Role of AP-1 in developmentally regulated lysosomal trafficking in Trypanosoma brucei.

African trypanosomes are the causative agents of human trypanosomiasis (sleeping sickness). The pathogenic stage of the parasite has unique adaptations to life in the bloodstream of the mammalian host, including upregulation of endocytic and lysosomal activities. We investigated stage-specific requirements for cytoplasmic adaptor/clathrin machinery in post-Golgi apparatus biosynthetic sorting to the lysosome using RNA interference silencing of the Tbmu1 subunit of adaptor complex 1 (AP-1), in conjunction with immunolocalization, kinetic analyses of reporter transport, and quantitative endocytosis assays. Tbmu1 silencing was lethal in both stages, indicating a critical function(s) for the AP-1 machinery. Transport of soluble and membrane-bound secretory cargoes was Tbmu1 independent in both stages. In procyclic parasites, trafficking of the lysosomal membrane protein, p67, was disrupted, leading to cell surface mislocalization. The lysosomal protease trypanopain was also secreted, suggesting a transmembrane-sorting receptor for this soluble hydrolase. In bloodstream trypanosomes, both p67 and trypanopain trafficking were unaffected by Tbmu1 silencing, suggesting that AP-1 is not necessary for biosynthetic lysosomal trafficking. Endocytosis in bloodstream cells was also unaffected, indicating that AP-1 does not function at the flagellar pocket. These results indicate that post-Golgi apparatus sorting to the lysosome is critically dependent on the AP-1/clathrin machinery in procyclic trypanosomes but that this machinery is not necessary in bloodstream parasites. We propose a simple model for stage-specific default secretory trafficking in trypanosomes that is consistent with the behavior of other soluble and glycosylphosphatidylinositol-anchored cargos and which is influenced by upregulation of endocytosis in bloodstream parasites as an adaptation to life in the mammalian bloodstream.

Chondroma of the Bladder: A Case Report and Review of the Literature.

Chondromas are benign tumors composed of mature hyaline cartilage (Bahnassy & Abdil-Khalik, 2009). Extraskeletal presentation of chondromas is extremely rare and mostly occurs in the soft tissues of the extremities, mainly the hands and feet (Bahnassy & Abdil-Khalik, 2009; Rapini et al, 2007). There are currently only 3 case reports in the literature of chondroma of the urinary bladder (Cho & Horvai, 2015; Reichard et al, 2014; Sloan & Rapoport, 1985). Here we present a case of urinary bladder chondroma with low proliferative potential managed by transurethral resection.

Neutrophil to Lymphocyte Ratio (NLR) at the Time of Transurethral Resection of Bladder Tumor: A Large Retrospective Study and Analysis of Racial Differences.

Introduction: Neutrophil/lymphocyte ratio (NLR) is an indicator of systemic inflammation and has been proven to be associated with an increased risk of extravesical disease, decreased cancer specific survival and overall survival in bladder cancer patients. A large proportion of healthy African Americans have a WBC count that is persistently lower than the normal range defined for individuals of European ancestry, this condition has been called "benign ethnic neutropenia". The purpose of our study was to determine if NLR was different in patients of African ancestry (AA) vs European ancestry (EA) across different tumor grades and stages at the time of transurethral resection of bladder tumor(s) (TURBT). Materials and Methods: The records of consecutive patients who underwent TURBT were reviewed from the University of Wisconsin and the Atlanta Veterans' Administration Medical Center (2000-2012). NLR was compared across tumor stage, tumor grade and ethnicity. Results: 297 consecutive patients met study criteria. 89% and 86%, were males and of European ancestry (EA) respectively. NLRs were different across T-stages (Ta-2.5, T1-3.9, T2-3.8; p = 0.001). but not across tumor grades in Ta (LG-2.5 vs HG-3.9, p = 0.57). EA had higher NLRs than AA (3.4 vs 1.9; p < 0.001). Conclusions: Higher NLRs appear to be associated with more advanced tumor stage at the time of TURBT. Patients of African ancestry have lower NLRs across all tumor stages compared to patients of European ancestry. Ethnicity should be taken into account when interpreting the NLR in patients with bladder cancer.

Lower extremity neuropathies after robot-assisted laparoscopic prostatectomy on a split-leg table.

BACKGROUND AND PURPOSE: Lower extremity neuropathies from prolonged lithotomy positioning have been well documented. When we initiated our robot-assisted laparoscopic prostatectomy (RALP) program in December 2002, we chose to use the split-leg table that allows patient support in a more anatomic position, hypothesizing that this would reduce risk of neurologic compression injuries. We report our incidence of lower extremity neuropathies associated with RALP using split-leg positioning and review patient and surgical variables associated with this complication. PATIENTS AND METHODS: We retrospectively reviewed records of 377 patients who underwent RALP using a split-leg table. Patient data including height, weight, body mass index, age, and smoking status; surgical variables such as surgeon operative experience and intraoperative times were also assessed. Intraoperative time was defined as anesthesia induction to anesthesia emergence to more accurately measure total time patients spent in the split-leg position. RESULTS: Of 377 patients, lower extremity neuropathies developed in 5 (1.3%) in the immediate postoperative period. Of all variables examined, only increased intraoperative time was identified as a potential risk factor for the development of this complication (496.2 ± 34.8 min vs 366.3 ± 96.1 min, P<0.001). Overall mean operative time for all patients was 368.0 ± 96.6 minutes. Three of the five patients had symptoms suggestive of a femoral mononeuropathy. CONCLUSIONS: Intraoperative time as defined in our study is a significant risk factor for development of postoperative neuropathy. We also found that split-leg positioning appears to put the femoral nerve at risk for injury, instead of the common peroneal nerve as has been previously reported from prolonged lithotomy positioning.

Multiple motifs regulate trafficking of the LAMP-like protein p67 in the ancient eukaryote Trypanosoma brucei.

p67 is a lysosome-associated membrane protein-like lysosomal type I transmembrane glycoprotein in African trypanosomes. The p67 cytoplasmic domain (CD) is both necessary and sufficient for lysosomal targeting in procyclic insect-stage parasites. The p67CD contains two [DE]XXXL[LI]-type dileucine motifs, which function as lysosomal targeting signals in mammalian cells. Using a green fluorescent protein fusion to the p67 transmembrane and cytoplasmic domains as a reporter system, we investigated the role of these motifs in lysosomal targeting in procyclic trypanosomes. Pulse-chase turnover studies, steady-state immunolocalization and quantitative flow cytometry all gave consistent results. Mutagenesis of the membrane-distal dileucine motif impairs lysosomal trafficking leading to partial appearance of the reporter on the cell surface. Mutagenesis of the membrane-proximal motif has little effect on proper targeting. Simultaneous mutagenesis of both motifs results in quantitative delivery to the cell surface. Thus, the distal motif plays a dominant role, but both dileucine motifs are necessary for maximal lysosomal targeting. Additional studies suggest that the upstream acidic residues in each motif influence lysosomal targeting and may also affect forward trafficking in the early secretory pathway. These results strongly suggest an evolutionary conservation in lysosomal trafficking mechanisms in the ancient eukaryote Trypanosoma brucei.

GPI valence and the fate of secretory membrane proteins in African trypanosomes.

Progression of GPI-anchored proteins in bloodstream African trypanosomes correlates with GPI-valence: homodimeric VSG (2 GPI) is a surface protein; heterodimeric transferrin receptor (1 GPI) localizes in the flagellar pocket; homodimeric GPI-minus VSG (0 GPI) is rapidly degraded in the lysosome. We test this relationship using three native secretory/endocytic proteins as monomeric GPI-plus and -minus reporters. GPI-minus procyclin trafficks to the lysosome and is degraded. GPI-plus procyclin trafficks to the flagellar pocket/cell surface and is released (approximately 50%) with an intact anchor, the remainder (approximately 50%) is degraded in the lysosome. GPI-plus BiPNHP, derived from the ER marker BiP, is released quantitatively (>80%), while GPI-plus p67HP, derived from the lysosomal marker p67, turns over by both release (approximately 15%) and lysosomal degradation (>50%). Turnover of endogenous transferrin receptor occurs primarily by lysosomal degradation (>90%). Thus shedding of monovalent GPI reporters correlates inversely with lysosomal targeting. We propose that mono-GPI reporters cycle through the flagellar pocket and endosome until they are disposed of by either shedding or lysosomal targeting. Partitioning between these fates may be a function of individual physical properties. Release is likely due to the exclusive use of C-14:0 myristate in the bloodstream stage GPI anchor. Up-regulation of transferrin receptor by culture in dog serum resulted in prominent cell surface localization, but not in elevated release. Surface receptor was non-functional for ligand binding suggesting that it may be bivalent homodimers of the GPI-anchored ESAG6 receptor subunit.