The human ABC transporter pseudogene family: Evidence for transcription and gene-pseudogene interference

<p>Abstract</p> <p>Background</p> <p>Pseudogenes are an integral component of the human genome. Little attention, however, has so far been paid to the phenomenon that some pseudogenes are transcriptionally active. Recently, we demonstrated that the human ortholog of the rodent testis-specific ATP-binding cassette (ABC) transporter Abca17 is a ubiquitously transcribed pseudogene (<it>ABCA17P</it>). The aim of the present study was to establish a complete inventory of all ABC transporter pseudogenes in the human genome and to identify transcriptionally active ABC transporter pseudogenes. Moreover, we tested the hypothesis that a regulatory interdependency exists between ABC transporter pseudogenes and their parental protein coding equivalents.</p> <p>Results</p> <p>Systematic bioinformatic analysis revealed the existence of 22 ABC transporter pseudogenes within the human genome. We identified two clusters on chromosomes 15 and 16, respectively, which harbor almost half of all pseudogenes (n = 10). Available information from EST and mRNA databases and RT-PCR expression profiling indicate that a large portion of the ABC transporter pseudogenes (45%, n = 10) are transcriptionally active and some of them are expressed as alternative splice variants. We demonstrate that both pseudogenes of the pseudoxanthoma elasticum gene <it>ABCC6</it>, <it>ABCC6P1 </it>and <it>ABCC6P2</it>, are transcribed. <it>ABCC6P1 </it>and <it>ABCC6 </it>possess near-identical promoter sequences and their tissue-specific expression profiles are strikingly similar raising the possibility that they form a gene-pseudogene dual transcription unit. Intriguingly, targeted knockdown of the transcribed pseudogene <it>ABCC6P1 </it>resulted in a significant reduction of <it>ABCC6 </it>mRNA expression levels.</p> <p>Conclusion</p> <p>The human genome contains a surprisingly small number of ABC transporter pseudogenes relative to other known gene families. They are unevenly distributed across the chromosomes. Importantly, a significant portion of the ABC transporter pseudogenes is transcriptionally active. The downregulation of <it>ABCC6 </it>mRNA levels by targeted suppression of the expression of its pseudogene <it>ABCC6P1 </it>provides evidence, for the first time, for a regulatory interdependence of a transcribed pseudogene and its protein coding counterpart in the human genome.</p

Persistent hypercoagulability in dogs envenomated by the European adder (Vipera berus berus)

Background Envenomation by the European adder, Vipera berus berus (Vbb), is a medical emergency. The overall in vivo haemostatic effects of pro- and anticoagulant components in Vbb venom, and the downstream effects of cellular injury and systemic inflammation, are unclear. Objectives To longitudinally describe the global coagulation status of dogs after Vbb envenomation and compare to healthy controls. A secondary aim was to investigate differences between dogs treated with and without antivenom. Methods Citrated plasma was collected at presentation, 12 hours (h), 24 h, 36 h and 15 days after bite from 28 dogs envenomated by Vbb, and from 28 healthy controls at a single timepoint. Thrombin generation (initiated with and without exogenous phospholipids and tissue factor), thrombin-antithrombin (TAT)-complexes and the procoagulant activity of phosphatidylserine (PS)-expressing extracellular vesicles (EVs), expressed as PS-equivalents, were measured. Results At presentation the envenomated dogs were hypercoagulable compared to controls, measured as increased thrombin generation, TAT-complexes and PS-equivalents. The hypercoagulability decreased gradually but compared to controls thrombin generation and PS-equivalents were still increased at day 15. The discrepancy in peak thrombin between envenomated dogs and controls was greater when the measurement was phospholipid-dependent, indicating that PS-positive EVs contribute to hypercoagulability. Lag time was shorter in non-antivenom treated dogs, compared to antivenom treated dogs <24 h after envenomation. Conclusions Hypercoagulability was measured in dogs up to 15 days after Vbb envenomation. Dogs treated with antivenom may be less hypercoagulable than their non-antivenom treated counterparts. Thrombin generation is a promising diagnostic and monitoring tool for Vbb envenomation.publishedVersio

Violence and abuse against women with disabilities in Malawi

This research project is a study of the nature of abuse, violence and neglect against women with disabilities in Malawi. The childhood as well as the present situation of 23 women with disabilities in Blantyre district is described. Data was collected in two ways:&nbsp;a) Through in-depth individual interviews with 19 women with visual-, mental- or physical disability, as well as women with albinism. b) Through a focus group discussion with four women with hearing impairment.&nbsp;The interviews and discussions were built on interview guides. Most of the informants reported that during childhood they were treated like the other children in the family. However, education was difficult. The schools and the school material were not adjusted to their needs. The informants stressed the need of more consideration in regards to their special needs in this relation and wanted the society to make education for women with disabilities a priority area. Many informants felt that men in the society took advantage of their vulnerable situation and promised to marry them. When the woman got pregnant, the man disappeared and left her to be a single mother. Many of the women regarded this as sexual abuse. The study was arranged as collaboration between SINTEF Health research, Norway, Federation of Disability Organisations in Malawi (FEDOMA) and Disabled Women in Development in Malawi (DIWODE). The Atlas Alliance and the Norwegian Department for Foreign Affairs have given economical support to the study.Violence and abuse against women with disabilities in Malaw

The effect of corn trypsin inhibitor, anti-tissue factor pathway inhibitor antibodies and phospholipids on microvesicle-associated thrombin generation in patients with pancreatic cancer and healthy controls

Circulating microvesicles (MVs) are suggested to be important contributors to cancer-associated thrombosis due to the presence of surface-bound procoagulant molecules like tissue factor (TF) and phosphatidylserine (PS). Pancreatic cancer is considered to be one of the most prothrombotic malignancies. The aim of this study was to describe the impact of analytical variables on MV-associated thrombin generation in patients with pancreatic cancer and in healthy controls. MVs were isolated from citrated plasma and added to pooled normal plasma (PNP). Thrombin generation was measured by the calibrated automated thrombogram. The impact of corn trypsin inhibitor (CTI), anti-tissue factor pathway inhibitor (TFPI) antibodies and phospholipids was described. Antibodies against TF were used to assess TF-dependency, and MV-bound PS activity was measured with the Zymuphen MP-activity kit. MVs from the pancreatic cancer patients displayed higher thrombin generation and higher PS-activity than MVs from the healthy control group, while TF-dependency was observed in only 1 out of 13 patient samples. Adequate thrombin generation-curves were only achieved when CTI was omitted and anti-TFPI antibodies were added to PNP prepared in low contact-activating tubes. Addition of phospholipids reduced the significant differences between the two groups, and should be omitted. This modified thrombin generation assay could be useful for measurement of procoagulant circulating MVs, allowing the contribution from MVs affecting both the intrinsic and the extrinsic pathway to be measured

Increased microvesicle-associated thrombin generation in patients with immune thrombocytopenia after initiation of thrombopoietin receptor agonists

Immune thrombocytopenia (ITP) patients have thrombocytopenia and increased bleeding risk, but, conversely, they also have increased thrombotic risk which appears to be exacerbated by thrombopoietin-receptor agonist (TPO-RA)-treatment. Microvesicles (MVs) released from activated/apoptotic cells are prothrombotic due to exposure of phosphatidylserine (PS) and tissue factor (TF). MVs are increased in ITP patients, but their prothrombotic effect, before and during treatment with TPO-RAs, is unclear. We studied the effect of TPO-RAs on the procoagulant activity of MVs in 11 ITP patients, before, and two and six weeks after initiation of treatment, and in 15 healthy controls. MV-associated PS-activity, TF-activity and the capacity of isolated MVs and plasma to generate thrombin in a phospholipid-dependent manner were measured. Before treatment with TPO-RAs, prothrombotic markers in ITP patients were comparable to levels found in healthy controls. After both two and six weeks of TPO-RA-treatment, ITP patients had higher MV-associated PS-activity and phospholipid-dependent thrombin generation in plasma than controls. In addition, ITP patients had increased phospholipid-dependent MV-associated thrombin generation two weeks after initiation of TPO-RA-treatment compared with controls and pre-treatment levels. MV-associated TF-activity was low in controls and in ITP patients before and after initiation of TPO-RA-treatment. In conclusion, TPO-RAs increase phospholipid-dependent MV-associated thrombin generation in ITP patients. This could contribute to or exacerbate a pre-existing hypercoagulable state. Phospholipid-dependent thrombin generation generated by isolated MVs, or measured directly in plasma, may be potential tools that could help in the risk-assessment of future thromboembolic events in ITP patients, both before and after initiation of TPO-RA-treatment

Extracellular vesicle-associated procoagulant activity is highest the first 3 hours after trauma and thereafter declines substantially: A prospective observational pilot study

BACKGROUND Trauma patients have high concentrations of circulating extracellular vesicles (EVs) following injury, but the functional role of EVs in this setting is only partly deciphered. We aimed to describe in detail EV-associated procoagulant activity in individual trauma patients during the first 12 hours after injury to explore their putative function and relate findings to relevant trauma characteristics and outcome. METHODS In a prospective observational study of 33 convenience recruited trauma patients, citrated plasma samples were obtained at trauma center admission and 2, 4, 6, and 8 hours thereafter. We measured thrombin generation from isolated EVs and the procoagulant activity of phosphatidylserine (PS)-exposing EVs. Correlation and multivariable linear regression analyses were used to explore associations between EV-associated procoagulant activity and trauma characteristics as well as outcome measures. RESULTS EV–associated procoagulant activity was highest in the first 3 hours after injury. EV–associated thrombin generation normalized within 7 to 12 hours of injury, whereas the procoagulant activity of PS-exposing EVs declined to a level right above that of healthy volunteers. Increased EV-associated procoagulant activity at admission was associated with higher New Injury Severity Score, lower admission base excess, higher admission international normalized ratio, prolonged admission activated partial thromboplastin time, higher Sequential Organ Failure Assessment score at day 0, and fewer ventilator-free days. CONCLUSION Our data suggest that EVs have a transient hypercoagulable function and may play a role in the early phase of hemostasis after injury. The role of EVs in trauma-induced coagulopathy and posttraumatic thrombosis should be studied bearing in mind this novel temporal pattern. LEVEL OF EVIDENCE Prognostic/epidemiologic, level V

The effect of inhibiting antibodies against tissue factor (TF) on microvesicle (MV)-associated thrombin generation in pooled normal plasma (PNP) with corn trypsin inhibitor (CTI).

<p>The thrombin generation (lag time) of MVs isolated from pancreatic cancer patients plasma (A) or of MVs isolated from plasma obtained from citrated whole blood that had been incubated with <i>Neisseria meningitidis</i> (10⁸/mL) (B). MVs were analyzed with (circles) or without (squares) preincubation with anti-TF abs, in the presence or absence of phospholipids (MP-reagent) and antibodies against TFPI (anti-TFPI abs). The patient sample with the shortest lag time and the most evident TF-dependent thrombin generation across the four conditions is marked in red.</p

MV-associated thrombin generation in pooled normal plasma (PNP) without corn trypsin inhibitor (CTI).

<p>MVs were isolated from plasma, washed (17,000 g, 30 min, RT) and added to PNP without CTI. Thrombin generation was measured -/+ MP-reagent (phospholipids) and -/+ anti-TFPI antibodies (abs). A) Thrombin generation curves of MVs from pancreatic cancer patients (black) and healthy controls (green). B) LT, ETP, peak and velocity index of MVs from pancreatic cancer patients (filled circles) and healthy controls (open circles) in PNP with anti-TFPI abs added. Lines indicate mean values, and * indicates significant differences between the pancreatic cancer group and the healthy control group (RM two-way ANOVA with Sidak’s multiple comparison test, adjusted p-values are shown).</p

Microvesicle (MV)-associated thrombin generation in pooled normal plasma (PNP) with corn trypsin inhibitor (CTI).

<p>MVs were isolated from plasma, washed (17,000 g, 30 min, RT) and added to CTI-containing PNP. Thrombin generation was measured -/+ MP-reagent (phospholipids) and -/+ anti-TFPI antibodies (abs). A) Thrombin generation curves of MVs from pancreatic cancer patients (black) and healthy controls (green). B) Lag time (LT) of MVs from pancreatic cancer patients (filled circles) and healthy controls (open circles). Lines indicate mean LT, and * indicates significant differences (RM two-way ANOVA with Sidak’s multiple comparison test, adjusted p-values are shown).</p

Whole-blood incubation with the Neisseria meningitidis lpxL1 mutant induces less pro-inflammatory cytokines than the wild type, and IL-10 reduces the MyD88-dependent cytokines

Levels of bacterial LPS, pro-inflammatory cytokines and IL-10 are related to the severity of meningococcal septicaemia. Patients infected with a Neisseria meninigitidis lpxL1 mutant (Nm-mutant) with penta-acylated lipid A present with a milder meningococcal disease than those infected with hexa-acylated Nm wild type (Nm-wt). The aim was to compare the pro-inflammatory responses after ex vivo incubation with the heat-inactivated Nm-wt or the Nm-mutant in citrated whole blood, and the modulating effects of IL-10. Concomitantly, we measured intracellular IL-6, IL-8 and TNF-α to elucidate which cell types were responsible for the pro-inflammatory responses. Incubation with Nm-wt (106/ml;107/ml;108/ml) resulted in a dose-dependent increase of the MyD88-dependent pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α), which were mainly derived from monocytes. In comparison, only 108/ml of the Nm-mutant significantly increased the concentration of these cytokines. The MyD88-independent cytokines (IP-10, RANTES) were evidently increased after incubation with the Nm-wt but were unaffected by the Nm-mutant. Co-incubation with IL-10 significantly reduced the concentrations of the MyD88-dependent cytokines induced by both the Nm-wt and the Nm-mutant, whereas the MyD88-independent cytokines were almost unaffected. In summary, the Nm-mutant is a weaker inducer of the MyD88-dependent/independent cytokines than the Nm-wt in whole blood, and IL-10 attenuates the Nm-stimulated increase in MyD88-dependent pro-inflammatory cytokines