From consistent subjective assessment of skin sensitivity severity to its accurate objective scoring.

BACKGROUND: Sensitive skin (SenS) is a syndrome leading to unpleasant sensations with little visible signs. Grading its severity generally relies on questionnaires or subjective ratings. MATERIALS AND METHODS: The SenS status of 183 subjects was determined by trained assessors. Answers from a four-item questionnaire were converted into numerical scores, leading to a 0-15 SenS index that was asked twice or thrice. Parameters from hyperspectral images were used as input for a multi-layer perceptron (MLP) neural network to predict the four-item questionnaire score of subjects. The resulting model was used to evaluate the soothing effect of a cosmetic cream applied to one hemiface, comparing it to that of a placebo applied to the other hemiface. RESULTS: The four-item questionnaire score accurately predicts SenS assessors' classification (92.7%) while providing insight into SenS severity. Most subjects providing repeatable replies are non-SenS, but accepting some variability in answers enables identifying subjects with consistent replies encompassing a majority of SenS subjects. The MLP neural network model predicts the SenS score of subjects with consistent replies from full-face hyperspectral images (R2 Validation set  = 0.969). A similar quality is obtained with hemiface images. Comparing the effect of applying a soothing cosmetic to that of a placebo revealed that subjects with the highest instrumental index (> 5) show significant SenS improvement. CONCLUSION: A four-item questionnaire enables calculating a SenS index grading its severity. Objective evaluation using hyperspectral images with an MLP neural network accurately predicts SenS severity and its favourable evolution upon the application of a soothing cream.

Variation of platelet function in clinical phenotypes of acute venous thromboembolism - Results from the GMP-VTE project.

BACKGROUND: The role of platelets in the pathogenesis of venous thromboembolism (VTE) is receiving increasing attention; however, limited information is available on platelet function in the acute phase of the disease. OBJECTIVE: To characterize platelet function according to VTE phenotypes. PATIENTS/METHODS: In total, 154 subjects (isolated pulmonary embolism [iPE], n = 28; isolated deep vein thrombosis [iDVT], n = 35; DVT+PE, n = 91) were included. In this study platelet function analyzer (PFA)-200, light transmission aggregometry (LTA), thrombin generation (TG) in presence (PRP) and absence (PFP) of platelets and platelet flow cytometry were investigated. LASSO regression was used to select clinical and platelet biomarkers that distinguish between VTE phenotypes. RESULTS: PFA-200 results did not differ between VTE phenotypes. LTA from DVT+PE subjects showed lowest maximum aggregation after epinephrine and adenosine diphosphate compared to iPE and iDVT. Lower % of PAC-1-positive platelets after in-vitro trigger were present in DVT+PE and iPE compared to iDVT. TG in PRP had lower peak height and velocity in DVT+PE and iPE against iDVT. The results of LASSO regression for the distinction between DVT+PE vs iDVT identified 18 variables (AUC =0.93) of which 72% were platelet biomarkers. For distinction between iPE and iDVT, 10 variables were selected (AUC = 0.96) of which 50% were platelet-related. Obesity was the only variable weakly discriminating between DVT+PE vs iPE (AUC = 0.66). CONCLUSION: This explorative study suggests an important distinction between PE-related phenotypes and iDVT when considering clinical and platelet function data. Lower platelet-dependent TG along with reduced platelet reactivity suggest higher platelet degranulation in PE-dependent phenotypes compared to iDVT.

Comprehensive platelet phenotyping supports the role of platelets in the pathogenesis of acute venous thromboembolism - results from clinical observation studies.

BACKGROUND: The pathogenesis of arterial and venous thrombosis is in large part interlaced. How much platelet phenotype relates to acute venous thromboembolism (VTE) independent of the underlying cardiovascular profile is presently poorly investigated. METHODS: Platelet count and mean platelet volume (MPV), platelet aggregation in whole blood and platelet rich plasma (PRP), platelet-dependent thrombin generation (TG) and platelet surface activation markers were measured under standardized conditions. Machine learning was applied to identify the most relevant characteristics associated with VTE from a large array (N = 58) of clinical and platelet-related variables. FINDINGS: VTE cases (N = 159) presented with lower platelet count and MPV vs controls (N = 140). Whole blood aggregation showed shorter collagen/Epinephrine closure times in cases, particularly within acetylsalicylic acid (ASA) users. Within ASA users, higher PRP aggregation after adenosine diphosphate (ADP), epinephrine, collagen and arachidonic acid was observed in cases vs controls. Within non-ASA and/or subjects on anticoagulants, cases presented with lower aggregation after ADP and collagen vs controls. Lower platelet-dependent TG, higher CD63 on resting and lower PAC-1 expression after collagen/ADP in-vitro stimulated platelets further characterized VTE cases vs controls, independent of therapy. Lasso regression analysis identified 26 variables associated with VTE of which 69% were platelet-related. INTERPRETATION: Comprehensive phenotyping of platelet function identified a large proportion of low responders to ASA in VTE cases. Lower platelet-dependent TG and lower platelet reactivity after ex-vivo stimulation characterized the "platelet exhausted syndrome" in cases. Finally, from a large array of covariates including clinical risk factors, platelet biomarkers comprised 69% of all selected variables differentiating VTE cases vs controls. FUNDING: German Federal Ministry of Education and Research, CTH-Mainz and Bayer AG.

Beyond detoxification: a role for mouse mEH in the hepatic metabolism of endogenous lipids.

Microsomal and soluble epoxide hydrolase (mEH and sEH) fulfill apparently distinct roles: Whereas mEH detoxifies xenobiotics, sEH hydrolyzes fatty acid (FA) signaling molecules and is thus implicated in a variety of physiological functions. These epoxy FAs comprise epoxyeicosatrienoic acids (EETs) and epoxy-octadecenoic acids (EpOMEs), which are formed by CYP epoxygenases from arachidonic acid (AA) and linoleic acid, respectively, and then are hydrolyzed to their respective diols, the so-called DHETs and DiHOMEs. Although EETs and EpOMEs are also substrates for mEH, its role in lipid signaling is considered minor due to lower abundance and activity relative to sEH. Surprisingly, we found that in plasma from mEH KO mice, hydrolysis rates for 8,9-EET and 9,10-EpOME were reduced by 50% compared to WT plasma. This strongly suggests that mEH contributes substantially to the turnover of these FA epoxides-despite kinetic parameters being in favor of sEH. Given the crucial role of liver in controlling plasma diol levels, we next studied the capacity of sEH and mEH KO liver microsomes to synthesize DHETs with varying concentrations of AA (1-30 µM) and NADPH. mEH-generated DHET levels were similar to the ones generated by sEH, when AA concentrations were low (1 µM) or epoxygenase activity was curbed by modulating NADPH. With increasing AA concentrations sEH became more dominant and with 30 µM AA produced twice the level of DHETs compared to mEH. Immunohistochemistry of C57BL/6 liver slices further revealed that mEH expression was more widespread than sEH expression. mEH immunoreactivity was detected in hepatocytes, Kupffer cells, endothelial cells, and bile duct epithelial cells, while sEH immunoreactivity was confined to hepatocytes and bile duct epithelial cells. Finally, transcriptome analysis of WT, mEH KO, and sEH KO liver was carried out to discern transcriptional changes associated with the loss of EH genes along the CYP-epoxygenase-EH axis. We found several prominent dysregulations occurring in a parallel manner in both KO livers: (a) gene expression of Ephx1 (encoding for mEH protein) was increased 1.35-fold in sEH KO, while expression of Ephx2 (encoding for sEH protein) was increased 1.4-fold in mEH KO liver; (b) Cyp2c genes, encoding for the predominant epoxygenases in mouse liver, were mostly dysregulated in the same manner in both sEH and mEH KO mice, showing that loss of either EH has a similar impact. Taken together, mEH appears to play a leading role in the hydrolysis of 8,9-EET and 9,10-EpOME and also contributes to the hydrolysis of other FA epoxides. It probably profits from its high affinity for FA epoxides under non-saturating conditions and its close physical proximity to CYP epoxygenases, and compensates its lower abundance by a more widespread expression, being the only EH present in several sEH-lacking cell types.

Thrombopoiesis is spatially regulated by the bone marrow vasculature.

In mammals, megakaryocytes (MKs) in the bone marrow (BM) produce blood platelets, required for hemostasis and thrombosis. MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Through imaging of MKs in the intact BM, here we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. By combining in vivo two-photon microscopy and in situ light-sheet fluorescence microscopy with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche. As MKs do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts.Megakaryocyte maturation is thought to occur as the cells migrate from a vessel-distant (endosteal) niche to the vessel within the bone. Here, the authors show that megakaryocytes represent largely sessile cells in close contact with the vasculature and homogeneously distributed in the bone marrow.

Proplatelet formation is selectively inhibited by collagen type I through Syk-independent GPVI signaling.

Collagen receptors GPVI (also known as GP6) and integrin α2ß1 are highly expressed on blood platelets and megakaryocytes, their immediate precursors. After vessel injury, subendothelial collagen becomes exposed and induces platelet activation to prevent blood loss. Collagen types I and IV are thought to have opposite effects on platelet biogenesis, directing proplatelet formation (PPF) towards the blood vessels to prevent premature release within the marrow cavity. We used megakaryocytes lacking collagen receptors or treated megakaryocytes with blocking antibodies, and could demonstrate that collagen-I-mediated inhibition of PPF is specifically controlled by GPVI. Other collagen types competed for binding and diminished the inhibitory signal, which was entirely dependent on receptor-proximal Src family kinases, whereas Syk and LAT were dispensable. Adhesion assays indicate that megakaryocyte binding to collagens is mediated by α2ß1, and that collagen IV at the vascular niche might displace collagen I from megakaryocytes and thus contribute to prevention of premature platelet release into the marrow cavity and thereby directionally promote PPF at the vasculature.

Dihydrodehydrodiisoeugenol enhances adipocyte differentiation and decreases lipolysis in murine and human cells.

Loss of subcutaneous fat is a hallmark of ageing usually starting in the face. Attempts to ameliorate cosmetically the appearance of subcutaneous fat loss have been of limited success as they fail to rebuild the missing subcutaneous tissue. Ageing-driven loss of subcutaneous fat results from (i) the reduced capacity of pre-adipocytes to differentiate into adipocytes and (ii) the fact that adipocytes of the elderly secrete increased amounts of TNFα, that in turn enhances lipolysis, inhibits pre-adipocyte differentiation and induces dedifferentiation of adipocytes. The neolignan dihydrodehydrodiisoeugenol (DDE) caused a 30% increase in lipid accumulation in murine 3T3-L1 cells. This effect was accompanied by an induction of the differentiation-associated transcription factors peroxisome proliferator-activated receptorγ (PPARγ2), CAAT/enhancer-binding protein α (C/EBPα), fatty acid binding protein 4 and adiponectin, and a loss of the pre-adipocyte marker Pref1. In addition, DDE diminished both basal and TNFα-induced lipolysis. Similar results were obtained in human subcutaneous (hsc) pre-adipocytes cultured in an age-adapted hormone mix with reduced levels of insulin and dexamethasone. In this system, DDE significantly increased lipid accumulation by 71% and 94% and was associated with an induction of PPARγ2 and adiponectin mRNA expression. DDE also reduced basal lipolysis in mature hsc adipocytes. DDE acted as a partial PPARγ agonist because (i) DDE displaced PPARγ ligand from the human PPAR ligand-binding site, (ii) DDE-induced lipid accumulation and (iii) DDE-induced adiponectin secretion could be overcome by the addition of PPARγ antagonists. Taken together, these studies identify DDE as a compound well suited to prevent and reverse loss of subcutaneous fat.

The hemolymph proteome of fed and starved Drosophila larvae.

The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics.

Defective tubulin organization and proplatelet formation in murine megakaryocytes lacking Rac1 and Cdc42.

Blood platelets are anuclear cell fragments that are essential for blood clotting. Platelets are produced by bone marrow megakaryocytes (MKs), which extend protrusions, or so-called proplatelets, into bone marrow sinusoids. Proplatelet formation requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases, such as RhoA, Rac1, and Cdc42, are important regulators of cytoskeletal rearrangements in platelets; however, the specific roles of these proteins during platelet production have not been established. Using conditional knockout mice, we show here that Rac1 and Cdc42 possess redundant functions in platelet production and function. In contrast to a single-deficiency of either protein, a double-deficiency of Rac1 and Cdc42 in MKs resulted in macrothrombocytopenia, abnormal platelet morphology, and impaired platelet function. Double-deficient bone marrow MKs matured normally in vivo but displayed highly abnormal morphology and uncontrolled fragmentation. Consistently, a lack of Rac1/Cdc42 virtually abrogated proplatelet formation in vitro. Strikingly, this phenotype was associated with severely defective tubulin organization, whereas actin assembly and structure were barely affected. Together, these results suggest that the combined action of Rac1 and Cdc42 is crucial for platelet production, particularly by regulating microtubule dynamics.

Altered microtubule equilibrium and impaired thrombus stability in mice lacking RanBP10.

The crucial function of blood platelets in hemostasis is to prevent blood loss by stable thrombus formation. This process is driven by orchestrated mechanisms including several signal transduction cascades and morphologic transformations. The cytoplasmic microtubule modulator RanBP10 is a Ran and ß1-tubulin binding protein that is essential for platelet granule release and mice lacking RanBP10 harbor a severe bleeding phenotype. In this study, we demonstrate that RanBP10-nullizygous platelets show normal adhesion on collagen and von Willebrand factor under flow conditions. However, using a ferric chloride-induced arterial thrombosis model, the formation of stable thrombi was significantly impaired, preventing vessel occlusion or leading to recanalization and thromboembolization. Delta-granule secretion was normal in mutant mice, whereas platelet shape change in aggregometry was attenuated. Lack of RanBP10 leads to increased ß1-tubulin protein, which drives α-monomers into polymerized microtubules. In mutant platelets agonists failed to contract the peripheral marginal band or centralize granules. Pretreatment of wild-type platelets with taxol caused microtubule stabilization and phenocopied the attenuated shape change in response to collagen, suggesting that RanBP10 inhibits premature microtubule polymerization of ß1-tubulin and plays a pivotal role in thrombus stabilization.

The microtubule modulator RanBP10 plays a critical role in regulation of platelet discoid shape and degranulation.

Terminally mature megakaryocytes undergo dramatic cellular reorganization to produce hundreds of virtually identical platelets. A hallmark feature of this process is the generation of an elaborate system of branched protrusions called proplatelets. We recently identified RanBP10 as a tubulin-binding protein that is concentrated along polymerized microtubules in mature megakaryocytes. RanBP10 depletion in vitro caused the disturbance of polymerized filaments. Here we study the function of RanBP10 in vivo by generating deficient mice using a gene-trap approach. Mutant mice show normal platelet counts, and fetal liver-derived megakaryocytes reveal only slightly reduced proplatelet formation. However, ultrastructural analysis unveiled a significantly increased geometric axis ratio for resting platelets, and many platelets exhibited disorders in microtubule filament numbers and localization. Mutant mice showed a markedly prolonged bleeding time. Granule release, a process that depends on internal contraction of the microtubule marginal coil, also was reduced. Flow cytometry analysis revealed reduced expression of CD62P and CD63 after PAR4-peptide stimulation. These data suggest that RanBP10 plays an essential role in hemostasis and in maintaining microtubule dynamics with respect to both platelet shape and function.

RanBP10 is a cytoplasmic guanine nucleotide exchange factor that modulates noncentrosomal microtubules.

Microtubule spindle assembly in mitosis is stimulated by Ran.GTP, which is generated along condensed chromosomes by the guanine nucleotide exchange factor (GEF) RCC1. This relationship suggests that similar activities might modulate other microtubule structures. Interphase microtubules usually extend from the centrosome, although noncentrosomal microtubules function in some differentiated cells, including megakaryocytes. In these cells, platelet biogenesis requires massive mobilization of microtubules in the cell periphery, where they form proplatelets, the immediate precursors of platelets, in the apparent absence of centrioles. Here we identify a cytoplasmic Ran-binding protein, RanBP10, as a factor that binds beta-tubulin and associates with megakaryocyte microtubules. Unexpectedly, RanBP10 harbors GEF activity toward Ran. A point mutation in the candidate GEF domain abolishes exchange activity, and our results implicate RanBP10 as a localized cytoplasmic Ran-GEF. RNA interference-mediated loss of RanBP10 in cultured megakaryocytes disrupts microtubule organization. These results lead us to propose that spatiotemporally restricted generation of cytoplasmic Ran.GTP may influence organization of the specialized microtubules required in thrombopoiesis and that RanBP10 might serve as a molecular link between Ran and noncentrosomal microtubules.

The hepatitis B virus PRE contains a splicing regulatory element.

The posttranscriptional regulatory element (PRE) is considered to enhance hepatitis B virus (HBV) gene expression by facilitating the nuclear export of intronless viral subgenomic RNAs. Its role in the RNA metabolism of the viral pregenomic RNA (pgRNA) is currently unknown. We identified a positively cis-acting splicing regulatory element (SRE-1) and present two lines of evidence for its functionality. Firstly, in a heterologous context SRE-1 functionally substitutes for a retroviral bidirectional exonic splicing enhancer (ESE). As expected, SRE-1 is a splicing enhancer also in its natural viral sequence context, since deletion of SRE-1 reduces splicing of pgRNA in cell culture experiments. Secondly, we show that stimulation of HBV RNA splicing by the splicing factor PSF was repressed by the PRE. Analysis of a variety of PSF mutants indicated that RNA-binding and protein-protein interaction were required to enhance splicing. In addition, we show that the PRE contributed to pgRNA stability, but has little influence on its nuclear export. Herein, we report for the first time that the PRE harbors splicing stimulating and inhibiting regulatory elements controlling processing of the viral pregenome. We discuss a model in which the regulation of pgRNA splicing depends on cellular factors interacting with the PRE.

A novel approach to describe a U1 snRNA binding site.

RNA duplex formation between U1 snRNA and a splice donor (SD) site can protect pre-mRNA from degradation prior to splicing and initiates formation of the spliceosome. This process was monitored, using sub-genomic HIV-1 expression vectors, by expression analysis of the glycoprotein env, whose formation critically depends on functional SD4. We systematically derived a hydrogen bond model for the complementarity between the free 5' end of U1 snRNA and 5' splice sites and numerous mutations following transient transfection of HeLa-T4+ cells with 5' splice site mutated vectors. The resulting model takes into account number, interdependence and neighborhood relationships of predicted hydrogen bond formation in a region spanning the three most 3' base pairs of the exon (-3 to -1) and the eight most 5' base pairs of the intron (+1 to +8). The model is represented by an algorithm classifying U1 snRNA binding sites which can or cannot functionally substitute SD4 with respect to Rev-mediated env expression. In a data set of 5' splice site mutations of the human ATM gene we found a significant correlation between the algorithmic classification and exon skipping (P = 0.018, chi2-test), showing that the applicability of the proposed model reaches far beyond HIV-1 splicing. However, the algorithmic classification must not be taken as an absolute measure of SD usage as it may be modified by upstream sequence elements. Upstream to SD4 we identified a fragment supporting ASF/SF2 binding. Mutating GAR nucleotide repeats within this site decreased the SD4-dependent Rev-mediated env expression, which could be balanced simply by artificially increasing the complementarity of SD4.