Application of ISSR-PCR for rapid strain typing of Debaryomyces hansenii isolated from dry-cured Iberian ham.

Yeast populations of dry-cured Iberian ham isolated from seven industries in the province of Badajoz were characterized by ISSR-PCR using the (CAG)4 primer and PCR-RFLP of the ITS1-5.8S rRNA-ITS2 fragment, and identified by DNA sequencing. A total of 242 isolates were analyzed, indicating the primary species present was Debaryomyces hansenii at 80.9% of the isolates followed by Candida zeylanoides at 10.3% of the isolates. The remainders of isolates were identified as Yamadazyma triangularis, Sporobolomyces roseus, Meyerozyma guilliermondii, Rhodotorula slooffiae, and Cryptococcus victoriae. The ISSR-PCR method was a fast and reliable method which was able to discriminate species at a level comparable to restriction analyses of the ITS1-5.8S rRNA-ITS2 region. This method allowed for strain typing of D. hansenii, yielding 29 different PCR patterns within 196 isolates. Moreover, ISSR-PCR using the (CAG)4 primer indicated that this technique could be a promising tool for rapid discrimination of yeast starter cultures and spoilage species in dry-cured Iberian ham.

An interspecies computational study on limb lengthening.

Distraction osteogenesis is a surgical technique that produces large volumes of new bone by gradually separating two osteotomized bone segments. A previously proposed mechanical-based model that includes the effect of pre-traction stresses (stress level in the gap tissue before each distraction step) during limb lengthening is used here. In the present work, the spatial and temporal patterns of tissue distribution during distraction osteogenesis in different species (sheep, rabbit) and in the human are compared numerically to predict experimental results. Interspecies differential characteristics such as size, distraction protocol, and rate of distraction, among others, are chosen according to experiments. Tissue distributions and reaction forces are then analysed as indicators of the healing pattern. The results obtained are in agreement with experimental findings regarding both tissue distribution and reaction forces. The ability of the model to qualitatively predict the two animal models and the human healing pattern in distraction osteogenesis indicates its potential in understanding the influence of mechanics in this complex process.

Effect of immunosuppressant blood levels on serum concentration of interleukin-17 and -23 in stable liver transplant recipients.

INTRODUCTION: T(H)17 cells have been recently described to be involved in inflammatory and immune-mediated diseases, but there is no evidence of their role in human liver transplantation. Interleukin (IL)-23 is considered an inducer cytokine, whereas IL-17 is the main cytokine released by T(H)17 cells. The aim of our study was to measure the serum levels of IL-17 and IL-23 in stable liver transplant recipients and examine the influence of immunosuppressant concentrations. MATERIALS AND METHODS: Serum levels of IL-23 and IL-17 were determined in 38 healthy subjects and 35 stable hepatic transplant recipients who were free of rejection episodes for at least 8 years. The results were analyzed according to the simultaneous blood levels of cyclosporine (n = 20) or tacrolimus (n = 15). RESULTS: No significant differences were observed in the serum levels of IL-17 and IL-23 between healthy subjects and transplanted patients. In addition, patients with low blood levels of tacrolimus (<6 ng/mL), but not cyclosporine, showed significantly lower serum levels of the 2 cytokines. CONCLUSION: These preliminary results suggested a lack of activation of the T(H)17 pathway, which was more pronounced among the patient subgroup treated with tacrolimus.

Modelling actin polymerization: the effect on confined cell migration.

The aim of this work is to model cell motility under conditions of mechanical confinement. This cell migration mode may occur in extravasation of tumour and neutrophil-like cells. Cell migration is the result of the complex action of different forces exerted by the interplay between myosin contractility forces and actin processes. Here, we propose and implement a finite element model of the confined migration of a single cell. In this model, we consider the effects of actin and myosin in cell motility. Both filament and globular actin are modelled. We model the cell considering cytoplasm and nucleus with different mechanical properties. The migration speed in the simulation is around 0.1 µm/min, which is in agreement with existing literature. From our simulation, we observe that the nucleus size has an important role in cell migration inside the channel. In the simulation the cell moves further when the nucleus is smaller. However, this speed is less sensitive to nucleus stiffness. The results show that the cell displacement is lower when the nucleus is stiffer. The degree of adhesion between the channel walls and the cell is also very important in confined migration. We observe an increment of cell velocity when the friction coefficient is higher.

T(H)17 versus Treg cells in renal transplant candidates: effect of a previous transplant.

INTRODUCTION: The T(H)1 and T(H)2 cells were described several years ago. However, this dichotomy has been disrupted by the description of other CD4(+) T cell subsets: the proinflammatory interleukin (IL)-17-producing T cells (T(H)17) and regulatory T cells (Tregs). The latter group inhibits the immune responses driven by T(H)1, T(H)2, and T(H)17 cells. IL-6 is involved in T(H)17 development, down-regulating Treg differentiation. Our hypothesis suggested that an imbalance between T(H)17 and Tregs enhances immune responses among renal transplant patients. MATERIALS AND METHODS: We studied 26 end-stage renal disease (ESRD) subjects and 10 patients awaiting a second renal transplant after previous graft dysfunction. We assessed the number of CD4(+)CD25(+)Foxp3(+) cells and serum levels of IL-17, the prototypic interleukin of T(H)17 cells. RESULTS: We observed a lower number of CD4(+)CD25(+)Foxp3(+) T cells among patients with previous graft dysfunction than those with ESRD (median 3.37 vs 8.63 cells/mm(3), P = .008). In contrast, IL-17 serum levels were augmented in graft dysfunction (median 4.45 pg/mL) compared with ESRD patients (1.39 pg/mL, P = .036), suggesting a proinflammatory state in patients awaiting a second renal transplant. CONCLUSION: The emerging alloresponse from a previous transplant favors the generation of T(H)17 instead of Treg cells. The enhanced activity of T(H)17 cells in retransplanted patients may down-regulate Treg cells, producing a proinflammatory environment that favors rejection of the next transplant.

Changes in the expression of the immunoglobulin-like transcript 3 (ILT3) and ILT4 receptors in renal allograft recipients: effect of donor and recipient aging.

INTRODUCTION: The aim of the present study was to investigate the number and phenotype of pre- and posttransplant peripheral blood dendritic cells (DCs) in kidney graft recipients to correlate with CD4(+)CD25(high) Treg and CD8(+)CD28(-) cells. Data were analyzed according to the age of the donor-recipient pairs. MATERIALS AND METHODS: A cohort of 49 cadaveric kidney transplant recipients was prospectively studied pretransplant and 6 months posttransplant by three-color flow cytometry with specific monoclonal antibodies. Patients were subgrouped according to age (elderly were considered above 60 years old and young below 55 years old) in the following donor-recipient pairs: aged/aged, young/aged, aged/young, young/young. RESULTS: At 6 months posttransplant, the proportion of cells tended to increase when the donor was young, regardless of the recipient. Importantly, there was a significant correlation between the numbers of immunoglobulin-like transcript 4(+) DCs and CD4(+)CD25(high) Treg cells before transplantation (r = .476, P = .004) and at 6 months (r = .408, P = .013). A significant association was also observed between ILT4(+) DCs and CD8(+)CD28(-) pretransplant (r = .540, P = .001) and at 12 months posttransplant (r = .609, P = .012). CONCLUSIONS: Renal grafts from young but not from aged donors seem to induce DC of a tolerogenic phenotype, both in aged and young recipients. These preliminary results suggested that donor age may have consequences in terms of tolerance induction.

Association between serum soluble CD30 and serum creatinine before and after renal transplantation.

OBJECTIVE: There is increasing evidence that circulating levels of soluble CD30 (sCD30) may represent a biomarker for outcome in kidney transplantation. The aim of this study was to measure the pre- and posttransplantation serum levels of sCD30 in cadaveric kidney transplant recipients and correlate them with serum creatinine. PATIENTS AND METHODS: Serum sCD30 was measured by a commercial enzyme-linked immunosorbent assay (ELISA) from prospective samples of 38 kidney allograft recipients serially transplanted at our center. Samples were collected at day 0 pretransplantation and at months 6, 12, 18, and 24 posttransplantation. We also studied sera from 29 patients with chronic kidney disease (CKD) at different stages of the K/DOQI guidelines, as a control group. RESULTS: Serum levels of sCD30 decreased significantly in samples posttransplantation compared with pretransplantation. The significant decrease after transplantation may be related to the improvement in renal function since we observed a significant correlation between serum levels of sCD30 and creatinine (sCr) at all times of the study. In addition, the patients with chronic renal failure showed a significant association between serum sCD30 and sCr (r = .454; P = .013). CONCLUSIONS: Our results did not suggest that the measurement of sCD30 may be used as a valuable biomarker in renal transplantation. Increased levels may be related to a decrease in its renal elimination.

Computational simulation of fracture healing: influence of interfragmentary movement on the callus growth.

Bone fractures heal through a complex process involving several cellular events. This healing process can serve to study factors that control tissue growth and differentiation from mesenchymal stem cells. The mechanical environment at the fracture site is one of the factors influencing the healing process and controls size and differentiation patterns in the newly formed tissue. Mathematical models can be useful to unravel the complex relation between mechanical environment and tissue formation. In this study, we present a mathematical model that predicts tissue growth and differentiation patterns from local mechanical signals. Our aim was to investigate whether mechanical stimuli, through their influence on stem cell proliferation and chondrocyte hypertrophy, predict characteristic features of callus size and geometry. We found that the model predicted several geometric features of fracture calluses. For instance, callus size was predicted to increase with increasing movement. Also, increases in size were predicted to occur through increase in callus diameter but not callus length. These features agree with experimental observations. In addition, spatial and temporal tissue differentiation patterns were in qualitative agreement with well-known experimental results. We therefore conclude that local mechanical signals can probably explain the shape and size of fracture calluses.

A damage model for the growth plate: application to the prediction of slipped capital epiphysis.

Despite slipped capital femoral epiphysis (SCFE) being one of the most common disorders of the adolescent hip, its early diagnosis is quite difficult. The main objective of this work is to apply an interface damage model to predict the failure of the bone-growth plate-bone interface. This model allows to evaluate the risk of development of SCFE and to investigate the range of mechanical properties of the physis that may cause slippage of the plate. This paper also studies the influence of different geometrical parameters and body weight of the patient on the development of SCFE. We have demonstrated, thanks to the proposed model, that higher physeal sloping and posterior sloping angles are associated to a higher probability of development of SCFE. In a similar way, increasing body weight results in a more probable slippage.

Computational model of mesenchymal migration in 3D under chemotaxis.

Cell chemotaxis is an important characteristic of cellular migration, which takes part in crucial aspects of life and development. In this work, we propose a novel in silico model of mesenchymal 3D migration with competing protrusions under a chemotactic gradient. Based on recent experimental observations, we identify three main stages that can regulate mesenchymal chemotaxis: chemosensing, dendritic protrusion dynamics and cell-matrix interactions. Therefore, each of these features is considered as a different module of the main regulatory computational algorithm. The numerical model was particularized for the case of fibroblast chemotaxis under a PDGF-bb gradient. Fibroblasts migration was simulated embedded in two different 3D matrices - collagen and fibrin - and under several PDGF-bb concentrations. Validation of the model results was provided through qualitative and quantitative comparison with in vitro studies. Our numerical predictions of cell trajectories and speeds were within the measured in vitro ranges in both collagen and fibrin matrices. Although in fibrin, the migration speed of fibroblasts is very low, because fibrin is a stiffer and more entangling matrix. Testing PDGF-bb concentrations, we noticed that an increment of this factor produces a speed increment. At 1 ng mL-1 a speed peak is reached after which the migration speed diminishes again. Moreover, we observed that fibrin exerts a dampening behavior on migration, significantly affecting the migration efficiency.

Calcineurin inhibitors affect circulating regulatory T cells in stable renal transplant recipients.

INTRODUCTION: Immunosuppression, although crucial for short-term management, has been described in renal transplantation to be a major hurdle for long-term graft survival. Efforts have been directed at achieving a true state of allotolerance, thereby reducing the load of immunosuppression. Recently, increased frequencies of CD4(+)CD25(high) regulatory T cells (Tregs) have been described as an additional mechanism to induce alloimmune tolerance. MATERIALS AND METHODS: We assessed 64 renal transplant recipients with stable renal function for at least 1 year, divided into two groups: one composed of patients receiving rapamycin but not calcineurin inhibitors (CNIs), and another, of those receiving CNIs but not rapamycin. RESULTS: We demonstrated that T cells with a regulatory phenotype were decreased in peripheral blood of renal transplant recipients under CNI therapy compared to those who were CNI-free. The Tregs in our patients showed a modest association with renal function as measured by the delta serum creatinine, which was not significant. CONCLUSIONS: CNIs, but not rapamycin, reduce the frequencies of circulating Tregs in renal transplant recipients. The use of rapamycin might be further exploited in strategies reducing immunosuppression in renal transplantation. Furthermore, quantification of blood Tregs may be a suitable tool to identify those recipients who are candidates for reducing immunosuppression.

Multi-scale finite element model of growth plate damage during the development of slipped capital femoral epiphysis.

Slipped capital femoral epiphysis (SCFE) is one of the most common disorders of adolescent hips. A number of works have related the development of SCFE to mechanical factors. Due to the difficulty of diagnosing SCFE in its early stages, the disorder often progresses over time, resulting in serious side effects. Therefore, the development of a tool to predict the initiation of damage in the growth plate is needed. Because the growth plate is a heterogeneous structure, to develop a precise and reliable model, it is necessary to consider this structure from both macro- and microscale perspectives. Thus, the main objective of this work is to develop a numerical multi-scale model that links damage occurring at the microscale to damage occurring at the macroscale. The use of this model enables us to predict which regions of the growth plate are at high risk of damage. First, we have independently analyzed the microscale to simulate the microstructure under shear and tensile tests to calibrate the damage model. Second, we have employed the model to simulate damage occurring in standardized healthy and affected femurs during the heel-strike stage of stair climbing. Our results indicate that on the macroscale, damage is concentrated in the medial region of the growth plate in both healthy and affected femurs. Furthermore, damage to the affected femur is greater than damage to the healthy femur from both the micro- and macrostandpoints. Maximal damage is observed in territorial matrices. Furthermore, simulations illustrate that little damage occurs in the reserve zone. These findings are consistent with previous findings reported in well-known experimental works.

Challenges in the Modeling of Wound Healing Mechanisms in Soft Biological Tissues.

Numerical models have become one of the most powerful tools in biomechanics and mechanobiology allowing highly detailed simulations. One of the fields in which they have broadly evolved during the last years is in soft tissue modeling. Particularly, wound healing in the skin is one of the processes that has been approached by computational models due to the difficulty of performing experimental investigations. During the last decades wound healing simulations have evolved from numerical models which considered only a few number of variables and simple geometries to more complex approximations that take into account a higher number of factors and reproduce more realistic geometries. Moreover, thanks to improved experimental observations, a larger number of processes, such as cellular stress generation or vascular growth, that take place during wound healing have been identified and modeled. This work presents a review of the most relevant wound healing approximations, together with an identification of the most relevant criteria that can be used to classify them. In addition, and looking towards the actual state of the art in the field, some future directions, challenges and improvements are analyzed for future developments.

Molecular cloning of verrucosidin-producing Penicillium polonicum genes by differential screening to obtain a DNA probe.

A differential molecular screening procedure was developed to obtain DNA clones enriched for verrucosidin-related genes that could be used as DNA probes to detect verrucosidin-producing Penicillium polonicum. Permissive and nonpermissive conditions for verrucosidin production were selected to obtain differentiated poly (A)+ RNA for the cloning strategy. P. polonicum yielded the highest amount of verrucosidin when cultured in malt extract broth at 25 degrees C without shaking. These conditions were selected as verrucosidin permissive conditions. When shaking was applied to the verrucosidin permissive conditions, verrucosidin was not detected. Approximately 5000 transformants were obtained for the library of DNA fragments from verrucosidin-producing P. polonicum and hybridized with cDNA probes obtained from poly (A)+ RNA of permissive and nonpermissive conditions. A total of 120 clones hybridized only with the permissive cDNA probes. From these, eight representative DNA inserts selected on the basis of size and labelled with fluorescein-dUTP were assayed as DNA probes in the second differential screening by Northern hybridization. Probe SVr1 gave a strong hybridization signal selectively with poly (A)+ RNAs from high verrucosidin production. When this probe was assayed by dot blot hybridization with DNA of different moulds species, hybridization was detected only with DNA from the verrucosidin-producing strain. The strategy used in this work has proved to be useful to detect unknown genes related to mycotoxins. In addition, the DNA probe obtained should be considered for the detection of verrucosidin-producing moulds.

Nonlinear finite element simulations of injuries with free boundaries: application to surgical wounds.

Wound healing is a process driven by biochemical and mechanical variables in which a new tissue is synthesised to recover original tissue functionality. Wound morphology plays a crucial role in this process, as the skin behaviour is not uniform along different directions. In this work, we simulate the contraction of surgical wounds, which can be characterised as elongated and deep wounds. Because of the regularity of this morphology, we approximate the evolution of the wound through its cross section, adopting a plane strain hypothesis. This simplification reduces the complexity of the computational problem; while allows for a thorough analysis of the role of wound depth in the healing process, an aspect of medical and computational relevance that has not yet been addressed. To reproduce wound contraction, we consider the role of fibroblasts, myofibroblasts, collagen and a generic growth factor. The contraction phenomenon is driven by cell-generated forces. We postulate that these forces are adjusted to the mechanical environment of the tissue where cells are embedded through a mechanosensing and mechanotransduction mechanism. To solve the nonlinear problem, we use the finite element method (FEM) and an updated Lagrangian approach to represent the change in the geometry. To elucidate the role of wound depth and width on the contraction pattern and evolution of the involved species, we analyse different wound geometries with the same wound area. We find that deeper wounds contract less and reach a maximum contraction rate earlier than superficial wounds.

Numerical modelling of the angiogenesis process in wound contraction.

Angiogenesis consists of the growth of new blood vessels from the pre-existing vasculature. This phenomenon takes place in several biological processes, including wound healing. In this work, we present a mathematical model of angiogenesis applied to skin wound healing. The developed model includes biological (capillaries and fibroblasts), chemical (oxygen and angiogenic growth factor concentrations) and mechanical factors (cell traction forces and extracellular matrix deformation) that influence the evolution of the healing process. A novelty from previous works, apart from the coupling of angiogenesis and wound contraction, is the more realistic modelling of skin as a hyperelastic material. Large deformations are addressed using an updated Lagrangian approach. The coupled non-linear model is solved with the finite element method, and the process is studied over two wound geometries (circular and elliptical) of the same area. The results indicate that the elliptical wound vascularizes two days earlier than the circular wound but that they experience a similar contraction level, reducing its size by 25 %.

[Identification of some characteristics of Primary Health Care physicians that influence pharmaceutical costs]. / Identificación de algunas características del médico de atención primaria que influyen en el gasto farmacéutico.

OBJECTIVE: Aging and medicalisation are leading to a progressive growth in pharmaceutical expenditure, which is difficult to measure. It is important to develop social studies of this drug spending in order to support the policies of containment. The aim of this study is to identify elements that influence pharmaceutical expenditure of Primary Care physicians in the Health Area of Cuenca (Spain). MATERIAL AND METHODS: A retrospective observational study using multilevel models of pharmaceutical expenditures by physicians between 2006 and 2009. It took into account the particular characteristics of each doctor and their patients. RESULTS: The average pharmaceutical expenditure by doctor and patient was € 277.13 year. In addition, a one-point of increase in the rate of referrals to specialised care increases pharmaceutical expenditure by 2.97 € per year and an increase in the percentage of generic drugs prescribed reduced in 2.54 € per year. These two variables and the percentage of retired patients (pensioners) are the most important factors to explain the variability in pharmaceutical expenditure. In contrast, the characteristics of physicians such as education, tenure, seniority, or age does not influence pharmaceutical costs. CONCLUSIONS: This study supports the importance of developing interventions in prescription policies. It will be of particular interest to those physicians with higher quotas of elderly patients. It also shows a significant relationship in pharmaceutical expenditures between primary and specialist physicians, which needs to be clarified by future studies.

A lattice-based approach to model distraction osteogenesis.

Distraction osteogenesis is a well-known technique in which new bone tissue is created when a distraction displacement is applied through an external frame. This orthopedic process is nowadays focus of intense research, both experimentally and numerically, as there are still many aspects not well understood. The aim of this study is to simulate bone distraction by means of a combined discrete-continuum approach based on a lattice formulation. Existing computational models simulate the main processes of distraction osteogenesis from a continuum perspective, considering as state variables the population of cells and tissue distributions. Results of the continuum and lattice-based approaches are similar with respect to the global evolution of the different cells but rather different in terms of the type of ossification process. Differences in the size of the soft interzone in the gap have also been found. In addition, the discrete-continuum formulation allows including a more realistic approach of the migration/proliferation process with a discrete random walk model instead of the Fick's law used in continuum approaches. Also, blood vessel growth can be simulated explicitly in this model with the inclusion of the endothelial cells. Further study is needed to provide additional insights to understand coupled phenomena at different scales in the cell-tissue interactions. However this work provides a first preliminary step for improving multiscale models.

Evaluation of residual stresses due to bone callus growth: a computational study.

Mechanical environment in callus is determinant for the evolution of bone healing. However, recent mechanobiological computational works have underestimated the effect that growth exerts on the mechanical environment of callus. In the present work, we computationally evaluate the significance of growth-induced stresses, commonly called residual stresses, in callus. We construct a mechanobiological model of a callus in the metatarsus of a sheep in two different stages: one week and four weeks after fracture. The magnitude of stresses generated during callus growth is compared with the magnitude of stresses when only external loads are applied to the callus. We predict that residual stresses are relevant in some areas, mainly located at the periosteal side far from the fracture gap. Therefore, the inclusion of these residual stresses could represent a significant impact on the callus growth and predict a different evolution of biological processes occurring during bone healing.

Tetrahydrohyperforin prevents cognitive deficit, Aß deposition, tau phosphorylation and synaptotoxicity in the APPswe/PSEN1ΔE9 model of Alzheimer's disease: a possible effect on APP processing.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deterioration of cognitive abilities, amyloid-ß peptide (Aß) accumulation and synaptic alterations. Previous studies indicated that hyperforin, a component of the St John's Wort, prevents Aß neurotoxicity and some behavioral impairments in a rat model of AD. In this study we examined the ability of tetrahydrohyperforin (IDN5607), a stable hyperforin derivative, to prevent the cognitive deficit and synaptic impairment in an in vivo model of AD. In double transgenic APPswe/PSEN1ΔE9 mice, IDN5706 improves memory and prevents the impairment of synaptic plasticity in a dose-dependent manner, inducing a recovery of long-term potentiation. In agreement with these findings, IDN5706 prevented the decrease in synaptic proteins in hippocampus and cortex. In addition, decreased levels of tau hyperphosphorylation, astrogliosis, and total fibrillar and oligomeric forms of Aß were determined in double transgenic mice treated with IDN5706. In cultured cells, IDN5706 decreased the proteolytic processing of the amyloid precursor protein that leads to Aß peptide generation. These findings indicate that IDN5706 ameliorates AD neuropathology and could be considered of therapeutic relevance in AD treatment.