Maf1 is a negative regulator of transcription in Trypanosoma brucei.

RNA polymerase III (Pol III) produces small RNA molecules that play essential roles in mRNA processing and translation. Maf1, originally described as a negative regulator of Pol III transcription, has been studied from yeast to human. Here we characterized Maf1 in the parasitic protozoa Trypanosoma brucei (TbMaf1), representing the first report to analyse Maf1 in an early-diverged eukaryote. While Maf1 is generally encoded by a single-copy gene, the T. brucei genome contains two almost identical TbMaf1 genes. The TbMaf1 protein has the three conserved sequences and is predicted to fold into a globular structure. Unlike in yeast, TbMaf1 localizes to the nucleus in procyclic forms of T. brucei under normal growth conditions. Cell lines that either downregulate or overexpress TbMaf1 were generated, and growth curve analysis with them suggested that TbMaf1 participates in the regulation of cell growth of T. brucei. Nuclear run-on and chromatin immunoprecipitation analyses demonstrated that TbMaf1 represses Pol III transcription of tRNA and U2 snRNA genes by associating with their promoters. Interestingly, 5S rRNA levels do not change after TbMaf1 ablation or overexpression. Notably, our data also revealed that TbMaf1 regulates Pol I transcription of procyclin gene and Pol II transcription of SL RNA genes.

Molecular characterization of 5S ribosomal RNA genes and transcripts in the protozoan parasite Leishmania major.

Eukaryotic 5S rRNA, synthesized by RNA polymerase III (Pol III), is an essential component of the large ribosomal subunit. Most organisms contain hundreds of 5S rRNA genes organized into tandem arrays. However, the genome of the protozoan parasite Leishmania major contains only 11 copies of the 5S rRNA gene, which are interspersed and associated with other Pol III-transcribed genes. Here we report that, in general, the number and order of the 5S rRNA genes is conserved between different species of Leishmania. While in most organisms 5S rRNA genes are normally associated with the nucleolus, combined fluorescent in situ hybridization and indirect immunofluorescence experiments showed that 5S rRNA genes are mainly located at the nuclear periphery in L. major. Similarly, the tandemly repeated 5S rRNA genes in Trypanosoma cruzi are dispersed throughout the nucleus. In contrast, 5S rRNA transcripts in L. major were localized within the nucleolus, and scattered throughout the cytoplasm, where mature ribosomes are located. Unlike other rRNA species, stable antisense RNA complementary to 5S rRNA is not detected in L. major.

Hospital reimbursement for carotid stenting and endarterectomy.

PURPOSE: To investigate the cost-effectiveness of carotid endarterectomy (CEA) vs. carotid artery stenting (CAS) in terms of hospital reimbursement. METHODS: A retrospective analysis was conducted of hospital reimbursement for patients undergoing CEA and CAS from 1 January 2008 through 30 September 2010 at a single tertiary referral institution. Hospital cost and reimbursement were assessed using patient-specific data gathered by the institution's cost accounting system. Professional fees were excluded. RESULTS: Hospital reimbursement data were extracted for a total of 301 cases (169 CEA and 132 CAS). Mean hospital reimbursement was 16% higher for CAS ($12,000±$7372) vs. CEA ($10,160±$6840, p=0.02). However, because of the significantly higher cost of materials necessary to perform CAS, the net revenue for the hospital was 29% greater in patients undergoing CEA ($3487) vs. CAS ($2603). The differences in hospital reimbursement and net revenue were consistent in asymptomatic (n=183), symptomatic (n=123), and urgent (n=36) subgroups. When focusing on cases by diagnosis-related group (DRG) codes vs. current procedural terminology (CPT) codes, the data shifted. Several patients were coded as an outpatient procedure (DRG 0): 28 (21%) of the 132 CAS patients and 7 (4%) of the 169 CEA patients, reducing their mean reimbursement to $4046 and $2513, respectively. If these patients were excluded, the mean hospital reimbursement differential widened between and CEA ($10,515) and CAS ($13,825). CONCLUSION: Hospital reimbursement for CAS is significantly higher than that for CEA. While both procedures created net positive income for the hospital, CEA was associated with a 29% higher net revenue due to the 40% cost premium of CAS when looking at all carotid procedures. However, proper DRG coding of CAS cases would have likely resulted in similar net revenue. Asymptomatic patients had the lowest cost and highest net revenue of all the subgroups. Per capita, significantly more healthcare resources were expended with CAS when compared to CEA. Given the lack of improved clinical outcome in most cases, CAS cannot be considered cost-effective for most patients.

Endovascular treatment of hepatic artery stenosis after liver transplantation.

BACKGROUND: Hepatic artery stenosis (HAS) after orthotopic liver transplantation is a significant risk factor for subsequent hepatic artery thrombosis (HAT). HAT is associated with a 30%-50% risk of liver failure culminating in retransplantation or death. Traditional treatment of hepatic artery complications has been surgical, with hepatic artery revision or retransplantation. Endovascular therapy of HAS, described primarily in the interventional radiology literature, may provide a less-invasive treatment option. METHODS: This was a retrospective review of all endovascular interventions performed for HAS after orthotopic liver transplantation over a 31-month period (August 2009 to January 2012). Patients with duplex ultrasound imaging evidence of severe main HAS (peak systolic velocity of >400 cm/s, resistive index of <.5) underwent endovascular treatment with either primary stent placement or percutaneous transluminal angioplasty (PTA) alone. Patients were followed with serial ultrasound imaging to assess for treatment success and late restenosis. Reintervention was performed if significant restenosis occurred. RESULTS: Thirty-five hepatic artery interventions were performed in 23 patients. Over the 31-month study period, 318 orthotopic liver transplantations were performed, yielding a 7.4% (23/318) rate of hepatic artery intervention. Primary technical success was achieved in 97% (34/35) of cases. Initial treatment was with PTA alone (n = 10) or primary stent placement (n = 13). The initial postintervention ultrasound images revealed improvements in hepatic artery peak systolic velocity (267 ± 118 [posttreatment] vs 489.9 ± 155 cm/s [pretreatment]; P < .0001) and main hepatic artery resistive index (0.61 ± 0.08 [posttreatment] vs 0.41 ± 0.07 [pretreatment]; P < .0001). At a mean follow-up of 8.2 ± 1.8 months (range, 0-29), there were 12 reinterventions in 10 patients for recurrent HAS. Thirty-one percent (n = 4/13) of patients undergoing initial stent placement required reintervention (at 236 ± 124 days of follow-up) compared with 60% (n = 6/10) of patients undergoing initial PTA (at 62.5 ± 44 days of follow-up). Primary patency rates (Kaplan-Meier) after primary stent placement were 92%, 85%, and 69% at 1, 3, and 6 months, respectively, compared with 70%, 60%, and 50% after PTA (P = .17). Primary-assisted patency for the entire cohort was 97% at 6 and 12 months. Major complications were one arterial rupture managed endovascularly and one artery dissection that precipitated HAT and required retransplantation. The overall rate of HAT in the entire cohort was 4.3% (1/23). CONCLUSIONS: Endovascular treatment of HAS can be performed with high technical success, excellent primary-assisted patency, and acceptable morbidity. Initial use of a stent may improve primary patency when compared with PTA. The need for reintervention is common, placing particular importance on aggressive surveillance. Longer follow-up and a larger cohort are needed to confirm these encouraging early results.

APEX2 Proximity Proteomics Resolves Flagellum Subdomains and Identifies Flagellum Tip-Specific Proteins in Trypanosoma brucei.

Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single flagellum (cilium) that plays critical roles in transmission and pathogenesis. An emerging concept is that the flagellum is organized into subdomains, each having specialized composition and function. The overall flagellum proteome has been well studied, but a critical knowledge gap is the protein composition of individual subdomains. We have tested whether APEX-based proximity proteomics could be used to examine the protein composition of T. brucei flagellum subdomains. As APEX-based labeling has not previously been described in T. brucei, we first fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, a well-characterized axonemal complex. We found that DRC1-APEX2 directs flagellum-specific biotinylation, and purification of biotinylated proteins yields a DRC1 "proximity proteome" having good overlap with published proteomes obtained from purified axonemes. Having validated the use of APEX2 in T. brucei, we next attempted to distinguish flagellar subdomains by fusing APEX2 to a flagellar membrane protein that is restricted to the flagellum tip, AC1, and another one that is excluded from the tip, FS179. Fluorescence microscopy demonstrated subdomain-specific biotinylation, and principal-component analysis showed distinct profiles between AC1-APEX2 and FS179-APEX2. Comparing these two profiles allowed us to identify an AC1 proximity proteome that is enriched for tip proteins, including proteins involved in signaling. Our results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve the proteome composition of flagellum subdomains that cannot themselves be readily purified.IMPORTANCE Sleeping sickness is a neglected tropical disease caused by the protozoan parasite Trypanosoma brucei The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility, transmission, and virulence depend on its flagellum (cilium), which consists of several different specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is need for approaches that enable proteomic analysis of individual subdomains. Our work establishes that APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei and has allowed identification of proximity proteomes for different flagellar subdomains that cannot be purified. This capacity opens the possibility to study the composition and function of other compartments. We expect this approach may be extended to other eukaryotic pathogens and will enhance the utility of T. brucei as a model organism to study ciliopathies, heritable human diseases in which cilium function is impaired.

Awake, percutaneous repair of a ruptured abdominal aortic aneurysm.

BACKGROUND: The rupture of an abdominal aortic aneurysm is a highly lethal event, claiming approximately 15,000 lives each year. Traditionally, open surgical repair has been the mainstay for treatment. However, this surgery is associated with almost a 50% perioperative mortality rate. Minimally invasive endovascular stent grafts have been used with great success in the elective repair of aortic aneurysms. This technology has subsequently been applied to the repair of ruptured abdominal aortic aneurysms with a substantial reduction in the periprocedural death rate and associated complications. CASE REPORT: We report a case of a patient with a ruptured abdominal aortic aneurysm and an acute ST elevation myocardial infarction who was treated with an endovascular stent graft in a totally percutaneous fashion using only conscious sedation and local anesthesia. CONCLUSION: Although the risk of mortality and complications remains high, endovascular repair of a ruptured abdominal aortic aneurysm offers the patient the best chance of survival.