Peat-based amendment of soils reduces the complexity of the volatile profile in cultivated black truffles.

BACKGROUND: Truffle cultivation is evolving rapidly and new agronomic practices such as 'truffle nests' (localized peat amendments of the orchard soil) are being developed. Truffle nests improve the shape of truffles and their depth in the soil and reduce the occurrence of insect damage but have also raised concerns about their impact on the ripeness and maturity of the harvested truffles. In this study, the effect of the nests on the volatile organic compounds profile and the aromatic profile of black truffles was evaluated, as well as the existence of perceptible sensorial differences in truffles. For this, truffles growing in nests were compared with truffles growing in the bulk soil of the same host tree. RESULTS: Gas chromatography showed that nest truffles had a less complex volatile organic compound profile than bulk-soil truffles. Olfactometry indicated that nest truffles were associated with higher modified frequency values of odorants corresponding to sulfur-containing compounds. Despite this, sensory evaluation with consumers could not clearly show that nest truffles can be distinguished sensorially from bulk-soil truffles. CONCLUSION: The results prove that soil conditions can influence the aromatic profile of truffles and thus suggest the possibility of managing truffle aroma using agronomic practices. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Intraspecific Competition Results in Reduced Evenness of Tuber melanosporum Mating-Type Abundance from the Nursery Stage.

The highly prized black truffle is a fungus mostly harvested in orchards planted with mycorrhizal seedlings. It is an obligatory outcrossing fungus with a single MAT locus containing two alternative mating-type idiomorphs. In the orchards, at the mycorrhizal level, these mating types are frequently spatially segregated. Some studies found that this segregation was pronounced from the nursery stage, whereas others did not find such a marked segregation. Besides, information on the host tree species and nursery conditions used in Spain, one of the main truffle-producing countries, are very scarce. In this study, we investigated the temporal dynamics of mating types in nursery seedlings of Quercus ilex and Quercus faginea, as well as the influence of cultural conditions in the nursery. Our results indicated that at the plant level, there was a trend for one of the mating types to dominate over the other from the first to the second year in the nursery, in both host species and both nursery conditions tested. However, this segregation process was not so sharp as previously reported. Our results support the hypothesis that intraspecific competition results in reduced evenness of mating-type abundance from the nursery stage, although almost all seedlings maintained both mating types and, at the seedling batch scale, the occurrence of both mating types was roughly balanced.

Glyphosate treatments for weed control affect early stages of root colonization by Tuber melanosporum but not secondary colonization.

The cultivation of the ectomycorrhizal fungus Tuber melanosporum has considerably spread in recent years throughout the world. During the first years of truffle cultivation, weed control is a key practice to improve the establishment of host trees and the proliferation of the fungus in the soil. Glyphosate is nowadays the most commonly used herbicide in Spanish truffle orchards. We explored the effect of glyphosate on the proliferation of T. melanosporum mycorrhizae, on extraradical mycelium and on the inoculum potential of T. melanosporum spores in greenhouse experiments using Quercus ilex seedlings as host plants. No detrimental effect on the secondary infection of T. melanosporum was found after three sequential glyphosate applications in young seedlings during one vegetative period. Instead, a change in the distribution of fine roots and T. melanosporum mycorrhizae along soil depth was observed. On the other hand, results indicate that high application rates of glyphosate hinder the infectivity of T. melanosporum spore inoculum, without apparent impact on the host performance. Our results suggest that glyphosate has the potential to jeopardise the role of the soil spore bank as inoculum source for the colonisation of new roots, also raising the question of whether glyphosate could hinder the presumed role of spores in sexual mating.

Short Leukocyte Telomere Length Is Associated With Cardioembolic Stroke Risk in Patients With Atrial Fibrillation.

BACKGROUND AND PURPOSE: The risk of cardioembolic stroke in patients with atrial fibrillation (AF) cannot be accurately assessed and novel tools are needed to improve prediction. We hypothesize that telomere shortening constitutes a novel risk factor for cardioembolic stroke in patients with AF. METHODS: The peripheral blood leukocyte telomere length (LTL) was determined by real-time polymerase chain reaction in 187 patients with AF, 93 of them without stroke history and 94 of them having suffered 1 cardioembolic stroke. Percentiles were calculated according to LTL values in the nonstroke group to estimate the cardioembolic stroke risk associated with LTL using logistic regression models. RESULTS: Short LTL values were independently and dose-dependently associated with an increased risk of cardioembolic stroke, with an odds ratio (95% confidence interval) of 2.93 (1.24-6.94) and 6.26 (2.01-19.52), respectively, for sex, hypertension, diabetes mellitus, heart failure, and age-adjusted models using the LTL 10th and 5th percentile cut-offs, respectively. CONCLUSIONS: Telomere shortening is associated with cardioembolic stroke risk in patients with AF. Prospective studies are encouraged to establish the value of LTL to improve prediction tools to categorize cardioembolic stroke risk in AF.

Receptor of activated protein C promotes metastasis and correlates with clinical outcome in lung adenocarcinoma.

RATIONALE: Efficient metastasis requires survival and adaptation of tumor cells to stringent conditions imposed by the extracellular milieu. Identification of critical survival signaling pathways in tumor cells might unveil novel targets relevant in disease progression. OBJECTIVES: To investigate the contribution of activated protein C (APC) and its receptor (endothelial protein C receptor [EPCR]) in animal models of lung cancer metastasis and in patients with lung adenocarcinoma. METHODS: Signaling pathway triggered by APC/EPCR and its relevance in apoptosis was studied in vitro. Functional significance was assessed by silencing and blocking antibodies in several in vivo models of lung cancer metastasis in athymic nude Foxn1(nu) mice. We examined EPCR levels using a microarray dataset of 107 patients. Immunohistochemical analysis was performed in an independent cohort of 295 patients with lung adenocarcinoma. MEASUREMENTS AND MAIN RESULTS: The effects of APC binding to EPCR rapidly triggered Akt and extracellular signal-regulated kinase signaling pathways, leading to attenuated in vitro apoptosis. In vivo, silencing of EPCR expression or blocking APC/EPCR interaction reduced infiltration in the target organ, resulting in impaired prometastatic activity. Moreover, overexpression of EPCR induced an increased metastatic activity to target organs. Analysis of clinical samples showed a robust association between high EPCR levels and poor prognosis, particularly in stage I patients. CONCLUSIONS: EPCR and its ligand APC promote cell survival that contributes to tumor cell endurance to stress favoring prometastatic activity of lung adenocarcinoma. EPCR/APC is a novel target of relevance in the clinical outcome of early-stage lung cancer.

Sequential application of inoculation methods improves mycorrhization of Quercus ilex seedlings by Tuber melanosporum.

The use of mycorrhized seedlings has been critical to the success of modern truffle cultivation, which nowadays supplies most European black truffles (Tuber melanosporum) to markets. Ascospore inoculation has been traditionally used to produce these seedlings, but little scientific information is publicly available on the inoculation methods applied or on the possibility of combining them. We evaluated the potential of sequential inoculation for the controlled colonization of holm oak fine roots by T. melanosporum, with two different nursery assays and a full factorial design. Three inoculation methods were sequentially applied: radicle inoculation, inoculation of the substrate in seedling trays and inoculation of the substrate in the final pot. Despite the differences in the results of the two assays, which suggest that cultivation conditions and/or the timing of nursery operations may influence the relative effectiveness of inoculation methods, the sequential application appeared as an effective and realistic alternative for commercial inoculation of holm oak seedlings with T. melanosporum. The increase in the amount of inoculum applied with each inoculation method improved the mycorrhizal colonization of seedlings, whereas separately none of the inoculation methods appeared clearly superior to the other ones. The depth distribution of truffle mycorrhizae pointed that the inoculation in the final pot was more effective than other methods in lower parts of the root system, whereas the early inoculation appeared more effective to reduce the occurrence of the opportunist ectomycorrhizal fungus Sphaerosporella brunnea.

VEGF121b and VEGF165b are weakly angiogenic isoforms of VEGF-A.

BACKGROUND: Different isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189) have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGF(xxx)b, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential. RESULTS: Recombinant VEGF121/165b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells with VEGF121/165b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1 empty vector, as control) and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGF(xxx)b isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGF(xxx)b and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033) between VEGF(xxx)b and total VEGF-A was found. CONCLUSIONS: Our results demonstrate that VEGF121/165b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGF(xxx)b isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken into account when considering a possible use of VEGF121/165b-based therapies in patients.

The functional properties of a truncated form of endothelial cell protein C receptor generated by alternative splicing.

BACKGROUND: A soluble form of endothelial cell protein C receptor (sEPCR) is generated by shedding of the cellular form. sEPCR binds to protein C and factor VIIa and inhibits both the activation of protein C and the activity of activated protein C and factor VIIa. High sEPCR levels may increase the risk of thrombosis. We wanted to explore the possibility of detecting soluble endothelial cell protein C receptor forms generated by alternative splicing. DESIGN AND METHODS: Reverse transcriptase polymerase chain reaction was used to look for new forms of endothelial cell protein C receptor. A yeast expression system was used to generate sufficient amounts of the distinct sEPCR forms. Surface plasmon resonance experiments, chromogenic assays, clotting assays and immunoassays were subsequently performed to characterize a new form of sEPCR that was found. RESULTS: We demonstrated, by reverse transcriptase polymerase chain reaction and sequencing, the existence of a new, soluble form of endothelial cell protein C receptor generated by alternative splicing, in which the transmembrane region is replaced by a 56-residue tail (tEPCR). Its cDNA was present in human umbilical vein endothelial cells and in most tissues as well as in lung cancer cells. tEPCR was not located in the membrane of transfected cells. We demonstrated that tEPCR binds to protein C and factor VIIa. tEPCR blocked the generation of activated protein C and inhibited the activity of both activated protein C and factor VIIa. tEPCR was detected, by immunoassays, in the supernatant of lung cancer cells and human umbilical vein endothelial cells. CONCLUSIONS: A truncated form of alternatively spliced endothelial cell protein C receptor was detected in the endothelium and cancer cells. tEPCR behaves as sEPCR generated by shedding of the cellular endothelial cell protein C receptor.

Inducing heat shock protein 70 expression provides a robust antithrombotic effect with minimal bleeding risk.

Antithrombotic medications target coagulation factors. Their use is associated with an increased bleeding risk. Safer drugs are needed. The heat shock protein 70 (Hsp70) exhibits antithrombotic properties that do not influence bleeding. By using murine models, we aimed to test the hypothesis that overexpressing Hsp70 with CM-695, a first in class dual inhibitor of HDAC6 and phosphodiesterase 9, protects against thrombosis while leaves bleeding tendency unaltered. CM-695 was used to induce Hsp70 overexpression. Hsp70 overexpressing mice were submitted to three thrombosis-triggering procedures. The ferric chloride carotid artery model was used to compare the antithrombotic role of CM-695 and rivaroxaban, a direct oral anticoagulant. The mouse tail transection model was used to compare the bleeding tendency upon CM-695 or rivaroxaban administration. Intraperitoneal (i. p.) 20 mg/kg CM-695 increased Hsp70 expression markedly in the murine aortic tissue. This treatment delayed thrombosis in the collagen/epinephrine [p=0.04 (Log-Rank test), n=10], Rose Bengal/laser [median vessel occlusion time (OT): 58.6 vs 39.0 minutes (min) in the control group (CG), p=0.008, n≥10] and ferric chloride (OT: 14.7 vs 9.2 min in the CG, p=0.032, n≥10) models. I.p. 80 mg/kg CM-695 (n≥9) and intravenous 3 mg/kg rivaroxaban (n≥8) significantly delayed thrombosis. CM-695 did not induce bleeding [median bleeding time (BT): 8.5 vs 7.5 min in the CG, n≥10]. However, BT was dramatically increased by rivaroxaban (30.0 vs 13.7 min in the CG, p=0.001, n=10). In conclusion, CM-695 is a new antithrombotic small molecule devoid of bleeding risk that may be envisioned as a useful clinical tool.

Hsp70 protects from stroke in atrial fibrillation patients by preventing thrombosis without increased bleeding risk.

AIMS: Atrial fibrillation (AF) is a major risk factor for cardio-embolic stroke. Anticoagulant drugs are effective in preventing AF-related stroke. However, the high frequency of anticoagulant-associated major bleeding is a major concern. This study sought to identify new targets to develop safer antithrombotic therapies. METHODS AND RESULTS: Here, microarray analysis in peripheral blood cells in eight patients with AF and stroke and eight AF subjects without stroke brought to light a stroke-related gene expression pattern. HSPA1B, which encodes for heat-shock protein 70 kDa (Hsp70), was the most differentially expressed gene. This gene was down-regulated in stroke subjects, a finding confirmed further in an independent AF cohort of 200 individuals. Hsp70 knock-out mice subjected to different thrombotic challenges developed thrombosis significantly earlier than their wild-type (WT) counterparts. Remarkably, the tail bleeding time was unchanged. Accordingly, both TRC051384 and tubastatin A, i.e. two Hsp70 inducers via different pathways, delayed thrombus formation in WT mice, the tail bleeding time still being unaltered. Most interestingly, Hsp70 inducers did not increase the bleeding risk even when aspirin was concomitantly administered. Hsp70 induction was associated with an increased vascular thrombomodulin expression and higher circulating levels of activated protein C upon thrombotic stimulus. CONCLUSIONS: Hsp70 induction is a novel approach to delay thrombus formation with minimal bleeding risk, and is especially promising for treating AF patients and in other situations where there is also a major bleeding hazard.

Total syntheses of four stereoisomers of 4alpha-hydroxy-1beta,7beta-peroxy- 10betaH-guaia-5-ene.

[reaction: see text]. The first total syntheses of four stereoisomers of 4alpha-hydroxy-1beta,7beta-peroxy-10betaH-guaia-5-ene are reported starting from the readily available (+)-dihydrocarvone. These compounds have been synthesized from dienes (-)-isoguaiene and (-)-10-epi-isoguaiene by tandem ene hydroperoxylation-[4 + 2] cycloaddition with O(2) followed by selective reduction. The structure of the natural 4alpha-hydroxy-1beta,7beta-peroxy-10betaH-guaia-5-ene isolated from Liabum floribundum has been confirmed.

Is EPCR a multi-ligand receptor? Pros and cons.

In the last decade, the endothelial cell protein C/activated protein C receptor (EPCR) has received considerable attention. The role initially attributed to EPCR, i.e. the enhancement of protein C (PC) activation by the thrombin-thrombomodulin complex on the surface of the large vessels, although important, did not go beyond the haemostasis scenario. However, the discovery of the cytoprotective, anti-inflammatory and anti-apoptotic features of the activated PC (APC) and the required involvement of EPCR for APC to exert such actions did place the receptor in a privileged position in the crosstalk between coagulation and inflammation. The last five years have shown that PC/APC are not the only molecules able to interact with EPCR. Factor VII/VIIa (FVII/VIIa) and factor Xa (FXa), two other serine proteases that play a central role in haemostasis and are also involved in signalling processes influencing wound healing, tissue remodelling, inflammation or metastasis, have been reported to bind to EPCR. These observations have paved the way for an exploration of unsuspected new roles for the receptor. This review aims to offer a new image of EPCR in the light of its extended panel of ligands. A brief update of what is known about the APC-evoked EPCR-dependent cell signalling mechanisms is provided, but special care has been taken to assemble all the information available about the interaction of EPCR with FVII/VIIa and FXa.

A bioinspired approach to tri-nor-guaianes. synthesis of (-)-clavukerin A.

A bioinspired approach to tri-nor-guaianes by degradation of the C-7 side chain of related guaia-11-enes is described. In this approach (-)-clavukerin A (1) is obtained by selective ozonolysis-Criegge rearrangement of (+)-1alphaH,7alphaH,10alphaH-guaia-4,11-dien-3-one (4) to afford 7beta-hydroxy and 7beta-acetoxy tri-nor-guaiane derivatives 6 and 7, respectively, which after elimination and deoxygenation give the title compound. The starting guaiadienone is readily obtained from commercially available santonin or (+)-dihydrocarvone.

Syntheses of (+)-alismoxide and (+)-4-epi-alismoxide.

The first total syntheses of (+)-alismoxide and (+)-4-epi-alismoxide are reported. Formal chemo-, regio-, and stereoselective addition of water to 10alpha-acetoxy-1alphaH,5betaH-guaia-3,6-diene afforded the target compounds after reduction. The absolute stereochemistry of (+)-alismoxide has been established. The low [alpha](D) +8.6 value indicates that significant amounts of alismoxide result from biosynthetic processes. Furthermore, the structure of the natural guaienediol isolated from Silphium perfoliatum has been corrected to (-)-alismoxide.

Differential effects of 2C9*3 and 2C9*2 variants of cytochrome P-450 CYP2C9 on sensitivity to acenocoumarol.

The 2C9*3 and 2C9*2 polymorphisms of cytochrome P-450 CYP2C9 are associated with hypersensitivity to warfarin and bleeding. The effect of these polymorphisms on sensitivity to acenocoumarol is unknown. Three groups of patients, with low, medium, or high acenocoumarol-dose requirements, were studied. Age influenced the acenocoumarol sensitivity. Bearing the 2C9*3 allele was associated with the need for a lower acenocoumarol dose (odds ratio [OR], 6.02; 95% confidence interval [CI], 1.50-24.18); 80% of carriers of the 2C9*3 allele required a low dose. The 2C9*2 allele was associated with a lower acenocoumarol-dose requirement (OR, 2.70; 95% CI, 1.11-6.58) because of a reduced risk of the need for a high acenocoumarol dose (4.8% of the patients in the high-dose group carried the 2C9*2 allele versus 34.1% and 30.2%, respectively, in the medium-dose and low-dose groups). Therefore, carriers of 2C9*3 may need a low initial loading dose of acenocoumarol. Because acenocoumarol sensitivity with the 2C9*2 variant does not seem to be clinically relevant, the drug could be an alternative to warfarin in 2C9*2 carriers.