Association between the skin microbiome and MHC class II diversity in an amphibian.

Microbiomes play an important role in determining the ecology and behaviour of their hosts. However, questions remain pertaining to how host genetics shape microbiomes, and how microbiome composition influences host fitness. We explored the effects of geography, evolutionary history and host genetics on the skin microbiome diversity and structure in a widespread amphibian. More specifically, we examined the association between bacterial diversity and composition and the major histocompatibility complex class II exon 2 diversity in 12 moor frog (Rana arvalis) populations belonging to two geographical clusters that show signatures of past and ongoing differential selection. We found that while bacterial alpha diversity did not differ between the two clusters, MHC alleles/supertypes and genetic diversity varied considerably depending on geography and evolutionary history. Bacterial alpha diversity was positively correlated with expected MHC heterozygosity and negatively with MHC nucleotide diversity. Furthermore, bacterial community composition showed significant variation between the two geographical clusters and between specific MHC alleles/supertypes. Our findings emphasize the importance of historical demographic events on hologenomic variation and provide new insights into how immunogenetic host variability and microbial diversity may jointly influence host fitness with consequences for disease susceptibility and population persistence.

Environmental and economic impacts of combining backfill materials for novel circular narrow trenches.

Over the last few years, several policies and new technological solutions have targeted the construction sector with the aim of reducing the sector's impacts on the environment. Among the different technological advances proposed, the reuse of materials in construction has been reported as a promising solution for an increase in sustainability and circularity. In particular, a type of cities' undergrounds assets for which materials' reuse is being explored are trenches for protecting services (i.e., water and gas transport pipelines, and optic fibre and other telecommunications services). Nonetheless, the economic and environmental benefits and impact of this type of system is still insufficiently quantified. In this research study, the economic and environmental impacts of four scenarios of trenches were assessed by using Life Cycle Costing (LCC) and Life Cycle Assessment (LCA). The four alternatives analysed consisted of: (1) the classical solution; (2) the classical solution with the reuse of soil; (3) the control low-strength material, and (4) the eco-trench. The results allowed concluding that in the eco-trench system, for which all material is reused, the environmental and economic impacts could be reduced by more than 80% and 50%, respectively. A parametric study for which the dimensions of the trenches were varied, permitted to reinforce these results and to quantify the impact's change along with the width and depth of the trench. Overall, this study provides a comprehensive view of the high-impact potential of reusing material for the construction of trenches in cities. The outcomes allow also remarking that the eco-trench system could be an attractive and advantageous solution for urban infrastructure stakeholders, both from an economic and environmental perspective.

Optimization and shelf life of a low-lactose yogurt with Lactobacillus rhamnosus HN001.

Lactose intolerance results in gastrointestinal discomfort and the malabsorption of certain nutrients, such as calcium. The replacement of milk with low-lactose and probiotic-enriched dairy products is an effective strategy of mitigating the symptoms of lactose intolerance. Lactobacillus rhamnosus HN001 (HN001) is a safe, immunity-stimulating probiotic. We have developed a process to increase the hydrolysis of lactose and HN001 growth in yogurt versus ß-galactosidase (ßG) concentration and enzymatic hydrolysis time (EHT) before bacterial fermentation. The objective of this study was to optimize the conditions by which yogurt is processed as a function of ßG and EHT using a multifactorial design, with lactose content, HN001 growth, process time, and sensory quality as dependent variables. Further, the shelf life of the optimized yogurt was evaluated. In the optimization study, polynomials explained the dependent variables. Based on Pearson correlation coefficients, HN001 growth correlated positively with the hydrolysis of lactose. However, low lactose content and high HN001 count increased the fermentation time and lowered the sensory quality. The optimized conditions-using polynomials to obtain yogurt with >1 × 10(7) cfu of HN001/mL, <10 g of lactose/L, and a minimum overall sensory quality of 7 on the Karlsruhe scale-yielded a theoretical value of 910 neutral lactose units/kg for ßG and 2.3h for EHT, which were validated in an industrial-scale assay. Based on a shelf-life study at 3 temperatures, the hydrolysis of lactose and the growth of HN001 continue during storage. Arrhenius equations were developed for the variables in the shelf-life study. Our results demonstrate that it is feasible to develop a low-lactose yogurt to which HN001 has been added for lactose-intolerant persons who wish to strengthen their immune system.

Transdermal delivery of alprazolam from a monolithic patch: formulation based on in vitro characterization.

Alprazolam, a benzodiazepine widely used for the treatment of psychiatric disorders, has been aimed to be formulated in a transdermal delivery system (TDS) prototype. A series of TDS prototypes dosed in all cases at 0.35 mg·cm(-2) of alprazolam were prepared as a monolithic drug in adhesive matrix using acrylic pressure-sensitive adhesives (PSA) of acrylate vinyl acetate (Duro-tack(®)). The effects of several permeation enhancers as azone, transcutol, propylene glycol, dodecyl alcohol, decyl alcohol, diethanolamine, N-methyl pyrrolidone and lauric acid were studied. Prototypes have been characterized based on adhesion parameters (peel adhesion and shear adhesion), in vitro human skin permeation and in vitro drug release according to European Pharmacopoeia for the selected prototype. Best results show that a combination of permeation enhancers from different chemical groups is able to provide almost a 33 fold increase in the transdermal alprazolam flux of an aqueous saturated dispersion (from 0.054 ± 0.019 to 1.76 ± 0.21 µg h.cm(-2)). Based on these in vitro flux data, a predictive simulation of the achievable plasmatic levels was performed assuming a constant systemic infusion of drug. In summary, it is possible to obtain a prototype of a TDS of alprazolam with adequate adhesive properties (peel adhesion and shear adhesion) and able to predict sustained therapeutic plasmatic levels.

Key parameters for the development of a NIR microscopic method for the quantification of processed by-products of animal origin in compound feedingstuffs.

The aim of this work is to show new advances in the analytical methods developed in the frame of the ban of processed animal by-products in compound feed that is currently applied within the European Union. With this aim, studies to develop a quantitative near infrared microscopy (NIRM) approach have been undertaken in order to fulfil future requirements of European legislation like the introduction of tolerance levels that would require for official control purposes the availability of specific quantitative methods. The capabilities of the NIRM method have been improved; no sample preparation is required and the acquisition parameters are optimised. Both the gross and the fine fractions of the samples are considered; the reflexion mode was used to analyse the gross raw fraction and the transmission mode was chosen to analyse the fine raw fraction. Parameters for reflexion analyses were already fixed in our previous studies while those of transmission mode have been determined in the present study. Because particles are too small, it is difficult to mark them; spectra were collected using the mapping technique. Quantitative analyses have been carried out for different percentages of adulteration (0.5, 1, 2 and 5%). Results were depending on the particle size distribution of the feed and of the fish meal which led to experimental values of adulteration varying between 0.13-0.92%, 0.93-3.7%, 2.42-5.83% and 1.95-9.39% for theoretical percentages of adulteration equal to 0.5, 1, 2 and 5%, respectively. The established protocol with the key parameters proposed has to be considered for the development of an accurate method of quantification.

[Calcaneal fractures: controversies and consensus]. / Fracturas de calcáneo: controversias y consensos.

Calcaneal fractures are the most common tarsal fractures and their treatment is still debated today. We intend in this update to highlight the points of controversy and clarify the consensus, especially in the treatment of intra-articular fractures, as well as to describe the management of major complications.

Las fracturas de calcáneo son las más frecuentes del tarso y su tratamiento sigue siendo hoy en día objeto de debate. Pretendemos en esta actualización destacar los puntos de controversia, así como clarificar los consensos, especialmente en el tratamiento de las fracturas intraarticulares, así como describir el manejo de las principales complicaciones.

Optimisation of asymmetric flow field-flow fractionation for the characterisation of nanoparticles in coated polydisperse TiO2 with applications in food and feed.

Titanium dioxide (TiO2) has various applications in consumer products and is also used as an additive in food and feeding stuffs. For the characterisation of this product, including the determination of nanoparticles, there is a strong need for the availability of corresponding methods of analysis. This paper presents an optimisation process for the characterisation of polydisperse-coated TiO2 nanoparticles. As a first step, probe ultrasonication was optimised using a central composite design in which the amplitude and time were the selected variables to disperse, i.e., to break up agglomerates and/or aggregates of the material. The results showed that high amplitudes (60%) favoured a better dispersion and time was fixed in mid-values (5 min). In a next step, key factors of asymmetric flow field-flow fraction (AF4), namely cross-flow (CF), detector flow (DF), exponential decay of the cross-flow (CFexp) and focus time (Ft), were studied through experimental design. Firstly, a full-factorial design was employed to establish the statistically significant factors (p < 0.05). Then, the information obtained from the full-factorial design was utilised by applying a central composite design to obtain the following optimum conditions of the system: CF, 1.6 ml min-1; DF, 0.4 ml min-1; Ft, 5 min; and CFexp, 0.6. Once the optimum conditions were obtained, the stability of the dispersed sample was measured for 24 h by analysing 10 replicates with AF4 in order to assess the performance of the optimised dispersion protocol. Finally, the recovery of the optimised method, particle shape and particle size distribution were estimated.

Intraoperative neurophysiological monitoring in peripheral nerve surgery: Technical description and experience in a centre.

INTRODUCTION: Intraoperative neurophysiological monitoring has experienced a spectacular development in the past 20 years, particularly in the fields of neurosurgery and spine surgery. it has become a useful, almost indispensable, tool in preventing nerve damage during surgery. The aim of this article is to describe the intraoperative technique and analyze its results in the field of peripheral nerve surgery. OBJECTIVE: To describe the usefulness of a technique in peripheral nerve surgery, the technique used and the experience in a centre. PATIENTS AND METHODS: A retrospective study was conducted on 30 cases of peripheral nerve surgery performed in this centre from 2009 to 2013, using the intraoperative monitoring technique. RESULTS: Of the total of 13 peripheral nerve tumors recorded, there were 11 excellent results and 2 good results, one temporary hypoesthesia and one with almost complete sensory, except for motor, recovery. Traumatic injury was recorded in 17 cases, of which 6 required performing a graft, and the remaining 11 cases only neurolysis was performed, with complete motor and sensory recovery. CONCLUSIONS: Intraoperative neurophysiological monitoring is a useful tool in the secondary surgery of peripheral nerve injury and the intraneural tumor pathology.

Contamination with Mycoplasma spp. induces interleukin-13 expression by human skin fibroblasts in culture.

The mycoplasmas comprise a discrete group of microorganisms that are known to exert a range of effects upon cells derived from the immune system. Some of these interactions turn out to be immunomodulatory, such as polyclonal stimulation of T and B cells or enhancement of the cytolytic potential of macrophages, NK cells and T lymphocytes. Immunologically committed cells, when infected with mycoplasmas, can also increase the production of cytokines (IL-1, IL-2, IL-4 and IL-6), interferon (IFN) gamma, tumor necrosis factor-alpha (TNF-alpha) and colony-stimulating factors (particularly GM-CSF). Moreover, mycoplasmas are potent inductors of cytokine secretion by fibroblasts in culture. Since growth factors are determinants for the activation and proliferation of immunocompetent cells in vitro, we decided to investigate if these effects are concordant with the finding of mycoplasma contamination. In order to address this question, we compared the pattern of lymphokine secretion by normal-derived human fibroblasts in culture with and without Mycoplasma spp. contamination. We found those human fibroblasts that have been contaminated with mycoplasma show production of IL-13 at the transcriptional level. This effect coincides with discrete morphological changes as compared to uncontaminated human fibroblasts. This is the first report to acknowledge that mycoplasma contamination can induce mRNA expression for IL-13 in cultured human fibroblasts.

Proliferation of peripheral blood mononuclear cells from rheumatoid arthritis patients to mycobacterial antigens.

Proliferation of rheumatoid and control peripheral blood mononuclear cells (PBMC) to an antigenic acetone-precipitable extract from mycobacterium tuberculosis (MTa) was investigated. Cells were also stimulated with the recall antigen tuberculin PPD (purified protein derivative) and the mitogen OKT3. Controls had a significantly higher response to both MTa and tuberculin PPD than RA patients. However, lymphocytes from patients who had had their disease for 3-10 years proliferated more vigorously to MTa than did PBMD from patients with longer disease duration. There was no difference in the proliferation to OKT3 between patients and controls. We were not able to confirm a previously found correlation between the HLA-DR4 phenotype and lymphocyte transformation to MTa.

[Hodgkin's disease complicated with autoimmune thrombocytopenic purpura and a nephrotic syndrome due to deposition of circulating immunocomplexes (author's transl)]. / Enfermedad de Hodgkin complicada con púrpura trombopénica autoimmune y síndrome nefrótico por depósito de immunocomplejos circulantes.

This report deals with a case of Hodgkin's disease presenting during its evolution with autoimmune thrombocytopenic purpura and a nephrotic syndrome with extramembranous glomerulonephritis. The histopathological study of renal tissue demonstrated the presence of IgG and the C3 fraction of complement in the extramembranous deposits. The same type of immunoglobulins and C3 existed in the serum as circulating immunocomplexes, suggesting that the glomerulonephritis was due to the deposition of such immunocomplexes in the glomerular membrane. A review of the literature discloses 32 cases of autoimmune thrombocytopenic purpura associated to Hodgkin's lymphoma, and 47 cases of Hodgkin's lymphoma associated to the nephrotic syndrome. However, the association of both complications in the same patient with lymphoma has been heretofore unreported. The possible etiopathogenic mechanisms of these autoimmune complications and its pathological features are discussed along with the response to treatment. Generally the activity of such disorders parallels that of the lymphoma, thus making them potentially useful parameters to appraise lymphomatous activity.

[Heat-shock proteins. Their clinical importance]. / Las proteínas de choque térmico. Su importancia clínica.

Heat-shock proteins (HSP) represents a protein superfamily comprising distinct molecular weights which are synthesized under conditions of cellular stress. The HSP constitute a phylogenetically-conserved response of cell preservation. Their functions are related to mechanisms such as thermotolerance and transport and scavenging of intracellular peptides. Some recent experimental evidence suggests that HSP participate directly in immune responses perhaps facilitating the recognition of exogenous antigens, and its levels are raised in inflammatory situations. Their pathogenic role in the chronic inflammation is still unclear, but the HSP could intervene as immunodominant antigens, mimicking structural proteins or evading the so-called immunological memory. In this review, we update the information concerning the relevance of HSP in medicine and discuss its potential role in autoimmune mechanisms.

Spontaneous cytokine gene expression by cultured skin fibroblasts of systemic sclerosis. Correlation with collagen synthesis.

OBJECTIVE: To investigate the spontaneous cytokine gene expression in fibroblasts from patients with diffuse cutaneous systemic sclerosis. Their pattern of expression was correlated with the production of collagen. METHODS: Fibroblasts were obtained from skin biopsies of nine patients diagnosed with systemic sclerosis (mean 16 +/- 8.7 years of disease duration) and ten control individuals. The cytokine gene expression was detected by coupled reverse transcriptase polymerase chain reaction for interleukins 1 beta, 6, 8, tumour necrosis factor-alpha, and transforming growth factor beta. In addition, collagen synthesis was measured by [14C]-proline uptake in fibroblast cultures. RESULTS: All fibroblast samples from patients expressed the interleukin-6 gene (p = 0.04 compared with controls). Eight of the nine patients expressed interleukin-8 (p = 0.02 compared with controls). Four of them expressed also transforming growth factor beta and two more weakly expressed the tumour necrosis factor-alpha gene. Only one patient showed transcription for the interleukin-1 beta gene. In accordance with such immune activation, collagen synthesis was higher in fibroblasts from patients with systemic sclerosis (p = 0.028) as compared with normal controls. Indeed, a positive correlation was found between the expression of IL-6 gene and collagen production (rs = 1). CONCLUSION: The constitutive expression of IL-6 and IL-8 genes by fibroblasts may play an important role in the perpetuation of local immune dysregulation, thus leading to a permanent fibroblast activation in patients with systemic sclerosis.

Fracturas de calcáneo: controversias y consensos / Calcaneal fractures: controversies and consensus

Resumen: Las fracturas de calcáneo son las más frecuentes del tarso y su tratamiento sigue siendo hoy en día objeto de debate. Pretendemos en esta actualización destacar los puntos de controversia, así como clarificar los consensos, especialmente en el tratamiento de las fracturas intraarticulares, así como describir el manejo de las principales complicaciones.

Abstract: Calcaneal fractures are the most common tarsal fractures and their treatment is still debated today. We intend in this update to highlight the points of controversy and clarify the consensus, especially in the treatment of intra-articular fractures, as well as to describe the management of major complications.

Validation of a near infrared microscopy method for the detection of animal products in feedingstuffs: results of a collaborative study.

The performance characteristics of a near infrared microscopy (NIRM) method, when applied to the detection of animal products in feedingstuffs, were determined via a collaborative study. The method delivers qualitative results in terms of the presence or absence of animal particles in feed and differentiates animal from vegetable feed ingredients on the basis of the evaluation of near infrared spectra obtained from individual particles present in the sample. The specificity ranged from 86% to 100%. The limit of detection obtained on the analysis of the sediment fraction, prepared as for the European official method, was 0.1% processed animal proteins (PAPs) in feed, since all laboratories correctly identified the positive samples. This limit has to be increased up to 2% for the analysis of samples which are not sedimented. The required sensitivity for the official control is therefore achieved in the analysis of the sediment fraction of the samples where the method can be applied for the detection of the presence of animal meal. Criteria for the classification of samples, when fewer than five spectra are found, as being of animal origin needs to be set up in order to harmonise the approach taken by the laboratories when applying NIRM for the detection of the presence of animal meal in feed.

Genetic variability in geographic populations of the natterjack toad (Bufo calamita).

Across altitudinal and latitudinal gradients, the proportion of suitable habitats varies, influencing the individual dispersal that ultimately can produce differentiation among populations. The natterjack toad (Bufo calamita) is distributed across a wide geographic range that qualifies the species as interesting for a geographic analysis of its genetic variability. Five populations of B. calamita in the Sierra de Gredos (Spain) were studied in an altitudinal gradient ranging from 750 to 2270 m using microsatellite markers. In addition, we analyzed the latitudinal genetic variation in B. calamita within a global European distribution using genetic diversity parameters (mean number of alleles per locus [M(a)] and expected heterozygosity [H(E)]) obtained from our results and those published in the literature. The low level of genetic differentiation found between populations of B. calamita (F(st) ranging from 0.0115 to 0.1018) and the decreases in genetic diversity with altitude (M(a) from 13.6 to 8.3, H(E) from 0.82 to 0.74) can be interpreted by the combined effects of discontinuous habitat, produced mainly by the high slopes barriers and geographic distance. In the latitudinal gradient, genetic diversity decreases from south to north as a consequence of the colonization of the species from the Pleistocene refugium. We conclude that the genetic variability in B. calamita along its wide altitudinal and latitudinal geographic distribution mainly reflects the colonization history of the species after the last glacial period.

Validation of an analytical method for the determination of glyceroltriheptanoate (GTH) in processed animal by-products: results of a collaborative study.

The performance characteristics of a method for the determination of the marker substance glycerol triheptanoate (GTH) in processed animal by-products (ABPs) based on gas chromatography (GC) coupled to mass spectrometry (MS) were determined via a collaborative study. Within the European Union, GTH needs to be added to the portion of processed ABPs that must not enter the feed and food chain (Categories 1 and 2) at a minimum concentration of 250 mg kg(-1) related to the fat fraction of the test samples analysed. The test materials included in the validation study consisted of three meat and bone meal (MBM) and three fat samples that contained GTH at different concentrations ranging from 61 to 455 mg kg(-1). The relative standard deviation of repeatability (RSD(r)) varied from 3.4 to 7.8% and the relative standard deviation of reproducibility (RSD(R)) varied from 9.0 to 16.5%, corresponding to HORRAT values that were, in all cases, equal or below the critical value of 2.0. The estimated trueness expressed in terms of average concentration compared to the target concentrations of GTH in all test materials varied from 95 to 107%, confirming acceptable values for the trueness of the method. Based on the acceptable values for precision and trueness, the method is fit for the intended purpose and can be used for official control purposes to determine GTH in processed animal by-products from Category 1 and Category 2.