Viability and Infectivity of Tuber borchii after Cryopreservation.

BACKGROUND: Truffles (Tuber spp.) are the most precious ectomycorrhizal edible mushrooms whose biodiversity is seriously endangered. OBJECTIVE: To develop a protocol for cryopreservation of Tuber spp. mycelia using T. borchii as a model species, verifying whether conservation in liquid nitrogen may affect viability, growth rate, hyphal morphology and infectivity. MATERIALS AND METHODS: Cryopreservation was performed using sorbitol, sucrose and DMSO as cryoprotectants. The morphological parameters analyzed were: hyphal diameter, septal distance and hyphal growth unit. Cryopreserved mycelium infectivity was assessed by inoculating Quercus robur seedlings. RESULTS: In T. borchii cryopreserved mycelium, the lag-phase lasted for 6-42 days but no differences in growth curve evolution, growth rate and hyphal morphology were observed except for hyphal growth unit. No differences in mycorrhizal colonization were observed between the seedlings inoculated with non-cryopreserved and cryopreserved mycelium. CONCLUSION: The established protocol is suitable for long-term conservation of Tuber mycelium and opens up the possibility of creating a Tuber spp. germplasm bank to preserve truffle diversity.

Ultrasound Characteristics of Calf Deep Vein Thrombosis and Residual Vein Obstruction After Low Molecular Weight Heparin Treatment.

OBJECTIVE/BACKGROUND: Calf deep vein thrombosis (CDVT) is frequently found in symptomatic outpatients, but CDVT ultrasound diagnostic criteria are still debated. It has been proposed that only clots with ≥5 mm maximum diameter can be considered as CDVT. AIMS: To assess clot diameters and characteristics of CDVT, and to assess the recanalization rate of CDVT after anticoagulant treatment. METHODS: In a prospective, multicenter cohort study symptomatic outpatients in whom CDVT was diagnosed by ultrasound were enrolled. Posterior tibial, fibular, medial and lateral gastrocnemius, and soleal veins were compressed transversally over their entire length. Clot diameter was measured during maximum compression and ultrasound was repeated after 6 weeks of low molecular weight heparin treatment. RESULTS: In 172 patients (age 70 ± 1 y, male 32%) CDVT was detected in 132 (76.7%) muscle veins only, and in 24 (14%) axial veins only, while 16 (9.3%) patients had both muscular and axial CDVT. A total of 212 clots were found with a diameter of 5.8 ± 1.8 mm (IQR 4.5-6.8 mm) with the 10th percentile being ≥3.5 mm. A cut off value of ≥5 mm had a sensitivity of 0.76 (95% CI 0.69-0.82), whereas a value of ≥3.5 mm had a sensitivity of 0.94 (95% CI 0.89-0.97). Recanalization (i.e. residual vein obstruction ≤2 mm) was found in 51% of patients and the recanalization rate was not correlated with clot diameter at enrolment (rho -0.128 p = 0.93) or with type of CDVT (axial vs. muscular thrombosis). Patients with significantly reduced mobility had lower probability of CDVT recanalization. CONCLUSION: A clot diameter ≥5 mm is found in only 76% of CDVT patients and a clot diameter ≥3.5 mm may be more appropriate as a threshold for CDVT. After 6 weeks of anticoagulant treatment, half of CDVT patients had recanalization and recanalization was not correlated with clot characteristics at enrolment, but with mobility of the patients.

Effects of soil tillage on Tuber magnatum development in natural truffières.

Tuber magnatum Pico, the Italian white truffle, commands the highest prices of any truffle. Despite its commercial value, it is the only edible European truffle which has not yet been successfully cultivated. Because of this, it is essential to safeguard natural truffières and to identify cultural practices to maximize their productivity. Soil tillage is successfully and extensively used in black truffle cultivation to enhance productivity, but its effects are not known on the development of T. magnatum. A recently developed real-time PCR assay was applied to evaluate the effects of tillage (10-15 cm depth) on T. magnatum mycelium in two different natural truffle grounds located in Tuscany and Emilia-Romagna. Tillage effects on bulk density, ectomycorrhizal fungal communities, and ascoma production were also assessed. Tilling significantly increased the quantity of T. magnatum mycelium which seemed to be related to an increase in soil porosity by up to 34%, and the diversity of ectomycorrhizal fungal communities. On the contrary, no significant effects were found on ascoma production. The results highlight that real-time PCR is the most reliable method for evaluating the effects of cultural practices on the development of T. magnatum in soil avoiding long-term studies on fruiting body production.

Assessment of ectomycorrhizal fungal communities in the natural habitats of Tuber magnatum (Ascomycota, Pezizales).

The ectomycorrhizal (ECM) fungal communities of four natural Tuber magnatum truffle grounds, located in different Italian regions (Abruzzo, Emilia-Romagna, Molise, and Tuscany), were studied. The main objective of this study was to characterize and compare the ECM fungal communities in the different regions and in productive (where T. magnatum ascomata were found) and nonproductive points. More than 8,000 (8,100) colonized root tips were counted in 73 soil cores, and 129 operational taxonomic units were identified using morphological and molecular methods. Although the composition of the ECM fungal communities studied varied, we were able to highlight some common characteristics. The most plentiful ECM fungal taxa belong to the Thelephoraceae and Sebacinaceae families followed by Inocybaceae and Russulaceae. Although several ectomycorrhizas belonging to Tuber genus were identified, no T. magnatum ectomycorrhizas were found. The putative ecological significance of some species is discussed.

Age-adjusted D-dimer to rule out deep vein thrombosis: findings from the PALLADIO algorithm.

Essentials The accuracy of the age-adjusted D-dimer in suspected venous thromboembolism is still debated. We assessed the performance of age-adjusted D-dimer combined with the PALLADIO algorithm. The age-adjusted threshold can reduce the need for imaging tests compared to the fixed cut-off. The safety of this approach should be confirmed in large management studies. SUMMARY: Background Age-adjusted D-dimer has been proposed to increase specificity for the diagnosis of venous thromboembolism (VTE). However, the accuracy of this threshold has been recently questioned. Objectives To assess the diagnostic performance of age-adjusted D-dimer combined with clinical pretest probability (PTP) in patients with suspected deep vein thrombosis (DVT). Methods PALLADIO (NCT01412242) was a multicenter management study that validated a new diagnostic algorithm, incorporating PTP, D-dimer (using the manufacturer's cut-off) and limited or extended compression ultrasonography (CUS) in outpatients with clinically suspected DVT. Patients with unlikely PTP and negative D-dimer had DVT ruled out without further testing (group 1); patients with likely PTP or positive D-dimer underwent limited CUS (group 2); patients with likely PTP and positive D-dimer underwent extended CUS (group 3). Patients with DVT ruled out at baseline had a 3-month follow-up. In this post-hoc analysis we evaluated age-adjusted D-dimer cut-off (defined as age times 10 µg L-1 , or age times 5 µg L-1 for D-dimers with a lower manufacturer's cut-off, in patients > 50 years). Results In total, 1162 patients were enrolled. At initial visit, DVT was detected in 4.0% of patients in group 2 and 53.0% in group 3. The age-adjusted D-dimer, compared with the fixed cut-off, resulted in 5.1% (95% CI, 4.0-6.5%) reduction of CUS. The incidence of symptomatic VTE during follow-up was: 0.24% (95% CI, 0.04-1.37) in group 1; 1.12% (95% CI, 0.44-2.85) in group 2; and 1.89% (95% CI, 0.64-5.40) in group 3. Conclusions The PALLADIO algorithm using age-adjusted D-dimer slightly decreased the number of required imaging tests, but this approach should be confirmed in large management studies.

Recolonization and recovery dynamics of the macrozoobenthos after sand extraction in relict sand bottoms of the Northern Adriatic Sea.

The long-term effects of sand extraction on macrozoobenthic communities were investigated in an offshore area in the Northern Adriatic Sea characterised by relict sands formed during the last Adriatic post-glacial transgression. Surveys were carried out before, during and 1, 6, 12, 18, 24 and 30 months after extraction at three impacted and seven reference stations. The operations did not influence the physical characteristics of the sediment, but they caused almost complete defaunation at dredged sites. Univariate and multivariate analyses highlighted that the macrozoobenthic community responses to the dredging operations were (1) a rapid initial recolonisation phase by the dominant taxa present before dredging, which took place 6-12 months after sand extraction; (2) a slower recovery phase, that ended 30 months after the operations, when the composition and structure of the communities were similar in the dredged and reference areas. This pattern of recolonisation-recovery fits well with the commonly encountered scenario where the substratum merely remains unchanged after marine aggregate extraction.

Venous thromboembolism and the risk of subsequent symptomatic atherosclerosis.

BACKGROUND: Recently, we reported an association between asymptomatic carotid atherosclerosis and venous thromboembolism (VTE) of unknown origin. We hypothesized that patients with VTE of unknown origin would be at a higher risk of developing symptomatic atherosclerosis than patients with VTE induced by known risk factors. METHODS: To examine this hypothesis, we studied 1,919 consecutive patients followed prospectively after their first VTE episode. The primary outcome was non-fatal and fatal symptomatic atherosclerotic disease in patients with VTE of unknown origin as compared to those with secondary VTE. An independent committee assessed all study outcomes, and adjusted hazard ratios (HR) were calculated using the Cox's proportional hazards model. RESULTS: After a median follow-up of 48 and 51 months, respectively, at least one symptomatic atherosclerotic complication was detected in 160 of the 1,063 patients (15.1%) with VTE of unknown origin, and in 73 of the 856 (8.5%) with secondary VTE. After adjusting for age and other risk factors of atherosclerosis, the HR for symptomatic atherosclerotic complications in patients with VTE of unknown origin compared to those with secondary VTE was 1.6 (95% confidence intervals; CI: 1.2-2.0). When the analysis was restricted to patients without previous symptomatic atherosclerosis, the HR became 1.7 (95% CI: 1.1-2.4). CONCLUSIONS: Patients with VTE of unknown origin have a 60% higher risk of developing symptomatic atherosclerotic disease than do patients with secondary venous thrombosis.

The effects of sand extraction on the macrobenthos of a relict sands area (northern Adriatic Sea): results 12 months post-extraction.

Sands for the nourishment of beaches along the Emilia-Romagna coast (northern Adriatic Sea) were dredged from an offshore area characterised by relict sands formed during the last Adriatic post-glacial transgression. The short-term effects of the sand extraction on macrozoobenthic communities were investigated before, during and 1, 6 and 12 months after dredging at three impacted stations and seven control stations. Sand extraction activities did not significantly influence the granulometry and %TOC in the sediment but caused almost complete defaunation at dredging stations. Yet, just 12 months after the extraction, the recolonisation of communities at the impacted stations was at an advanced stage. Unlike other studies on the effects of extraction of marine sand, no significant settlement of opportunistic species was observed. The limited impact of the sand extraction operation on the physical characteristics of the sediment and hydrological-sedimentary characteristics in the relict sand area should aid its rapid recovery and the restoration of the original community in a short period of time (2-4 years after dredging).

Effects of long-term dumping of harbor-dredged material on macrozoobenthos at four disposal sites along the Emilia-Romagna coast (Northern Adriatic Sea, Italy).

Sediment from harbors of the Emilia-Romagna (Northern Adriatic Sea) were dredged and dumped in four disposal areas characterized by muddy bottoms. The long-term effects of the dumping on macrozoobenthic communities were investigated before and after 6 month, 8 month, 2 years and 4 years. The disposal of dredged material did not influence the granulometry and %TOC in the sediment, and no alterations in the structure of the macrobenthic communities were observed in the four areas. The lack of impact could be ascribed to the environmental characteristics and precautionary measures taken to minimize the effects of the dumping. It appears that: (1) the communities of the dumping areas are well adapted to unstable environments; (2) the sediments were disposed gradually and homogeneously over relatively large areas; Other factors that help to reduce the impact of sediment disposal are the low concentrations of contaminants in dredged materials and the similarity of sediment in the dredged and disposal areas. Off-shore discharge appears a sustainable strategy for the management of uncontaminated dredged sediments from the Northern Adriatic Sea harbors.

Morphological and molecular characterization of mycelia of some Tuber species in pure culture.

• Pure cultures of Tuber maculatum , Tuber melanosporum , Tuber aestivum , Tuber macrosporum , Tuber rufum and Tuber brumale were isolated and characterized by morphological and molecular methods. • The Tuber mycelia were isolated from fruit bodies and molecular identification was performed using specific primers, restriction fragment length polymorphism and/or sequence analyses of the ITS region. • The species grew between 1.1 mm wk -1 and 14 mm wk -1 on the selected medium. The mycelium of different Tuber species showed several common morphological features such as hyphal anastomoses, vesicle formation and hyphal aggregation. Differences were found in the frequency of these morphological features and in the hyphal pattern. The isolated mycelia also showed differences in the hyphal branch angle, septal distance, hyphal diameter and rate of growth of the hyphae. • This result opens the possibility of using pure mycelial cultures of Tuber spp. for experimental purposes and for the commercial production of infected truffle plants.

Effect of nutrient ingestion on glucagon-like peptide 1 (7-36 amide) secretion in human type 1 and type 2 diabetes.

Exogenous glucagon-like peptide 1(GLP-1) bioactivity is preserved in type 2 diabetic patients, resulting the peptide administration in a near-normalization of plasma glucose mainly through its insulinotropic effect. GLP-1 also reduces meal-related insulin requirement in type 1 diabetic patients, suggesting an impairment of the entero-insular axis in both diabetic conditions. To investigate this metabolic dysfunction, we evaluated endogenous GLP-1 concentrations, both at fasting and in response to nutrient ingestion, in 16 type 1 diabetic patients (age = 40.5 +/- 14yr, HbA1C = 7.8 +/- 1.5%), 14 type 2 diabetics (age = 56.5 +/- 13yr, HbA1C = 8.1 +/- 1.8%), and 10 matched controls. In postabsorptive state, a mixed breakfast (230 KCal) was administered to all subjects and blood samples were collected for plasma glucose, insulin, C-peptide and GLP-1 determination during the following 3 hours. In normal subjects, the test meal induced a significant increase of GLP-1 (30', 60': p < 0.01), returning the peptide values towards basal concentrations. In type 2 diabetic patients, fasting plasma GLP-1 was similar to controls (102.1 +/- 1.9 vs. 97.3 +/- 4.01 pg/ml), but nutrient ingestion failed to increase plasma peptide levels, which even decreased during the test (p < 0.01). Similarly, no increase in postprandial GLP-1 occurred in type 1 diabetics, in spite of maintained basal peptide secretion (106.5 +/- 1.5 pg/ml). With respect to controls, the test meal induced in both diabetic groups a significant increase in plasma glucagon levels at 60' (p < 0.01). In conclusion, either in condition of insulin resistance or insulin deficiency chronic hyperglycemia, which is a common feature of both metabolic disorders, could induce a progressive desensitization of intestinal L-cells with consequent peptide failure response to specific stimulation.

Urinary excretion of glucagon-like peptide 1 (GLP-1) 7-36 amide in human type 2 (non-insulin-dependent) diabetes mellitus.

The urinary excretion of insulinotropic glucagon-like peptide 1 (GLP-1) was investigated as an indicator of renal tubular integrity in 10 healthy subjects and in 3 groups of type 2 diabetic patients with different degrees of urinary albumin excretion rate. No significant difference emerged between the groups with respect to age of the patients, known duration of diabetes, metabolic control, BMI, or residual beta-cell pancreatic function. Endogenous creatinine clearance was significantly reduced under conditions of overt diabetic nephropathy, compared with normo and microalbuminuric patients (p < 0.01). Urinary excretion of GLP-1 was significantly higher in normoalbuminuric patients compared to controls (490.4 +/- 211.5 vs. 275.5 +/- 132.1 pg/min; p < 0.05), with further increase under incipient diabetic nephropathy conditions (648.6 +/- 305 pg/min; p < 0.01). No significant difference resulted, in contrast, between macroproteinuric patients and non-diabetic subjects. Taking all patients examined into account, a significant positive relationship emerged between urinary GLP-1 and creatinine clearance (p = 0.004). In conclusion, an early tubular impairment in type 2 diabetes would occur before the onset of glomerular permeability alterations. The tubular dysfunction seems to evolve with the development of persistent microalbuminuria. Finally, the advanced tubular involvement, in terms of urinary GLP1 excretion, under overt diabetic nephropathy conditions would be masked by severe concomitant glomerular damage with the coexistence of both alterations resulting in a peptide excretion similar to control subjects.