Lymphatic clearance of the human skin in patients with acute deep vein thrombosis using a novel fluorescent technique

The purpose of this study was to investigate lymphatic clearance of the human skin in patients with acute deep thrombosis of the femoral vein. In 13 patients with deep vein thrombosis and no other cause for swelling of the limbs, lymphatic clearance of the skin at the foot was measured. Ten microliters of fluorescein isothiocyanatedextran 150,000 were injected intradermally and the fluorescent light intensity of the deposit measured 10 min and 24 hours after injection by window densitometry. In addition, intralymphatic pressure was measured by the servo-nulling system. The results were compared with a sex- and age-matched control group. Fluorescent light intensity decreased by 23.8 +/- 12.3 arbitrary units or by a factor of 1.8 +/- 0.5 in patients with DVT after 24 hours, which was significantly less than in healthy controls (33.7 +/- 8.9 arbitrary units or by factor 5.0 +/- 4.1, p < 0.013). Intralymphatic pressure was not different between the two groups. These results indicate that lymphatic clearance is significantly reduced in the acute phase of deep venous thrombosis.

Characterisation of six novel A-subunit mutations leading to congenital factor XIII deficiency and molecular analysis of the first diagnosed patient with this rare bleeding disorder

In 1960, the first case report on factor XIII deficiency was published describing a seven-year-old Swiss boy with a so far unknown bleeding disorder. Today, more than 60 mutations in the factor XIIIA- and B-subunit genes are known leading to congenital factor XIII deficiency. In the present study, we describe six novel mutations in the factor XIII A-subunit gene. Additionally, we present the molecular characterisation of the first described patient with congenital factor XIII deficiency. The six novel mutations include a small deletion, Glu202 delG, leading to a premature stop codon and truncation of the protein, and a splice site mutation at the exon 10/intron 10 boundary, +1G/A, giving rise to an incorrect spliced mRNA lacking exons 10 and 11. The remaining four mutations are characterised by the single amino acid changes Met159Arg, Gly215Arg, Trp375Cys, and His716Arg, and were expressed in COS-1 cells. Antigen levels and activity of the mutants were significantly reduced compared to the wild-type. The patient described in 1960 also shows a single amino acid change, Arg77Cys. Structural analysis of all mutant enzymes suggests several mechanisms leading to destabilisation of the protein.

Keratinocyte transcriptional regulation of the human c-Myc promoter occurs via a novel Lef/Tcf binding element distinct from neoplastic cells

The proto-oncogene c-Myc is involved in early neoplastic transformations. Two consensus Lef/Tcf binding elements (TBE) were found to be prerequisite for transcriptional transactivation by the armadillo proteins beta-catenin and plakoglobin (PG) together with Tcf4 in human neoplastic cells. In epidermal keratinocytes, c-Myc was reported to be repressed by Lef-1 and PG. Using reporter gene assays, here we demonstrate that deletion of the two consensus TBE fails to abrogate transcriptional regulation by Lef-1/PG in wildtype and beta-catenin-/- keratinocytes, while it reduces transcription in pre-neoplastic PG-/- keratinocytes. We identified a TBE sequence variant downstream of the major transcriptional initiation site that binds Lef-1 in vitro and in vivo, and its mutation compromised transcriptional regulation by Lef-1/PG. Collectively, this study demonstrates that the two consensus TBE's reported in neoplastic cells are dispensable for c-Myc regulation in normal keratinocytes, which instead use a novel TBE sequence variant. This unprecedented finding may have important implications for armadillo target genes involved in carcinogenesis.

Evaluation of a novel biphasic calcium phosphate in standardized bone defects: a histologic and histomorphometric study in the mandibles of minipigs

OBJECTIVE: A novel biphasic calcium phosphate (CaP) granulate consisting of hydroxyapatite (HA) and beta-tricalciumphosphate (TCP) was compared with pure HA and pure TCP and with autograft as positive control. MATERIALS AND METHODS: Four standardized bone defects were prepared in both mandibular angles of 16 minipigs and grafted with autogenous bone chips, HA, HA/TCP (60% : 40%), or TCP. Histologic and histomorphometric analysis of bone formation and graft degradation followed healing periods of 2, 4, 8, and 24 weeks. RESULTS: 2 weeks: more bone formation in defects filled with autograft than with the three CaP materials (P<0.05). 4 weeks: bone formation differed significantly (P<0.05) between all four materials (autograft>TCP>HA/TCP>HA). 8 weeks: more bone formation in defects with autograft and TCP than with HA/TCP (P<0.05), and HA/TCP had more bone formation than HA (P<0.05). 24 weeks: no difference in bone formation between the groups. Autograft and TCP resorbed quickly and almost completely over 8 weeks, whereas HA/TCP and HA showed limited degradation over 24 weeks. CONCLUSION: All defects healed with mature lamellar bone and intimate contact between bone and the remaining graft material. The rate of bone formation corresponded to the content of TCP in the CaP materials.

Unilateral focal polymicrogyria in a patient with classical Aarskog-Scott syndrome due to a novel missense mutation in an evolutionary conserved RhoGEF domain of the faciogenital dysplasia gene FGD1

Faciogenital dysplasia or Aarskog-Scott syndrome (AAS) is an X-linked disorder characterized by craniofacial, skeletal, and urogenital malformations and short stature. Mutations in the only known causative gene FGD1 are found in about one-fifth of the cases with the clinical diagnosis of AAS. FGD1 is a guanine nucleotide exchange factor (GEF) that specifically activates the Rho GTPase Cdc42 via its RhoGEF domain. The Cdc42 pathway is involved in skeletal formation and multiple aspects of neuronal development. We describe a boy with typical AAS and, in addition, unilateral focal polymicrogyria (PMG), a feature hitherto unreported in AAS. Sequencing of the FGD1 gene in the index case and his mother revealed the presence of a novel mutation (1396A>G; M466V), located in the evolutionary conserved alpha-helix 4 of the RhoGEF domain. M466V was not found in healthy family members, in >300 healthy controls and AAS patients, and has not been reported in the literature or mutation databases to date, indicating that this novel missense mutation causes AAS, and possibly PMG. Brain cortex malformations such as PMG could be initiated by mutations in the evolutionary conserved RhoGEF domain of FGD1, by perturbing the signaling via Rho GTPases such as Cdc42 known to cause brain malformation.

Introduction of a novel dose saving acquisition mode for the PortalVision aS500 EPID to facilitate on-line patient setup verification

In external beam radiotherapy, electronic portal imaging becomes more and more an indispensable tool for the verification of the patient setup. For the safe clinical introduction of high dose conformal radiotherapy like intensity modulated radiation therapy, on-line patient setup verification is a prerequisite to ensure that the planned dosimetric coverage of the tumor volume is actually realized in the patient. Since the direction of setup fields often deviates from the direction of the treatment beams, extra dose is delivered to the patient during the acquisition of these portal images which may reach clinical relevance. The aim of this work was to develop a new acquisition mode for the PortalVision aS500 electronic portal imaging device from Varian Medical Systems that allows one to take portal images with reduced dose while keeping good image quality. The new acquisition mode, called RadMode, selectively enables and disables beam pulses during image acquisition allowing one to stop wasting valuable dose during the initial acquisition of "reset frames." Images of excellent quality can be taken with 1 MU only. This low dose per image facilitates daily setup verification with considerably reduced extra dose.

Molecular interactions underlying the unusually high adenosine affinity of a novel Trypanosoma brucei nucleoside transporter

Trypanosoma brucei encodes a relatively high number of genes of the equilibrative nucleoside transporter (ENT) family. We report here the cloning and in-depth characterization of one T. brucei brucei ENT member, TbNT9/AT-D. This transporter was expressed in Saccharomyces cerevisiae and displayed a uniquely high affinity for adenosine (Km = 0.068 +/- 0.013 microM), as well as broader selectivity for other purine nucleosides in the low micromolar range, but was not inhibited by nucleobases or pyrimidines. This selectivity profile is consistent with the P1 transport activity observed previously in procyclic and long-slender bloodstream T. brucei, apart from the 40-fold higher affinity for adenosine than for inosine. We found that, like the previously investigated P1 activity of long/slender bloodstream trypanosomes, the 3'-hydroxy, 5'-hydroxy, N3, and N7 functional groups contribute to transporter binding. In addition, we show that the 6-position amine group of adenosine, but not the inosine 6-keto group, makes a major contribution to binding (DeltaG0 = 12 kJ/mol), explaining the different Km values of the purine nucleosides. We further found that P1 activity in procyclic and long-slender trypanosomes is pharmacologically distinct, and we identified the main gene encoding this activity in procyclic cells as NT10/AT-B. The presence of multiple P1-type nucleoside transport activities in T. brucei brucei facilitates the development of nucleoside-based treatments for African trypanosomiasis and would delay the onset of uptake-related drug resistance to such therapy. We show that both TbNT9/AT-D and NT10/AT-B transport a range of potentially therapeutic nucleoside analogs.

Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

Improving the performance of GIS/spatial analysts though novel applications of the Emotiv EPOC EEG headset

Geospatial information systems are used to analyze spatial data to provide decision makers with relevant, up-to-date, information. The processing time required for this information is a critical component to response time. Despite advances in algorithms and processing power, we still have many “human-in-the-loop” factors. Given the limited number of geospatial professionals, analysts using their time effectively is very important. The automation and faster humancomputer interactions of common tasks that will not disrupt their workflow or attention is something that is very desirable. The following research describes a novel approach to increase productivity with a wireless, wearable, electroencephalograph (EEG) headset within the geospatial workflow.

The crystal structure of the platelet activator aggretin reveals a novel (alphabeta)2 dimeric structure

Aggretin is a C-type lectin purified from Calloselasma rhodostoma snake venom. It is a potent activator of platelets, resulting in a collagen-like response by binding and clustering platelet receptor CLEC-2. We present here the crystal structure of aggretin at 1.7 A which reveals a unique tetrameric quaternary structure. The two alphabeta heterodimers are arranged through 2-fold rotational symmetry, resulting in an antiparallel side-by-side arrangement. Aggretin thus presents two ligand binding sites on one surface and can therefore cluster ligands in a manner reminiscent of convulxin and flavocetin. To examine the molecular basis of the interaction with CLEC-2, we used a molecular modeling approach of docking the aggretin alphabeta structure with the CLEC-2 N-terminal domain (CLEC-2N). This model positions the CLEC-2N structure face down in the "saddle"-shaped binding site which lies between the aggretin alpha and beta lectin-like domains. A 2-fold rotation of this complex to generate the aggretin tetramer reveals dimer contacts for CLEC-2N which bring the N- and C-termini into the proximity of each other, and a series of contacts involving two interlocking beta-strands close to the N-terminus are described. A comparison with homologous lectin-like domains from the immunoreceptor family reveals a similar but not identical dimerization mode, suggesting this structure may represent the clustered form of CLEC-2 capable of signaling across the platelet membrane.

Integrin alphavbeta6 is a marker of the progression of biliary and portal liver fibrosis and a novel target for antifibrotic therapies

BACKGROUND/AIMS: The integrin alphavbeta6 promotes proliferation of specialized epithelia and acts as a receptor for the activation of latent TGFbeta1. We studied alphavbeta6 expression in experimental and human liver fibrosis and the potential of its pharmacological inhibition for treatment of hepatic fibrosis. METHODS: alphavbeta6 expression was studied by quantitative PCR and immunohistochemistry in rats with cirrhosis due to bile duct ligation (BDL), administration of thioacetamide (TAA), in Mdr2(Abcb4)(-/-) mice with spontaneous biliary fibrosis, and in livers of patients with chronic hepatitis C (n=79) and end-stage liver disease due to various etiologies (n=18). The effect of a selective alphavbeta6 inhibitor was evaluated in Mdr2(Abcb4)(-/-) mice with ongoing fibrogenesis. RESULTS: Integrin beta6 mRNA increased with fibrosis stage in hepatitis C and was upregulated between 25- and 100-fold in TAA- and BDL-induced cirrhosis, in Mdr2(Abcb4)(-/-) mice and in human end-stage liver disease. alphavbeta6 protein was absent in normal livers and expressed de novo on (activated) bile duct epithelia and transitional hepatocytes. A single dose of the alphavbeta6 inhibitor injected into Mdr2(Abcb4)(-/-) mice significantly induced profibrolytic matrix metalloproteinases (MMP)-8 and -9 after 3 h, with a corresponding increase in extracellular matrix-degrading activities. In parallel profibrogenic transcripts (procollagen alpha1(I), TGFbeta2, and MMP-2) showed a trend of downregulation. CONCLUSIONS: (1) Integrin alphavbeta6 is induced de novo in rodent and human liver fibrosis, where it is expressed on activated bile duct epithelia and (transitional) hepatocytes during fibrosis progression. (2) In vivo a single dose of a small molecule alphavbeta6 inhibitor induced antifibrogenic and profibrolytic genes and activities, suggesting alphavbeta6 is a unique target for treatment of liver fibrosis.

A novel mutation of the epsilon-sarcoglycan gene in a Chinese family with myoclonus-dystonia syndrome

In a Chinese myoclonus-dystonia syndrome (MDS) family presented with a phenotype including a typical MDS, cervical dystonia, and writer's cramp, genetic analyses revealed a novel 662 + 1insG heterozygous mutation in exon 5 in the epsilon-sarcoglycan (SGCE) gene, leading to a frameshift with a down stream stop codon. Low SGCE mRNA levels were detected in the mutation carriers by real-time PCR, suggesting that the nonsense mutation might interfere with the stability of SGCE mRNA. This is the first report on Chinese with a SGCE mutation leading to MDS. Our data support the fact that same mutation of SGCE gene can lead to a varied phenotype, even in the same family.

A novel FIP1L1-PDGFRA mutant destabilizing the inactive conformation of the kinase domain in chronic eosinophilic leukemia/hypereosinophilic syndrome

BACKGROUND: The Fip1-like-1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRA) gene fusion is a common cause of chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome (HES), and patients suffering from this particular subgroup of CEL/HES respond to low-dose imatinib therapy. However, some patients may develop imatinib resistance because of an acquired T674I mutation, which is believed to prevent drug binding through steric hindrance. METHODS: In an imatinib resistant FIP1L1-PDGFRA positive patient, we analyzed the molecular structure of the fusion gene and analyzed the effect of several kinase inhibitors on FIP1L1-PDGFRA-mediated proliferative responses in vitro. RESULTS: Sequencing of the FIP1L1-PDGFRA fusion gene revealed the occurrence of a S601P mutation, which is located within the nucleotide binding loop. In agreement with the clinical observations, imatinib did not inhibit the proliferation of S601P mutant FIP1L1-PDGFRA-transduced Ba/F3 cells. Moreover, sorafenib, which has been described to inhibit T674I mutant FIP1L1-PDGFRA, failed to block S601P mutant FIP1L1-PDGFRA. Structural modeling revealed that the newly identified S601P mutated form of PDGFRA destabilizes the inactive conformation of the kinase domain that is necessary to bind imatinib as well as sorafenib. CONCLUSIONS: We identified a novel mutation in FIP1L1-PDGFRA resulting in both imatinib and sorafenib resistance. The identification of novel drug-resistant FIP1L1-PDGFRA variants may help to develop the next generation of target-directed compounds for CEL/HES and other leukemias.