IQGAP1 Interacts with Components of the Slit Diaphragm Complex in Podocytes and Is Involved in Podocyte Migration and Permeability In Vitro

IQGAP1 is a scaffold protein that interacts with proteins of the cytoskeleton and the intercellular adhesion complex. In podocytes, IQGAP1 is associated with nephrin in the glomerular slit diaphragm (SD) complex, but its role remains ill-defined. In this work, we investigated the interaction of IQGAP1 with the cytoskeleton and SD proteins in podocytes in culture, and its role in podocyte migration and permeability. Expression, localization, and interactions between IQGAP1 and SD or cytoskeletal proteins were determined in cultured human podocytes by Western blot (WB), immunocytolocalization (IC), immunoprecipitation (IP), and In situ Proximity Ligation assay (IsPL). Involvement of IQGAP1 in migration and permeability was also assessed. IQGAP1 expression in normal kidney biopsies was studied by immunohistochemistry. IQGAP1 expression by podocytes increased during their in vitro differentiation. IC, IP, and IsPL experiments showed colocalizations and/or interactions between IQGAP1 and SD proteins (nephrin, MAGI-1, CD2AP, NCK 1/2, podocin), podocalyxin, and cytoskeletal proteins (α-actinin-4). IQGAP1 silencing decreased podocyte migration and increased the permeability of a podocyte layer. Immunohistochemistry on normal human kidney confirmed IQGAP1 expression in podocytes and distal tubular epithelial cells and also showed an expression in glomerular parietal epithelial cells. In summary, our results suggest that IQGAP1, through its interaction with components of SD and cytoskeletal proteins, is involved in podocyte barrier properties

Obliteration dynamics in cerebral arteriovenous malformations after cyberknife radiosurgery: quantification with sequential nidus volumetry and 3-tesla 3-dimensional time-of-flight magnetic resonance angiography. Neurosurgery 2009; 64

OBJECTIVE: To investigate the time-dependent obliteration of cerebral arteriovenous malformations (cAVM) after CyberKnife radiosurgery (CKRS) (Accuray, Inc., Sunnyvale, CA) by means of sequential 3-T, 3-dimensional (3D), time-of-flight (TOF) magnetic resonance angiography (MRA), and volumetry of the arteriovenous malformation (AVM) nidus. METHODS: In this prospective study, 3D TOF MRA was performed on 20 patients with cAVMs treated by single-fraction CKRS. Three-dimensional TOF MRA was performed on a 3-T, 32-channel magnetic resonance scanner (Magnetom TIM Trio; Siemens Medical Solutions, Erlangen, Germany) with isotropic voxel size at a spatial resolution of 0.6 ϫ 0.6 ϫ 0.6 mm 3 . The time-dependent relative decay of the transnidal blood flow evidenced by 3D TOF MRA was referred to as "obliteration dynamics." Volumetry of the nidus size was performed with OsiriX imaging software (OsiriX Foundation, Geneva, Switzerland). All patients had 3 to 4 follow-up examinations at 3-to 6-month intervals over a minimum follow-up period of 9 months. Subtotal obliteration was determined if the residual nidus volume was 5% or less of the initial nidus volume. Stata/IC software (Version 10.0; Stata Corp., College Station, TX) was used for statistical analysis and to identify potential factors of AVM obliteration. RESULTS: Regarding their clinical status, case history, and pretreatments, the participants of this study represent difficult-to-treat cAVM patients. The median nidus volume was 1.8 mL (range, 0.4-12.5 mL); the median minimum dose prescribed to the nidus was 22 Gy (range, 16-24 Gy) delivered to the 67% isodose line (range, 55-80%). CKRS was well tolerated, with complications in 2 patients. No further hemorrhages occurred after RS, except 1 small and clinically inapparent incident. The median follow-up period after RS was 25.0 months (range, 11.7-36.8 months). After RS, a statistically significant obliteration was observed in all patients. However, the obliteration dynamics of the cAVMs showed a pronounced variability, with 2 types of post-therapeutic behavior identified. cAVMs of Group A showed a faster reduction of transnidal blood flow than cAVMs in Group B. The median time to subtotal obliteration was 23.8 months for all patients, 11.6 months for patients in Group A, and 27.8 months for patients in Group B (P ϭ 0.05). Logistic regression analysis revealed dose homogeneity and the circumscribed isodose to be the only variables (P Ͻ 0.01) associated with the obliteration dynamics in this study. The cumulative complete angiographic obliteration rate was 67% (95% confidence interval, 32-95%) 2 years after RS. CONCLUSION: The use of sequential 3D TOF MRA at 3 T and nidus volumetry enables a noninvasive quantitative assessment of the dynamic obliteration process induced by CKRS in cAVMs. This method may be helpful to identify factors related to AVM obliteration after RS when larger patient cohorts become available

Production of high-titer lentiviral particles for stable genetic modification of mammalian cells

Lentiviral gene transfer technologies exploit the natural efficiency of viral transduction to integrate exogenous genes into mammalian cells. This provides a simple research tool for inducing transgene expression or endogenous gene knockdown in both dividing and nondividing cells. This chapter describes an improved protocol for polyethylenimine (PEI)-mediated multi-plasmid transfection and polyethylene glycol (PEG) precipitation to generate and concentrate lentiviral vectors

Early tumor shrinkage and response assessment according to mRECIST predict overall survival in hepatocellular carcinoma patients under sorafenib

Background: The aim of this study was to explore the relationship between follow-up imaging characteristics and overall survival (OS) in advanced hepatocellular carcinoma (HCC) patients under sorafenib treatment. Methods: Associations between OS and objective response (OR) by mRECIST or early tumor shrinkage (ETS; ≥20% reduction in enhancing tumor diameter at the first follow-up imaging) were analyzed in HCC patients treated with sorafenib within a multicenter phase II trial (SORAMIC). 115 patients were included in this substudy. The relationship between survival and OR or ETS were explored. Landmark analyses were performed according to OR at fixed time points. Cox proportional hazards models with OR and ETS as a time-dependent covariate were used to compare survival with factors known to influence OS. Results: The OR rate was 29.5%. Responders had significantly better OS than non-responders (median 30.3 vs. 11.4 months; HR, 0.38 [95% CI, 0.22–0.63], p < 0.001), and longer progression-free survival (PFS; median 10.1 vs. 4.3 months, p = 0.015). Patients with ETS ≥ 20% had longer OS (median 22.1 vs. 11.4 months, p = 0.002) and PFS (median 8.0 vs. 4.3 months, p = 0.034) than patients with ETS < 20%. Besides OR and ETS, male gender, lower bilirubin and ALBI grade were associated with improved OS in univariate analysis. Separate models of multivariable analysis confirmed OR and ETS as independent predictors of OS. Conclusion: OR according to mRECIST and ETS in patients receiving sorafenib treatment are independent prognostic factors for OS. These parameters can be used for assessment of treatment benefit and optimal treatment sequencing in patients with advanced HCC

Assessment of selected media supplements to improve F/HN lentiviral vector production yields

The development of lentiviral-based therapeutics is challenged by the high cost of current Good Manufacturing Practices (cGMP) production. Lentiviruses are enveloped viruses that capture a portion of the host cell membrane during budding, which then constitutes part of the virus particle. This process might lead to lipid and protein depletion in the cell membrane and affect cell viability. Furthermore, growth in suspension also causes stresses that can affect virus production yields. To assess the impact of these issues, selected supplements (Cholesterol Lipid Concentrate, Chemically Defined Lipid Concentrate, Lipid Mixture 1, Gelatin Peptone N3, N-Acetyl-L-Cysteine and Pluronic F-68) were assayed in order to improve production yields in a transient transfection production of a Sendai virus F/HN-pseudotyped HIV-1-based third generation lentiviral vector in FreeStyle 293 (serum-free media) in suspension. None of the supplements tested had a significant positive impact on lentiviral vector yields, but small non-significant improvements could be combined to increase vector production in a cell line where other conditions have been optimised