The burn severity and plant recovery relationship affect the biological and chemical soil properties of Pinus halepensis Mill. stands in the short and mid-terms after wildfire.

In the Mediterranean Basin, changes in climate and fire regime (increased recurrence and severity) reduce ecosystem services after wildfires by increasing soil degradation and losses in plant diversity. Our study was a biological approach to relate soil properties to vegetation recovery and burn severity. We focused our study on the natural recovery of the soil-plant interphase in Pinus halepensis Mill. forests located in the SE of Iberian Peninsula, a semiarid climate. We included some chemical properties 3 years after fire (available phosphorus (P) and soil organic carbon (Corg), among others), and biological soil indicators 3 and 5 years after fire (i.e. basal soil respiration (BSR), microbial biomass carbon (Cmic), carbon mineralization coefficient (Cmineral), metabolic quotient (qCO2) and microbial quotient (Cmic:Corg)). We analyzed the activity of three different enzymes: urease (UR), phosphatase (PHP) and ß-glucosidase (GLU). The changes in most chemical properties were ephemeral, but P and Corg showed higher values in burned areas, and the highest were found for low-moderate severity. Plant recovery was the triggering factor for the recovery of Corg and biological soil function. Burn severity and time after fire influenced Cmic and the Cmic:Corg, which were higher for moderate-high severity 3 years later, but were below the unburned values 5 years after fire. The microbial activities of GLU and UR were recovered in burned areas 5 years after fire. The PHP values lowered according to higher burn severity and time after fire. The soil ecological trends obtained by a principal component analysis revealed a relationship linking GLU, BSR and qCO2 that explained soil response to burn severity. PHP, Cmic and Cmic:Corg explained most of the variability related to time after fire. Our results provide insights into how burn severity, in Mediterranean fire-prone Aleppo pine stands, modulated the natural plant recovery linked to soil biochemical and microbiological response to fire. High burn severity limited natural vegetation recovery, and both reduced biological soil functionality. This knowledge can be implemented in post-fire planning to apply post-fire management (for mitigation and restoration) in which the "no intervention" tool should be contemplated. These findings provide information to be applied in adaptive forest management to improve the resilience of vulnerable ecosystems and to reduce burn severity in future fire events.

Five Years of Multidisciplinary Care in Hereditary Cancer: Our Experience in a Spanish University Hospital.

OBJECTIVE: To analyse the evolution of a multidisciplinary heredofamilial cancer unit (HFCU) in a university hospital. METHODS: This was a retrospective analysis of the activity of our HFCU in its first 5 years of existence. RESULTS: Between July 2010 and July 2015, 1,518 patients from 1,318 families attended our HFCU. Genetic testing was offered to 862 patients. Of those, 833 (96.6%) accepted testing, with available results for 636 (76.4%). Pathogenic mutations in BRCA1 and BRCA2 were found in 175 patients. Lynch syndrome and adenomatous polyposis were the most frequent syndromes diagnosed (151/175, 86.3%) among 17 different syndromes studied. Of the 404 patients without a previous genetic diagnosis in the family, 62 (15.3%) were found to have mutations in disease-causing genes. Prophylactic surgery and follow-up (33.7%) or follow-up only (66.3%) was proposed for mutation carriers according to international guidelines and patients' preferences. CONCLUSION: We have a high mutation detection rate, genetic test acceptance, and compliance with risk reduction strategies. However, there is room for improvement, especially in genetic testing timing, considering that an increase in the indications for genetic testing is expected.

Primary dermal melanoma: a case report and a review of the literature.

Patients with cutaneous metastatic melanoma of unknown primary origin (stage IV M1a disease according to the American Joint Committee on Cancer melanoma staging system) have an estimated 5-year survival rate of between 5% and 17.9% and a median survival of 6 months. However, certain patients with stage IV M1a disease have much higher survival rates. The existence of this subpopulation has given rise to the term primary dermal melanoma to describe such cases. We report a case of melanoma with characteristics consistent with primary dermal melanoma and review the relevant literature. A diagnosis of primary dermal melanoma requires careful clinical and pathologic correlation and should be considered in all patients with a solitary melanoma confined to the dermis and subcutaneous tissue when there is no evidence of a primary tumor or disease at other sites following appropriate staging studies. We believe that familiarity with this subtype of melanoma is essential in order to provide patients with optimal care and better prognostic information.

High resolution melting analysis for the identification of novel mutations in DKC1 and TERT genes in patients with dyskeratosis congenita.

Dyskeratosis congenita (DC) is a rare inherited bone-marrow failure syndrome with high clinical heterogeneity. Cells derived from DC patients present short telomeres at early ages, as a result of mutations in genes encoding components of the telomerase complex (DKC1, TERC, TERT, NHP2 and NOP10), or the shelterin complex (TINF2). However, mutations have been identified only in around 50% of the cases, indicating that other genes could be involved in the development of this disease. Indeed, mutations in TCBA1 or chromosome segment C16orf57 have been described recently. We have used HRM technology to perform genetic analysis in the above mentioned genes, in Spanish patients showing both, some clinical features of DC and short telomeres. The mutations have been identified by PCR amplification of DC genes followed by high resolution melting (HRM) and direct DNA sequencing analysis. We have identified seven new families with DC, three with X-linked DC and four with autosomal dominant DC, in which we have found two novel mutations in DKC1 (p.His68Arg and p.Lys390del) and four novel mutations in TERT gene (p.Pro530Leu, p.Arg698Trp, p.Arg971His and p.Arg698Gln). The results show that the use of HRM analysis enables a rapid and inexpensive identification of mutations in dyskeratosis congenita associated genes.

Changes in soil microbial community structure following the abandonment of agricultural terraces in mountainous areas of Eastern Spain.

In Eastern Spain, almond trees have been cultivated in terraced orchards for centuries, forming an integral part of the Mediterranean forest scene. In the last decades, orchards have been abandoned due to changes in society. This study investigates effects of changes in land use from forest to agricultural land and the posterior land abandonment on soil microbial community, and the influence of soil physico-chemical properties on the microbial community composition (assessed as abundances of phospholipids fatty acids, PLFA). For this purpose, three land uses (forest, agricultural and abandoned agricultural) at four locations in SE Spain were selected. Multivariate analysis showed a substantial level of differentiation in microbial community structure according to land use. The microbial communities of forest soils were highly associated with soil organic matter content. However, we have not found any physical or chemical soil property capable of explaining the differences between agricultural and abandoned agricultural soils. Thus, it was suggested that the cessation of the perturbation caused by agriculture and shifts in vegetation may have led to changes in the microbial community structure. PLFAs indicative of fungi and ratio of fungal to bacterial PLFAs were higher in abandoned agricultural soils, whereas the relative abundance of bacteria was higher in agricultural soils. Actinomycetes were generally lower in abandoned agricultural soils, while the proportions of vesicular-arbuscular mycorrhyzal fungi were, as a general trend, higher in agricultural and abandoned agricultural soils than in forests. Total microbial biomass and richness increased as agricultural < abandoned agricultural < forest soils.

Near infrared spectroscopy for determination of various physical, chemical and biochemical properties in Mediterranean soils.

The potential of near infrared (NIR) reflectance spectroscopy to predict various physical, chemical and biochemical properties in Mediterranean soils from SE Spain was evaluated. Soil samples (n=393) were obtained by sampling thirteen locations during three years (2003-2005 period). These samples had a wide range of soil characteristics due to variations in land use, vegetation cover and specific climatic conditions. Biochemical properties also included microbial biomarkers based on phospholipid fatty acids (PLFA). Partial least squares (PLS) regression with cross validation was used to establish relationships between the NIR spectra and the reference data from physical, chemical and biochemical analyses. Based on the values of coefficient of determination (r(2)) and the ratio of standard deviation of validation set to root mean square error of cross validation (RPD), predicted results were evaluated as excellent (r(2)>0.90 and RPD>3) for soil organic carbon, Kjeldahl nitrogen, soil moisture, cation exchange capacity, microbial biomass carbon, basal soil respiration, acid phosphatase activity, ß-glucosidase activity and PLFA biomarkers for total bacteria, Gram positive bacteria, actinomycetes, vesicular-arbuscular mycorrhizal fungi and total PLFA biomass. Good predictions (0.81

Temporal characterisation of soil-plant natural recovery related to fire severity in burned Pinus halepensis Mill. forests.

Despite Mediterranean ecosystems' high resilience to fire, both climate and land use change, and alterations in fire regimes increase their vulnerability to fire by affecting the long-term natural recovery of ecosystem services. The objective of this work is to study the effects of fire severity on biochemical soil indicators, such as chemical composition or enzymatic activity, related to time after fire and natural vegetation recovery (soil-plant interphase). Soil samples from three wildfires occurring 3, 15 and 21 years ago were taken in the south-eastern Iberian Peninsula (semiarid climate). Sampling included three fire severity levels in naturally regenerated (and changing to shrublands) Pinus halepensis Mill. forests. In the short-term post-fire period, phosphorus concentration, electrical conductivity and urease activity were positively linked to fire severity, and also influenced ß-glucosidade activity in a negative relationship. During the 15-21-year post-fire period, the effects related to medium-high fire severity were negligible and soil quality indicators were linked to natural regeneration success. The results showed that most soil properties recovered in the long term after fire (21 years). These outcomes will help managers and stakeholders to implement management tools to stabilise soils and to restore burned ecosystems affected by medium-high fire severity. Such knowledge can be considered in adaptive forest management to reduce the negative effects of wildfires and desertification, and to improve the resilience of vulnerable ecosystems in a global change scenario.

Effect of irrigation on the survival of total coliforms in three semiarid soils after amendment with sewage sludge.

Sewage sludges are increasingly used in soil amendment programmes, although not without risk since they contain, among other potential hazards, high concentrations of total coliform bacteria. In this paper we have studied the effect of irrigation on the survival of total coliforms in three semiarid degraded soils with different agricultural practices. Fresh sewage sludge was added at 50 g kg(-1) soil, and incubated in both the presence and absence of irrigation. The absence of irrigation led to a sharp decrease in the number of total coliforms in all soils, with the bacteria disappearing in 40 days. Irrigation produced a substantial initial increase in the number of coliforms in the three soils, although after 80 days there was none growing in any of the soils. The results showed that there were significant differences in the survival of coliform bacteria due to the presence or absence of irrigation.

Effects of salvage logging on soil properties and vegetation recovery in a fire-affected Mediterranean forest: A two year monitoring research.

Post-fire management can have an additional impact on the ecosystem; in some cases, even more severe than the fire. Salvage logging (SL) is a common practice in most fire-affected areas. The management of burnt wood can determine microclimatic conditions and seriously affect soil properties. In some cases, the way of doing it, using heavy machinery, and the vulnerability of soils to erosion and degradation can make this management potentially aggressive to soil. Research was done in "Sierra de Mariola Natural Park" (E Spain). A forest fire (>500ha) occurred in July 2012. In February 2013, SL treatment was applied in a part of the affected forest. Plots for monitoring this effect were installed in this area and in a similar nearby area where no treatment was done, used as control (C). Soil samplings were done immediately after treatment and every 6months during two years. Some soil properties were analysed, including organic matter (OM) content, nitrogen (N) available phosphorous (P) basal soil respiration (BSR), microbial biomass carbon (Cmic), bulk density (BD), water repellency (WR), aggregate stability (AS) and field capacity (FC). SL treatment caused an increase in BD, a decrease of AS, FC, OM and N. In the control area, in general the soil properties remained constant across the 2years of monitoring, and the microbial parameters (BSR and Cmic), initially affected by the fire, recovered faster in C than in the SL area. Plant recovery also showed some differences between treatments. No significant differences were observed in the number of plant species recorded (richness) comparing C versus SL plots, but the number of individuals of each species (evenness) was significantly higher in C plots. In conclusion, we can affirm that for the conditions of this study case, SL had a negative effect on the soil-plant system.

Temporal changes in soil water repellency after a forest fire in a Mediterranean calcareous soil: Influence of ash and different vegetation type.

Forest fires usually modify soil water repellency (SWR), and its persistence and intensity show a high variability both in space and time. This research studies the evolution of SWR in a Mediterranean calcareous soil affected by a forest fire, which occurred in Gorga (SE Spain) in July 2011, comparing the effect of the main vegetation cover between pine (Pinus halepensis) and shrubs species (Quercus coccifera, Rosmarinus officinalis, Cistus albidus, Erica arborea and Brachypodium retusum) and the relationship with soil moisture content (SMC). Also the study analyzed the effect of ash on SWR dynamics under field conditions. Six plots were established on the fire-affected area and the unburned-control-adjacent area to monitoring SWR with the water drop penetration time (WDPT) test, SMC through moist sensors (5cm depth) and three different ash treatments: ash presence, ash absence and incorporation of ash into the soil. An immediate increase of SWR was observed in the fire-affected area, mainly in pine plots. SWR changes in control (unburned) plots were quite similar between different types of vegetation influence, despite higher SWR values being observed on pine plots during the study period. A noticeable decrease of SWR was observed during the first months after fire in the affected areas, especially after the first rainy period, both in pine and shrubs plots. SWR increase was registered in all plots, and the highest levels were in March 2012 in burned pine plots. SWR decrease was higher in plots where ash was removed. Fire-affected soils became wettable 1year and a half after the fire.

Real-time PCR detection chemistry.

Real-time PCR is the method of choice in many laboratories for diagnostic and food applications. This technology merges the polymerase chain reaction chemistry with the use of fluorescent reporter molecules in order to monitor the production of amplification products during each cycle of the PCR reaction. Thus, the combination of excellent sensitivity and specificity, reproducible data, low contamination risk and reduced hand-on time, which make it a post-PCR analysis unnecessary, has made real-time PCR technology an appealing alternative to conventional PCR. The present paper attempts to provide a rigorous overview of fluorescent-based methods for nucleic acid analysis in real-time PCR described in the literature so far. Herein, different real-time PCR chemistries have been classified into two main groups; the first group comprises double-stranded DNA intercalating molecules, such as SYBR Green I and EvaGreen, whereas the second includes fluorophore-labeled oligonucleotides. The latter, in turn, has been divided into three subgroups according to the type of fluorescent molecules used in the PCR reaction: (i) primer-probes (Scorpions, Amplifluor, LUX, Cyclicons, Angler); (ii) probes; hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon, MGB-Pleiades, MGB-Eclipse, ResonSense, Yin-Yang or displacing); and (iii) analogues of nucleic acids (PNA, LNA, ZNA, non-natural bases: Plexor primer, Tiny-Molecular Beacon). In addition, structures, mechanisms of action, advantages and applications of such real-time PCR probes and analogues are depicted in this review.

Targeted screening for the detection of Pompe disease in patients with unclassified limb-girdle muscular dystrophy or asymptomatic hyperCKemia using dried blood: A Spanish cohort.

We aimed to screen for Pompe disease in patients with unclassified limb-girdle muscular dystrophy (LGMD) or asymptomatic hyperCKemia using dried blood spot (DBS) assays. Subsequently, we aimed to calculate the diagnostic delay between initial symptom presentation and the diagnosis. A prospective, multicenter, observational study was conducted in 348 patients: 146 with unclassified LGMD and 202 with asymptomatic or paucisymptomatic hyperCKemia. We quantified levels of acid alpha-glucosidase (GAA) from dried blood spots analyzed fluorometrically. The test was positive in 20 patients, and Pompe disease was confirmed by genetic testing in 16. Undiagnosed Pompe disease was detected in 7.5% of patients with LGMD and in 2.5% of patients with persistent, idiopathic elevation of serum creatine kinase. The c.-32-13 T > G mutation was found most commonly. The diagnostic delay was 15 years on average. In conclusion, DBS tests are useful and reliable screening tools for Pompe disease. We recommend the dried blood spot test to be included in the diagnostic work-up of patients with unclassified myopathies with proximal weakness and/or hyperCKemia of unknown cause and, when positive, to define the diagnosis, it will have to be confirmed by biochemical and/or molecular genetic analysis.

PanGen-Fam: Spanish registry of hereditary pancreatic cancer.

PURPOSE: To describe the organisation of the registry and the preliminary results in terms of characteristics of high-risk pancreatic ductal adenocarcinoma (PDAC) families recruited to date and findings of the screening programme. To compare early onset sporadic cases (⩽50 years), sporadic cases (>50 years) and cases with family history of cancer, for PDAC possible risk factors. METHODS/PATIENTS: Families with hereditary cancer syndromes predisposing to PDAC were recruited from two main sources: Spanish hospitals participating in PanGenEU, a pan-European multicentre case-control study, and their genetic counseling unit. Individuals at high-risk of PDAC were enrolled into a screening programme, consisting of Endoscopic ultrasound, computerised tomography, magnetic resonance imaging. Genetic testing of candidate genes was offered according to each patient's risk. RESULTS: Among 577 consecutive PDAC cases, recruited via PanGenEU, 36 (6%) had ⩾2 first-degree relative with PDAC: Familial pancreatic cancer (FPC). So far PanGen-Fam has recruited 42 high-risk PDAC families; 25 (60%) had FPC. Five index cases with cancer were positive for BRCA2 and one for BRCA1 germline mutations. In the second year of prospective PDAC screening, one neuroendocrine tumour and a high-grade dysplasia lesion suspicious of carcinoma were diagnosed among 41 high-risk individuals. Furthermore EUS detected chronic-pancreatitis-like parenchymal changes in 15 patients. CONCLUDING STATEMENT: The identification and recruitment of PDAC high-risk families into the PanGen-Fam registry provides an opportunity to detect early onset cancer and precursor pancreatic cancer lesions at a potentially curative stage and to increase the knowledge of the natural history of the disease.

Persistent delivery of factor IX in mice: gene therapy for hemophilia using implantable microcapsules.

Severe hemophilia B is a life-threatening, life long condition caused by absence of or defective coagulation factor IX. Gene therapy could provide an alternative treatment to repeated injection of plasma-derived concentrate or recombinant factor IX. We have previously described the use of implantable microcapsules containing recombinant myoblasts to deliver human factor IX in mice. This study reports the generation of improved myoblast-specific expression vectors. Mouse myoblast clones transfected with the various vectors secreted factor IX in vitro, at rates between 70 and 1000 ng/10(6) cells/day. The recombinant myoblast clones were then encapsulated and implanted into mice. Immunocompetent mice implanted with encapsulated myoblasts had up to 65 ng of factor IX per milliliter in their plasma for up to 14 days, after which antibodies to human factor IX became detectable, and this coincided with decreased factor IX in mouse plasma. In immunodeficient mice, however, factor IX delivery was maintained at a constant level for at least 6 weeks (end of experiment). Interestingly, the highest-secreting myoblast clone in vitro did not deliver the highest level of hFIX in vivo. This discrepancy observed between performance in vitro and in vivo may have important implications for the development of gene therapy protocols based on recombinant cells.

Epididymoorchitis due to Brucella mellitensis: a retrospective study of 59 patients.

Epididymoorchitis is a focal form of human brucellosis described in 2%-20% of patients with brucellosis. We assessed 59 cases of Brucella epididymoorchitis (BEO) between 1991 and 1999. The median age of patients was 34 years (range, 15-75 years). The onset of symptoms was acute in 46 patients (78%). Scrotal pain and swelling (100% of patients), fever (88%), and sweating (73%) were the most common symptoms. Brucella species was isolated from blood cultures in 41 patients (69%) and from epididymal aspiration in 4 patients. Treatment consisted of a combination of a doxycycline and an aminoglycoside (n=39) or rifampin (n=10); trimethoprim-sulfamethoxazole with rifampin (n=3); or trimethoprim-sulfamethoxazole as monotherapy (n=7). The median duration of therapy was 45 days (range, 21-90 days). The infections of 9 patients (15%) failed to respond to therapy, and 15 patients relapsed (25%). Three patients with necrotizing orchitis whose infections were unresponsive to antibiotics required an orchiectomy. In general, classical brucellosis therapy is adequate for BEO.

Recognition and optimum treatment of brucellosis.

Brucellosis (infection with Brucella spp.) is a common zoonosis in many parts of the world. Human brucellosis is a multisystem disease that may present with a broad spectrum of clinical manifestations. Treatment of brucellosis must effectively control acute illness and prevent complications and relapse. The choice of regimen and duration of antimicrobial therapy should be based on the presence of focal disease and underlying conditions which contraindicate certain specific antibiotics. The regimen of first choice is combination therapy with doxycycline for 45 days and streptomycin for 14 days. Gentamicin or netilmicin for the first 7 days may be substituted for streptomycin. Second-choice regimens consist of combinations of doxycycline and rifampicin (rifampin) for 45 days, or monotherapy with doxycycline for 45 days. Surgery should be considered for patients with endocarditis, cerebral or epidural abscess, spleen abscess or other abscesses which are antibiotic-resistant. Tetracyclines are generally contraindicated for pregnant patients and children < 8 years old. Rifampicin 900 mg once daily for 6 weeks is considered the drug of choice for treating brucellosis in pregnant women. In children < 8 years old the preferred regimen is rifampicin with cotrimoxazole (trimethoprim-sulfamethoxazole) for 45 days. An alternative regimen consists of a combination of rifampicin for 45 days with gentamicin 5 to 6 mg/kg/day for the first 5 days.

Clinical utility of a dipstick assay in patients with brucellosis: correlation with the period of evolution of the disease.

To examine the clinical utility of a dipstick assay for the detection of Brucella-specific IgM antibodies, and the correlation with the evolution of the disease. Twenty-six patients who were admitted to the General Hospital of Albacete (Spain) over a 2-year period and diagnosed with brucellosis were included in the study. One hundred and twenty-five serum samples collected at the time of diagnosis and at intervals during and after treatment were tested by the Coombs test, the standard seroagglutination test (SAT), the SAT in the presence of dithiothreitol (DTT-SAT), and a dipstick assay for the detection of Brucella-specific immunoglobulin M (IgM) antibodies. The sensitivity of the dipstick assay at the moment of the diagnosis was similar to that of the SAT (62% and 73%, respectively), somewhat higher than that of the DTT-SAT (50%), and lower than that of the Coombs test (100%). Patients with a negative dipstick test at the moment of diagnosis displayed a period of evolution of the disease longer than that of the dipstick-positive patients. After the beginning of therapy, the detection rate of the dipstick assay decreased faster than those of the SAT, the DTT-SAT, and the Coombs test. Thirty days after the start of therapy, the detection rate of the dipstick assay had decreased to 7%, whereas that of the SAT and DTT-SAT was 46%, and that of the Coombs test was still 92%. The dipstick assay could be used as a rapid diagnostic test for patients in the early stages of illness. Patients with a long period of illness will probably have a negative dipstick test, and could be diagnosed with the aid of the Coombs test and classical clinical findings.

Comparison of three different PCR methods for detection of Brucella spp in human blood samples.

In the diagnosis of human brucellosis, PCR could be a more sensitive technique than blood cultures and more specific than conventional serological tests. We compared three different PCR methods for the detection of Brucella spp. and we studied whether human genomic DNA affect the sensitivity of three primer pairs for the detection of Brucella DNA in a peripheral-blood PCR assay. These three pairs of primers amplified three different fragments included in: (i). a gene encoding a 31-kDa Brucella abortus antigen (primers B4/B5), (ii). a sequence 16S rRNA of B. abortus (primers F4/R2), and (iii). a gene encoding an outer membrane protein (omp-2) (primers JPF/JPR). The three primers assayed showed a difference in sensitivity for detecting purified Brucella DNA, ranging between 8 fg and 20 pg. However, the sensitivity of the primers F4/R2 and B4/B5 was affected by the presence of human DNA while the primers JPF/JPR were not. Therefore, although the sensitivity of PCR using primers F4/R2 is affected by human DNA, they are still the most sensitive and they could provide a useful tool for the diagnosis of human brucellosis.