paratuberculosis) that was repeated 8 times over the period, and segregation of animals into two herds. The IDTB test detected 65.2% and the IFN-γ test detected 69.6% of the Mycobacterium bovis culture-positive cattle. However, the IDTB test performed better during the first part of the trial, while the IFN-γ test was the only method that detected infected animals during the following three samplings. The number of false positive reactors with the IDTB and/or the IFN-γ tests was remarkably high compared to other reports, and could be caused by cross-reactivity with M. avium subsp. paratuberculosis. Also, the M. bovis isolates from cattle and wildlife from the same property were characterised using molecular techniques to disclose an epidemiological link. The IDTB test may not be appropriate to eradicate bovine tuberculosis in herds with dual mycobacterial infections. This report highlights the need to use several diagnostic techniques for the accurate detection of M. bovis infected animals in these herds.

ABSTRACTMycobacterium caprae, a recently defined member of the Mycobacterium tuberculosiscomplex, causes tuberculosis among animals and, to a limited extent, in humans in several European countries. To characterize M. capraein comparison with other Mycobacterium tuberculosiscomplex members and to evaluate genotyping methods for this species, we analyzed 232 M. capraeisolates by mycobacterial interspersed repetitive unit (MIRU) genotyping and by spoligotyping. The isolates originated from 128 distinct epidemiological settings in 10 countries, spanning a period of 25 years. We found 78 different MIRU patterns (53 unique types and 25 clusters with group sizes from 2 to 9) but only 17 spoligotypes, giving Hunter-Gaston discriminatory indices of 0.941 (MIRU typing) and 0.665 (spoligotyping). For a subset of 103 M. capraeisolates derived from outbreaks or endemic foci, MIRU genotyping and IS6110restriction fragment length polymorphism were compared and shown to provide similar results. MIRU loci 4, 26, and 31 were most discriminant in M. caprae, followed by loci 10 and 16, a combination which is different than those reported to discriminate M. bovisbest. M. capraeMIRU patterns together with published data were used for phylogenetic inference analysis employing the neighbor-joining method. M. capraeisolates were grouped together, closely related to the branches of classical M. bovis, M. pinnipedii, M. microti, and ancestral M. tuberculosis, but apart from modern M. tuberculosis.The analysis did not reflect geographic patterns indicative of origin or spread of M. caprae.Altogether, our data confirm M. capraeas a distinct phylogenetic lineage within the Mycobacterium tuberculosiscomplex.

ABSTRACTSpoligotyping is a major tool for molecular typing of Mycobacterium tuberculosiscomplex organisms. For epidemiological purposes, strains are considered clonal only when their spoligotyping patterns are identical. We report a change in the spoligotyping profiles of truly isogenic strains (a clinical isolate and a subculture derived in the laboratory) caused by deletion of a direct variable repeat. Without the information about the relationship between them, a link between these strains would have gone unnoticed. Evolutionary events should be taken into account in the interpretation of spoligotyping results and in the design of databases.

ABSTRACTMycobacterium bovisinfection in wildlife and feral species is a potential source of infection for livestock and a threat to protected and endangered species. The aim of this study was to identify Spanish wild animal species infected with M. bovisthrough bacteriological culture and spacer oligonucleotide typing (spoligotyping) of isolates for epidemiological purposes. This study included samples from red deer (Cervus elaphus), fallow deer (Dama dama), wild boar (Sus scrofa), Iberian lynx (Lynx pardina), hare (Lepus europaeus), and cattle (Bos taurus). They were collected in several geographical areas that were selected for their unique ecological value and/or known relationships between wildlife and livestock. In the areas included in this survey, M. bovisstrains with the same spoligotyping pattern were found infecting several wild species and livestock, which indicates an epidemiological link. A locally predominant spoligotype was found in these areas. Better understanding of the transmission and distribution of disease in these populations will permit more precise targeting of control measures.

Samples from four dogs and four cats suspected of having tuberculosis were processed for histopathology, bacterial culture and polymerase chain reaction (PCR). A simple, rapid method for the extraction of DNA from tissue samples was used in two PCR assays designed to confirm the diagnosis of tuberculosis. The PCR assays detected all the culture‐positive samples from these animals and no false positive results were obtained. The PCR technique was successful for the direct detection of organisms from the Mycobacterium tuberculosiscomplex and reduced the time needed for a diagnosis to two days.

One of the components of the quality of care is the 'goodness' of interpersonal relationships. This 'goodness' is a determinant of patient satisfaction, and it is in turn determined by the health care providers' satisfaction. It is therefore important to study both types of satisfaction and their interrelation. This paper focuses on health care providers and reports preliminary results on their job satisfaction. Pilot research on health-care providers was conducted on a representative sample of professionals from six general hospitals of Alicante province (Spain). Age and job appeared as the best predictors of job satisfaction; a permanent job was the best predictor of work overload and to be male was the best predictor of job-related tension. Females reported more job satisfaction and less job-related burnout than males; casual workers reported more satisfaction and less tension, less burnout level, less perceived responsibility and less work overload than permanent workers. Finally, physicians appeared more tense and reported more perceived responsibility than other groups.

ABSTRACTDNA fingerprinting techniques were used to type 273 isolates ofMycobacterium bovisfrom Australia, Canada, the Republic of Ireland, and Iran. The results of restriction fragment length polymorphism (RFLP) analysis with DNA probes from IS6110, the direct repeat (DR), and the polymorphic GC-rich sequence (PGRS) were compared with those of a new PCR-based method called spacer oligonucleotide typing (spoligotyping) developed for the rapid typing of Mycobacterium tuberculosis(J. Kamerbeek et al., J. Clin. Microbiol. 35:907–914, 1997). Eighty-five percent of the isolates harbored a single copy of IS6110, and 81.5% of these carried IS6110on the characteristic 1.9-kb restriction fragment. RFLP analysis with IS6110identified 23 different types, RFLP analysis with the DR probe identified 35 types, RFLP analysis with the PGRS probe identified 77 types, and the spoligotyping method identified 35 types. By combining all results, 99 different strains could be identified. Isolate clusters were frequently associated within herds or were found between herds when epidemiological evidence confirmed animal movements. RFLP analysis with IS6110was sufficiently sensitive for the typing of isolates with more than three copies of IS6110, but RFLP analysis with the PGRS probe was the most sensitive typing technique for strains with only a single copy of IS6110. Spoligotyping may have advantages for the rapid typing of M. bovis, but it needs to be made more sensitive.

The spacer oligonucleotide typing (spoligotyping) method was evaluated for its ability to differentiate Mycobacterium bovis strains. This method detects the presence or absence of spacers of the direct repeat locus of the M. bovis genome. The spacers in the direct repeat locus are amplified by PCR and are detected by hybridization of the biotin-labelled PCR product with a membrane containing oligonucleotides derived from spacer sequences that have previously been bound to a membrane. One hundred eighty-two M. bovis isolates from domestic animals (cattle, goat, sheep, and cats) and wild animals (deer and wild boar) were spoligotyped, and the results were compared with those obtained by IS6110 restriction fragment length polymorphism analysis. Two rather homogeneous clusters of isolates containing 20 and 4 types, respectively, were identified by spoligotyping. The first cluster included isolates from cattle, cats, and feral animals. By spoligotyping, isolates from the Spanish wild boar and deer had the same pattern as some bovine isolates, suggesting transmission between these animals and cattle and highlighting the importance of the study of these reservoirs. The second cluster included all the caprine and ovine isolates. Within each cluster, the patterns of the different strains differed only slightly, suggesting that the spoligotypes may be characteristic of strains from particular animal species. Spoligotyping proved to be useful for studying the epidemiology of bovine M. bovis isolates, especially of those isolates containing only a single copy of IS6110. In view of our results, we suggest fingerprinting all M. bovis strains by the spoligotyping method initially and then by IS6110 restriction fragment length polymorphism typing of the strains belonging to the most common spoligotypes.

Current methods for detection and identification of mycobacteria from clinical specimens, with particular interest in M avium and other nontuberculous mycobacteria, are reviewed. Traditional microbiological procedures, including microscopy, comparison of the different pretreatment methods for decontamination and culture media, and DNA probes and high-performance liquid chromatography techniques are evaluated. New methods include the polymerase chain reaction, a new tool available for quick identification of mycobacteria. The protocol used for detection of the etiologic agent of six cases of bird mycobacteriosis is briefly described. Authors recommend this technique as a rapid and reliable diagnosic method.

Mycobacterium bovis is a slowly growing microorganism, and confirmation of the diagnosis by conventional culture is a lengthy process. A simple, rapid method for the extraction of DNA from bovine tissue samples was developed and used in a PCR designed for the diagnosis of tuberculosis. Tissues from 81 cattle from tuberculosis-infected herds (group 1) and 19 cattle from tuberculosis-free herds (group 2) were tested in this PCR, and the results were compared with those of conventional culture. The PCR assay detected 71.4% of the culture-positive animals from group 1. Tissue from all animals in group 2 were negative in the PCR assay and by culture. The described method could be used as a rapid screening technique which would be complementary to culture of tissue specimens for the routine diagnosis of bovine tuberculosis. The PCR technique is much faster than culture and reduces the time for diagnosis from several months to 2 days. It also provides for the detection of M. bovis when rapidly growing Mycobacterium spp. are present in the sample and may be able to detect the presence of M. bovis in samples even when organisms have become nonviable.

ObjectiveTo evaluate the usefulness of the gamma‐interferon assay in the diagnosis of caprine tuberculosis in comparison with a single intradermal tuberculin test, and to obtain a group of animals free from this infection in a herd with a high prevalence.

It is important to correctly identify species within the Mycobacterium tuberculosis complex because of the zoonotic implications of bovine tuberculosis, especially in developing countries. We assessed the use of various genetic markers for species-specific identification of mycobacteria from the M. tuberculosis complex. A multiplex PCR designed for detection of the mtp40 and IS1081 elements was optimized and evaluated in 339 mycobacterial strains from different animal and geographic origins. The host range of the IS6110, MPB70, and 16S rRNA genes was also studied by PCR in all the strains. Finally, the usefulness of the genetic markers was compared by an immunoperoxidase test for specific identification of Mycobacterium bovis strains. The mtp40 sequence was detected in 87 of the 91 strains of M. tuberculosis and in 9 of the 11 Mycobacterium africanum strains but not in any of the M. bovis or Mycobacterium microti strains, indicating that the mtp40 element was also found in all of the M. tuberculosis complex strains isolated from seals. This organism is considered to be a true seal pathogen, but its origin is essentially unknown. The finding of the mtp40 element in the strains from seals suggests a closer relationship of these strains with a human origin than to an animal origin. The mtp40 element was not found in any other mycobacterial species included in the study. As a result of this study, we suggest that biochemical tests or alternate genetic markers are still needed to differentiate M. tuberculosis from M. africanum when these species coexist as causative agents of tuberculosis. The immunoperoxidase test worked well for the identification of M. bovis strains. We also report, for the first time, PCR amplification of the repetitive element IS6110 in an isolate of Mycobacterium ulcerans and an isolate of Mycobacterium gilvum, which emphasizes the need for further investigation of the host range of this sequence.

When studying mutations in DNA samples, determining whether novel sequence changes are somatic mutations or germline polymorphisms can be difficult. Here we describe a novel and very simple approach for identification of somatic mutations and loss of heterozygosity (LoH) events in DNA samples where no matched tissue sample is available. Our method makes use of heterozygous polymorphisms that are located near the putative mutation to trace both germinal alleles.

Osteoblasts orchestrate bone formation through the secretion of type I collagen and other constituents of the matrix on which hydroxyapatite crystals mineralize. Here, we show that TENT5A, whose mutations were found in congenital bone disease osteogenesis imperfecta patients, is a cytoplasmic poly(A) polymerase playing a crucial role in regulating bone mineralization. Direct RNA sequencing revealed that TENT5A is induced during osteoblast differentiation and polyadenylates mRNAs encoding Col1α1, Col1α2, and other secreted proteins involved in osteogenesis, increasing their expression. We postulate that TENT5A, possibly together with its paralog TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization. Importantly, the Tent5aknockout (KO) mouse line displays bone fragility and skeletal hypomineralization phenotype resulting from quantitative and qualitative collagen defects. Thus, we report a biologically relevant posttranscriptional regulator of collagen production and, more generally, bone formation.

Estudiar la distribución espacio-temporal de los casos de enfermedad neumocócica invasora (ENI) por serotipos resistentes a eritromicina y su relación con el consumo comunitario de macrólidos y la cobertura vacunal infantil.

Selection during varietal improvement has been shown to reduce genetic diversity in several different crop species. A reduction in genetic diversity can be detrimental to future breeding efforts and increase susceptibility to biotic stresses. The purpose of this study was to analyze changes in levels of allelic diversity at the gene and population levels in 40 zoysiagrass (Zoysiaspp. Willd.) cultivars released between 1910 and 2016 using simple sequence repeat (SSR) markers. Fifty‐six SSR primer pairs were used to determine whether allelic diversity has changed among cultivars released over a century of modern plant breeding. While no significant differences were observed for total numbers of alleles or genetic similarity values between the initial and most recent time periods, our results demonstrate that genetic diversity among zoysiagrass cultivars has fluctuated over time. Allelic diversity declined during the 1990s and then recovered during 2000s. STRUCTURE analysis revealed five subpopulations with varying levels of admixture and that, for the most part, good representation of these subpopulations has been maintained over the different decades of release. These findings illustrate that plant breeding has not resulted in a substantial decline in zoysiagrass genetic diversity, but also that the potential exists for increasing diversity through future germplasm collections and the inclusion of less‐used Zoysiaspecies to create new combinations of alleles.

Review: 189 refs.