Sugar beet PMT5a and STP13 carriers suitable for proton-driven plasma membrane sucrose and glucose import in taproots.

Sugar beet (Beta vulgaris) is the major sugar-producing crop in Europe and Northern America, as the taproot stores sucrose at a concentration of around 20%. Genome sequence analysis together with biochemical and electrophysiological approaches led to the identification and characterization of the TST sucrose transporter driving vacuolar sugar accumulation in the taproot. However, the sugar transporters mediating sucrose uptake across the plasma membrane of taproot parenchyma cells remained unknown. As with glucose, sucrose stimulation of taproot parenchyma cells caused inward proton fluxes and plasma membrane depolarization, indicating a sugar/proton symport mechanism. To decipher the nature of the corresponding proton-driven sugar transporters, we performed taproot transcriptomic profiling and identified the cold-induced PMT5a and STP13 transporters. When expressed in Xenopus laevis oocytes, BvPMT5a was characterized as a voltage- and H+-driven low-affinity glucose transporter, which does not transport sucrose. In contrast, BvSTP13 operated as a high-affinity H+/sugar symporter, transporting glucose better than sucrose, and being more cold-tolerant than BvPMT5a. Modeling of the BvSTP13 structure with bound mono- and disaccharides suggests plasticity of the binding cleft to accommodate the different saccharides. The identification of BvPMT5a and BvSTP13 as taproot sugar transporters could improve breeding of sugar beet to provide a sustainable energy crop.

Hypothermic sepsis in time since death estimation - a case report.

Both hyper- and hypothermia are problematic in temperature based forensic time since death estimation. Hyperthermia may occur in infection, traumatic brain injury, and intoxication. Hypothermia is encountered predominantly in exposure. Sepsis may present itself clinically as hypothermic. Sepsis is not uncommon in the forensic setting and mostly occurs in the context of malpractice accusations. There is usually little overlap between sepsis and typical forensic time since death estimation scenarios of violent or otherwise suspicious deaths. In the presented case, hypothermia and time since death estimations did collide. An inmate was found dead in his jail cell. Wardens claimed they had visually approached him alive relatively shortly prior. Rectal temperature measurements, using two separate crime scene thermometers as well as temperature loggers, revealed low rectal temperature at relatively high ambient temperature. These findings suggested a much longer postmortem interval and consequently raised doubts about the stated timeline. The wardens' claims were however confirmed by camera recordings, which also allowed a reasonable estimate of the true time of death. The cause of death was confirmed as septic organ failure at autopsy, which explained low rectal temperature. The presence of WISCHNEWSKI-spots was noted. When the PRISM-method was applied to the temperature recordings, low rectal temperature at the time of death was detected successfully. However, adaptation of the underlying equation for lower "starting temperature" did not produce satisfactory results. It is concluded that even though hypothermia at the time of death may possibly be detected from temperature data, attempts at time since death estimation for cases of hypothermia by adaptation of the equation should be avoided.

"Scalping" in the context of criminal dismemberment and mutilation-a case report and review of the literature.

We report on a case of criminal dismemberment and attempted scalping of a homicide victim with a "Mohawk" haircut. Case findings are presented. A review of the literature was performed for scalping in its historical and cultural context and particularly in criminal dismemberment and mutilation: Historically, scalping was prevalent in many ancient cultures around the world, where scalps were taken as trophies or "proof of kill", much like shrunken heads, trophy skulls, and other artefacts. Scalping was particularly widespread in Northern America in the context of tribal warfare, both before and after colonization. The iconic "Mohawk" haircut is closely linked with scalping, as it was meant to taunt the enemy. In the modern forensic context, scalping constitutes a form of criminal mutilation. However, cases of criminal dismemberment and mutilation are rare in forensic casework. Our literature review revealed a low number of scalping in criminal dismemberment and mutilation cases. The documentation was overall poor. Positioning scalping within the classification of criminal mutilation and dismemberment was difficult. In literature, even though case numbers were small, the majority of "textbook scalping" cases were German. The presented case, to our best knowledge, is the first modern-day photo-documented case of (attempted) scalping, even more so of a person wearing a "Mohawk".

Extended Coagulation Profiling in Isolated Traumatic Brain Injury: A CENTER-TBI Analysis.

BACKGROUND: Trauma-induced coagulopathy in traumatic brain injury (TBI) remains associated with high rates of complications, unfavorable outcomes, and mortality. The underlying mechanisms are largely unknown. Embedded in the prospective multinational Collaborative European Neurotrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study, coagulation profiles beyond standard conventional coagulation assays were assessed in patients with isolated TBI within the very early hours of injury. METHODS: Results from blood samples (citrate/EDTA) obtained on hospital admission were matched with clinical and routine laboratory data of patients with TBI captured in the CENTER-TBI central database. To minimize confounding factors, patients with strictly isolated TBI (iTBI) (n = 88) were selected and stratified for coagulopathy by routine international normalized ratio (INR): (1) INR < 1.2 and (2) INR ≥ 1.2. An INR > 1.2 has been well adopted over time as a threshold to define trauma-related coagulopathy in general trauma populations. The following parameters were evaluated: quick's value, activated partial thromboplastin time, fibrinogen, thrombin time, antithrombin, coagulation factor activity of factors V, VIII, IX, and XIII, protein C and S, plasminogen, D-dimer, fibrinolysis-regulating parameters (thrombin activatable fibrinolysis inhibitor, plasminogen activator inhibitor 1, antiplasmin), thrombin generation, and fibrin monomers. RESULTS: Patients with iTBI with INR ≥ 1.2 (n = 16) had a high incidence of progressive intracranial hemorrhage associated with increased mortality and unfavorable outcome compared with patients with INR < 1.2 (n = 72). Activity of coagulation factors V, VIII, IX, and XIII dropped on average by 15-20% between the groups whereas protein C and S levels dropped by 20%. With an elevated INR, thrombin generation decreased, as reflected by lower peak height and endogenous thrombin potential (ETP), whereas the amount of fibrin monomers increased. Plasminogen activity significantly decreased from 89% in patients with INR < 1.2 to 76% in patients with INR ≥ 1.2. Moreover, D-dimer levels significantly increased from a mean of 943 mg/L in patients with INR < 1.2 to 1,301 mg/L in patients with INR ≥ 1.2. CONCLUSIONS: This more in-depth analysis beyond routine conventional coagulation assays suggests a counterbalanced regulation of coagulation and fibrinolysis in patients with iTBI with hemostatic abnormalities. We observed distinct patterns involving key pathways of the highly complex and dynamic coagulation system that offer windows of opportunity for further research. Whether the changes observed on factor levels may be relevant and explain the worse outcome or the more severe brain injuries by themselves remains speculative.

Endothelial glycocalyx in traumatic brain injury associated coagulopathy: potential mechanisms and impact.

Traumatic brain injury (TBI) remains one of the leading causes of death and disability worldwide; more than 10 million people are hospitalized for TBI every year around the globe. While the primary injury remains unavoidable and not accessible to treatment, the secondary injury which includes oxidative stress, inflammation, excitotoxicity, but also complicating coagulation abnormalities, is potentially avoidable and profoundly affects the therapeutic process and prognosis of TBI patients. The endothelial glycocalyx, the first line of defense against endothelial injury, plays a vital role in maintaining the delicate balance between blood coagulation and anticoagulation. However, this component is highly vulnerable to damage and also difficult to examine. Recent advances in analytical techniques have enabled biochemical, visual, and computational investigation of this vascular component. In this review, we summarize the current knowledge on (i) structure and function of the endothelial glycocalyx, (ii) its potential role in the development of TBI associated coagulopathy, and (iii) the options available at present for detecting and protecting the endothelial glycocalyx.

Global Characterisation of Coagulopathy in Isolated Traumatic Brain Injury (iTBI): A CENTER-TBI Analysis.

BACKGROUND: Trauma-induced coagulopathy in patients with traumatic brain injury (TBI) is associated with high rates of complications, unfavourable outcomes and mortality. The mechanism of the development of TBI-associated coagulopathy is poorly understood. METHODS: This analysis, embedded in the prospective, multi-centred, observational Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study, aimed to characterise the coagulopathy of TBI. Emphasis was placed on the acute phase following TBI, primary on subgroups of patients with abnormal coagulation profile within 4 h of admission, and the impact of pre-injury anticoagulant and/or antiplatelet therapy. In order to minimise confounding factors, patients with isolated TBI (iTBI) (n = 598) were selected for this analysis. RESULTS: Haemostatic disorders were observed in approximately 20% of iTBI patients. In a subgroup analysis, patients with pre-injury anticoagulant and/or antiplatelet therapy had a twice exacerbated coagulation profile as likely as those without premedication. This was in turn associated with increased rates of mortality and unfavourable outcome post-injury. A multivariate analysis of iTBI patients without pre-injury anticoagulant therapy identified several independent risk factors for coagulopathy which were present at hospital admission. Glasgow Coma Scale (GCS) less than or equal to 8, base excess (BE) less than or equal to - 6, hypothermia and hypotension increased risk significantly. CONCLUSION: Consideration of these factors enables early prediction and risk stratification of acute coagulopathy after TBI, thus guiding clinical management.

Insights into early ontogenesis: characterization of stress and development key genes of pikeperch (Sander lucioperca) in vivo and in vitro.

There are still numerous difficulties in the successful farming of pikeperch in the anthropogenic environment of various aquaculture systems, especially during early developmental steps in the hatchery. To investigate the physiological processes involved on the molecular level, we determined the basal expression patterns of 21 genes involved in stress and immune responses and early ontogenesis of pikeperch between 0 and 175 days post hatch (dph). Their transcription patterns most likely reflect the challenges of growth and feed conversion. The gene coding for apolipoprotein A (APOE) was strongly expressed at 0 dph, indicating its importance for yolk sac utilization. Genes encoding bone morphogenetic proteins 4 and 7 (BMP4, BMP7), creatine kinase M (CKM), and SRY-box transcription factor 9 (SOX9) were highly abundant during the peak phases of morphological changes and acclimatization processes at 4-18 dph. The high expression of genes coding for peroxisome proliferator-activated receptors alpha and delta (PPARA, PPARD) at 121 and 175 dph, respectively, suggests their importance during this strong growth phase of juvenile stages. As an alternative experimental model to replace further in vivo investigations of ontogenetically important processes, we initiated the first approach towards a long-lasting primary cell culture from whole pikeperch embryos. The present study provides a set of possible biomarkers to support the monitoring of pikeperch farming and provides a first basis for the establishment of a suitable cell model of this emerging aquaculture species.

A Modified Tripeptide Motif of RS1 (RSC1A1) Down-Regulates Exocytotic Pathways of Human Na+-d-glucose Cotransporters SGLT1, SGLT2, and Glucose Sensor SGLT3 in the Presence of Glucose.

A domain of protein RS1 (RSC1A1) called RS1-Reg down-regulates the plasma membrane abundance of Na+-d-glucose cotransporter SGLT1 by blocking the exocytotic pathway at the trans-Golgi. This effect is blunted by intracellular glucose but prevails when serine in a QSP (Gln-Ser-Pro) motif is replaced by glutamate [RS1-Reg(S20E)]. RS1-Reg binds to ornithine decarboxylase (ODC) and inhibits ODC in a glucose-dependent manner. Because the ODC inhibitor difluoromethylornithine (DFMO) acts like RS1-Reg(S20E), and DFMO and RS1-Reg(S20E) are not cumulative, we raised the hypothesis that RS1-Reg(S20E) down-regulates the exocytotic pathway of SGLT1 at the trans-Golgi by inhibiting ODC. We investigated whether QEP down-regulates human SGLT1 (hSGLT1) like hRS1-Reg(S20E) and whether human Na+-d-glucose cotransporter hSGLT2 and the human glucose sensor hSGLT3 are also addressed. We expressed hSGLT1, hSGLT1 linked to yellow fluorescent protein (hSGLT1-YFP), hSGLT2-YFP and hSGLT3-YFP in oocytes of Xenopus laevis, injected hRS1-Reg(S20E), QEP, DFMO, and/or α-methyl-d-glucopyranoside (AMG), and measured AMG uptake, glucose-induced currents, and plasma membrane-associated fluorescence after 1 hour. We also performed in vitro AMG uptake measurements into small intestinal mucosa of mice and human. The data indicate that QEP down-regulates the exocytotic pathway of SGLT1 similar to hRS1-Reg(S20E). Our results suggests that both peptides also down-regulate hSGLT2 and hSGLT3 via the same pathway. Thirty minutes after application of 5 mM QEP in the presence of 5 mM d-glucose, hSGLT1-mediated AMG uptake into small intestinal mucosa was decreased by 40% to 50%. Thus oral application of QEP in a formulation that optimizes uptake into enterocytes but prevents entry into the blood is proposed as novel antidiabetic therapy.

Effect of adipocyte-derived IGF-I on adipose tissue mass and glucose metabolism in the Berlin Fat Mouse.

Besides liver, IGF-I is expressed in adipose tissue. However, the effects of this local IGF-I on adipose tissue and metabolism are unclear. We generated adipocyte-specific knock-out mice on the background of the Berlin Fat Mouse Inbred (BFMI) line to evaluate the contribution of adipocyte-IGF-I on glucose metabolism and adipose tissue development. BFMI mice are obese, non-diabetic with elevated plasma insulin and IGF-I concentration. The knock-out in adipocytes led to a total white adipose tissue expression of 50-60% due to unaltered Igf-1 expression in stromavascular cells. The lack of IGF-I from adipocytes did not alter plasma IGF-I concentration. BFMIChr3-Igf-I-KOQ-AT mice had reduced adipose tissue mass in most depots. During oral glucose tolerance tests, BFMIChr3-Igf-I-KOQ-AT mice showed an impaired glucose clearance (p = .03). Interestingly, insulin action was enhanced during insulin tolerance tests (p = .05). In conclusion, adipocyte-specific IGF-I ablation in obese BFMI mice results in reduced adipose tissue mass and thereby alters glucose metabolism.

The desert plant Phoenix dactylifera closes stomata via nitrate-regulated SLAC1 anion channel.

Date palm Phoenix dactylifera is a desert crop well adapted to survive and produce fruits under extreme drought and heat. How are palms under such harsh environmental conditions able to limit transpirational water loss? Here, we analysed the cuticular waxes, stomata structure and function, and molecular biology of guard cells from P. dactylifera. To understand the stomatal response to the water stress phytohormone of the desert plant, we cloned the major elements necessary for guard cell fast abscisic acid (ABA) signalling and reconstituted this ABA signalosome in Xenopus oocytes. The PhoenixSLAC1-type anion channel is regulated by ABA kinase PdOST1. Energy-dispersive X-ray analysis (EDXA) demonstrated that date palm guard cells release chloride during stomatal closure. However, in Cl- medium, PdOST1 did not activate the desert plant anion channel PdSLAC1 per se. Only when nitrate was present at the extracellular face of the anion channel did the OST1-gated PdSLAC1 open, thus enabling chloride release. In the presence of nitrate, ABA enhanced and accelerated stomatal closure. Our findings indicate that, in date palm, the guard cell osmotic motor driving stomatal closure uses nitrate as the signal to open the major anion channel SLAC1. This initiates guard cell depolarization and the release of anions together with potassium.

Protein RS1 (RSC1A1) Downregulates the Exocytotic Pathway of Glucose Transporter SGLT1 at Low Intracellular Glucose via Inhibition of Ornithine Decarboxylase.

Na+-d-glucose cotransporter 1 (SGLT1) is rate-limiting for glucose absorption in the small intestine. Shortly after intake of glucose-rich food, SGLT1 abundance in the luminal membrane of the small intestine is increased. This upregulation occurs via glucose-induced acceleration of the release of SGLT1-containing vesicles from the trans-Golgi network (TGN), which is regulated by a domain of protein RS1 (RSC1A1) named RS1-Reg. Dependent on phosphorylation, RS1-Reg blocks release of vesicles containing SGLT1 or concentrative nucleoside transporter 1. The hypothesis has been raised that RS1-Reg binds to different receptor proteins at the TGN, which trigger release of vesicles with different transporters. To identify the presumed receptor proteins, two-hybrid screening was performed. Interaction with ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme of polyamine synthesis, was observed and verified by immunoprecipitation. Binding of RS1-Reg mutants to ODC1 was characterized using surface plasmon resonance. Inhibition of ODC1 activity by RS1-Reg mutants and the ODC1 inhibitor difluoromethylornithine (DFMO) was measured in the absence and presence of glucose. In addition, short-term effects of DFMO, RS1-Reg mutants, the ODC1 product putrescine, and/or glucose on SGLT1 expressed in oocytes of Xenopus laevis were investigated. High-affinity binding of RS1-Reg to ODC1 was demonstrated, and evidence for a glucose binding site in ODC1 was provided. Binding of RS1-Reg to ODC1 inhibits the enzymatic activity at low intracellular glucose, which is blunted at high intracellular glucose. The data suggest that generation of putrescine by ODC1 at the TGN stimulates release of SGLT1-containing vesicles. This indicates a biomedically important role of ODC1 in regulation of glucose homeostasis.

Comparison of hemostatic dressings for superficial wounds using a new spectrophotometric coagulation assay.

BACKGROUND: Due to demographical changes the number of elderly patients depending on oral anticoagulation is expected to rise. Prolonged bleeding times in case of traumatic injuries represent the drawback of these medications, not only in major trauma, but also in superficial wounds. Therefore, dressings capable of accelerating coagulation onset and shortening bleeding times are desirable for these patients. METHODS: The hemostatic potential and physical properties of different types of superficial wound dressings (standard wound pad, two alginates, chitosan, collagen (Lyostypt(®)), oxidized cellulose, and QuikClot(®)) were assessed in vitro. For this purpose the clotting times of blood under the influence of the named hemostatics from healthy volunteers were compared with Marcumar(®) or ASS(®) treated patients. For that, a newly developed coagulation assay based on spectrophotometric extinction measurements of thrombin activity was used. RESULTS: The fastest coagulation onset was observed for oxidized cellulose (Ø 2.47 min), Lantor alginate-L (Ø 2.50 min) and QuikClot(®) (Ø 3.01 min). Chitosan (Ø 5.32 min) and the collagen Lyostypt(®) (Ø 7.59 min) induced clotting comparatively late. Regarding physical parameters, QuikClot(®) showed the lowest absorption capacity and speed while chitosan and both alginates achieved the highest. While oxidized cellulose displayed the best clotting times, unfortunately it also revealed low absorption capacity. CONCLUSIONS: All tested specimens seem to induce clotting independently from the administered type of oral anticoagulant, providing the possibility to neglect the disadvantage in clotting times arising from anticoagulation on a local basis. QuikClot(®), oxidized cellulose and unexpectedly alginate-L were superior to chitosan and Lyostypt(®). Due to its additional well-known positive effect on wound healing alginate-L should be considered for further investigations.

Relationship between obesity phenotypes and genetic determinants in a mouse model for juvenile obesity.

Obesity, a state of imbalance between lean mass and fat mass, is important for the etiology of diseases affected by the interplay of multiple genetic and environmental factors. Although genome-wide association studies have repeatedly associated genes with obesity and body weight, the mechanisms underlying the interaction between the muscle and adipose tissues remain unknown. Using 351 mice (at 10 wk of age) of an intercross population between Berlin Fat Mouse Inbred (BFMI) and C57BL/6NCrl (B6N) mice, we examined the causal relationships between genetic variations and multiple traits: body lean mass and fat mass, adipokines, and bone mineral density. Furthermore, evidence from structural equation modeling suggests causality among these traits. In the BFMI model, juvenile obesity affects lean mass and impairs bone mineral density via adipokines secreted from the white adipose tissues. While previous studies have indicated that lean mass has a causative effect on adiposity, in the Berlin Fat Mouse model that has been selected for juvenile obesity (at 9 wk of age) for >90 generations, however, the causality is switched from fat mass to lean mass. In addition, linkage studies and statistical modeling have indicated that quantitative trait loci on chromosomes 5 and 6 affect both lean mass and fat mass. These lines of evidence indicate that the muscle and adipose tissues interact with one another and the interaction is modulated by genetic variations that are shaped by selections. Experimental examinations are necessary to verify the biological role of the inferred causalities.

Infrastructure, logistics and clinical practice management of acute trauma hemorrhage and coagulopathy: a survey across German trauma centers.

PURPOSE: Early detection and management of acute trauma hemorrhage and coagulopathy have been associated with improved outcomes, but local infrastructure, logistics and clinical strategies may differ. METHODS: To assess local differences in infrastructure, logistics and clinical management of acute trauma hemorrhage and coagulopathy we have conducted a web-based survey amongst clinicians working in DGU®-certified supraregional, regional and local trauma centers. RESULTS: 137/1875 respondents completed the questionnaire yielding a response rate of 7.3%. The majority specified to work as head of department or senior consultant (95%) in trauma/orthopedic surgery (80%) of supraregional (38%), regional (34%) or local (27%) trauma centers. Conventional coagulation assays are most frequently used to monitor bleeding trauma patients. Only half of the respondents (53%) rely on extended coagulation tests, e.g. viscoelastic hemostatic assays. Tests to assess preinjury use of direct oral anticoagulants and platelet inhibitors are still not widely available and vary according to level of care. Conventional blood products are widely available but there remain differences between trauma centers of different level of care to access other hemostatic therapies, e.g. coagulation factor concentrates. Trauma centers of higher level of care are more likely to implement treatment protocols. CONCLUSION: This survey confirms still existing differences in infrastructure, logistics and clinical practice management for the detection of acute trauma hemorrhage and coagulopathy amongst DGU®-certified supraregional, regional and local trauma centers. Further work is recommended to locally implement diagnostics, therapies and treatment algorithms compliant to current guidelines to ensure the best possible outcomes in bleeding trauma patients.

Plasmatic coagulation profile after major traumatic injury: a prospective observational study.

PURPOSE: Uncontrolled hemorrhage is still the major cause of preventable death after trauma and is aggravated by trauma-induced coagulopathy (TIC). The underlying pathophysiology of TIC is still elusive, but several key effectors such as the thrombin-generation capacity, the protein C (PC) pathway, and the fibrinolytic activity could be identified. The aim of this prospective observational study was to investigate plasma coagulation markers attributed to reflect the course of TIC and to identify the mechanisms being responsible for the coagulopathy after major trauma. METHODS: Seventy-three consecutive patients after major trauma and admission to a level-1-trauma unit were included to the study. During early trauma management, extended coagulation testing including the measurement of circulating thrombin markers and activated PC (APC) was performed and correlated with standard shock parameters and the patients' clinical course and outcome. RESULTS: In contrast to standard coagulation parameters, thrombin markers and APC were found to be increased in correlation with injury severity. Even in patients with lower impact mechanisms, early endogenous accumulation of thrombin markers and APC (ISS < 16: 0.5 ng/ml; ISS ≥ 16-26: 1.5 ng/ml; ISS > 26: 4.1 ng/ml) were observed. Furthermore, APC showed ISS- and injury-dependent patterns while ROC curve analysis revealed that especially APC plasma levels were predictive for coagulopathy and general patient outcome. CONCLUSION: Increased levels of APC and thrombin markers in patients after major trauma were positively correlated with injury severity. APC showed an ISS- and injury-dependent kinetic and might serve as candidate biomarker to identify patients at risk for developing TIC.

IGF-I contributes to glucose homeostasis in the Berlin Fat Mouse Inbred line.

This study aimed to investigate the tissue-specific role of the insulin-like growth factor 1 (IGF-I) on glucose homeostasis in the high-fatness selected Berlin Fat Mouse Inbred (BFMI) line. Therefore, the expression of different IGF-I transcripts and IGF-I protein, IGF-binding proteins, insulin as well as glucose tolerance was analyzed in BFMI in comparison with that in lean mice. In addition, dietary effects were investigated. The BFMI line showed normal blood glucose clearance on standard diet, but on high-fat diet the clearance was impaired, indicating the beginning of insulin resistance. Circulating IGF-I and insulin levels were elevated in BFMI than in lean mice on both diets along with a down-regulation of three IGF-I binding proteins in BFMI mice. Serum IGF-I levels corresponded with the expression pattern for both hepatic and one class II splice variants in reproductive adipose tissue, but not in muscle. High insulin and high IGF-I levels likely prevent BFMI mice from diabetes.

Ectomycorrhizas from a Lower Eocene angiosperm forest.

The development of mycorrhizal associations is considered a key innovation that enabled vascular plants to extensively colonize terrestrial habitats. Here, we present the first known fossil ectomycorrhizas from an angiosperm forest. Our fossils are preserved in a 52 million-yr-old piece of amber from the Tadkeshwar Lignite Mine of Gujarat State, western India. The amber was produced by representatives of Dipterocarpaceae in an early tropical broadleaf forest. The ectomycorrhizas were investigated using light microscopy and field emission scanning electron microscopy. Dissolving the amber surrounding one of the fossils allowed ultrastructural analyses and Raman spectroscopy. Approx. 20 unramified, cruciform and monopodial-pinnate ectomycorrhizas are fossilized adjacent to rootlets, and different developmental stages of the fossil mycorrhizas are delicately preserved in the ancient resin. Compounds of melanins were detectable in the dark hyphae. The mycobiont, Eomelanomyces cenococcoides gen. et spec. nov., is considered to be an ascomycete; the host is most likely a dipterocarp representative. An early ectomycorrhizal association may have conferred an evolutionary advantage on dipterocarps. Our find indicates that ectomycorrhizas occurred contemporaneously within both gymnosperms (Pinaceae) and angiosperms (Dipterocarpaceae) by the Lower Eocene.

Plasmatic and cell-based enhancement by microparticles originated from platelets and endothelial cells under simulated in vitro conditions of a dilutional coagulopathy.

BACKGROUND: Aggressive fluid management and other external factors may lead to hypothermia, acidosis and hemodilution (defined as Lethal Triad, LT) contributing to a trauma-induced coagulopathy (TIC) that worsens patients' outcomes. Procoagulant microparticles (MP) are crucial players at the interface of cellular and plasmatic coagulation. However, their functions remain largely unexplored. This study aimed to characterize effects of MP subtypes and concentrations on functional coagulation under in vitro simulated conditions. METHODS: Blood from eleven volunteers were collected to simulate in vitro conditions of hemodilution (HD) and LT, respectively. HD was induced by replacing a blood volume of 33% by crystalloids and for LT, samples were further processed by reducing the temperature to 32 °C and lowering the pH to 6.8. MP were obtained either from platelet concentrates (platelet-derived MP, PDMP) or from cell culture (ECV304 cells for endothelial-derived MP, EDMP) by targeted stimulation. After introducing MP to in vitro conditions, we measured their concentration-dependent effects (1.000, 10.000 and 15.000 MP/µl blood) on coagulation compared to whole blood (WB). For each condition, coagulation was characterized by flow cytometric platelet activation and by quantification of fibrin clot propagation using Thrombodynamics® technology. RESULTS: MP originated from platelets and endothelial cells affected blood coagulation in a concentration-dependent manner. Particularly, high PDMP quantities (10.000 and 15.000 PDMP/µl blood) significantly induced platelet activation and fibrin clot growth and size in HD conditions. In LT conditions as well, only high PDMP concentration induced platelet activation, clot growth and size. In contrast, EDMP did not induce platelet activation, but resulted in enhanced formation of spontaneous clots, irrespective of simulated condition. With increasing EDMP concentration, the time until the onset of spontaneous clotting decreased in both HD and LT conditions. DISCUSSION: The study demonstrates an essential role of MP within the coagulation process under simulated coagulopathic conditions. PDMP affected platelets promoting clot formation likely by providing a surface enlargement. EDMP presumably affected clotting factors of the plasmatic coagulation resulting in an increased formation of spontaneous clots. CONCLUSION: Under simulated conditions of a dilutional coagulopathy, MP from different cellular origin indicate a divergent but both procoagulant mechanism within the coagulation process.