Faculty Bibliography

Background: We prepared an anti-p21Ras scFv which could specifically bind with mutant and wild-type p21Ras. However, it cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate the scFv penetration into tumor cells and produce antitumor effects.
Method(s): RGD4C-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed to express fusion proteins in E. coli, then the fusion proteins were purified with HisPur Ni-NTA. RGD4C-EGFP was used as reporter to test the factors affecting RGD4C penetration into tumor cell. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. The ability of RGD4C-p21Ras-scFv to penetrate SW480 cells and colocalization with Ras protein was detected by immunocytochemistry and immunofluorescence. The antitumor activity of the RGD4C-p21Ras-scFv was assessed with the MTT, TUNEL, colony formation and cell migration assays. Chloroquine (CQ) was used an endosomal escape enhancing agent to enhance endosomal escape of RGD4C-scFv.
Result(s): RGD4C-p21Ras-scFv fusion protein were successfully expressed and purified. We found that the RGD4C fusion protein could penetrate into tumor cells, but the tumor cell entry of was time and concentration dependent. Endocytosis inhibitors and a low temperature inhibited RGD4C fusion protein endocytosis into cells. The change of the cell membrane potential did not affect penetrability. RGD4C-p21Ras-scFv could penetrate SW480 cells, effectively inhibit the growth, proliferation and migration of SW480 cells and promote this cells apoptosis. In addition, chloroquine (CQ) could increase endosomal escape and improve antitumor activity of RGD4C-scFv in SW480 cells.
Conclusion(s): The RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of ras-driven cancers.
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(1) Background: The King-Devick (KD) rapid number naming test is sensitive for concussion diagnosis, with increased test time from baseline as the outcome measure. Eye tracking during KD performance in concussed individuals shows an association between inter-saccadic interval (ISI) (the time between saccades) prolongation and prolonged testing time. This pilot study retrospectively assesses the relation between ISI prolongation during KD testing and cognitive performance in persistently-symptomatic individuals post-concussion. (2) Results: Fourteen participants (median age 34 years; 6 women) with prior neuropsychological assessment and KD testing with eye tracking were included. KD test times (72.6 ± 20.7 s) and median ISI (379.1 ± 199.1 msec) were prolonged compared to published normative values. Greater ISI prolongation was associated with lower scores for processing speed (WAIS-IV Coding, r = 0.72, p = 0.0017), attention/working memory (Trails Making A, r = -0.65, p = 0.006) (Digit Span Forward, r = 0.57, p = -0.017) (Digit Span Backward, r= -0.55, p = 0.021) (Digit Span Total, r = -0.74, p = 0.001), and executive function (Stroop Color Word Interference, r = -0.8, p = 0.0003). (3) Conclusions: This pilot study provides preliminary evidence suggesting that cognitive dysfunction may be associated with prolonged ISI and KD test times in concussion.

INTRODUCTION/BACKGROUND:Genetic and imaging studies demonstrate a link between vascular morphology and migraine with aura (MA). We examined the relationship between basilar artery (BA) curvature and MA in a population-based cohort of stroke-free participants. METHODS:This cross-sectional study included participants from the MRI substudy of the Northern Manhattan Study. Participants had structured migraine assessments at enrollment and underwent brain MR angiography. BA curvature was defined as the sum of the total BA horizontal deviation from midline at the distal tip, mid-pons, and vertebrobasilar junction, and was the primary independent variable in logistic regression analyses. BA measurements were obtained blinded to migraine status. We compared groups of all migraine vs no migraine, migraine without aura (MwoA) vs no migraine, and MA vs no migraine. RESULTS:Of 880 participants, 146 had MwoA and 32 had MA. Average BA curvatures were 15.2 ± 8.9 mm in non-migraineurs, 15.8 ± 9.3 mm in MwoA, and 18.5 ± 11.4 mm in MA. In an adjusted model, greater BA curvature was associated with MA (OR 1.042 per mm, 95% CI 1.006-1.080) but not with MwoA (OR 1.014 per mm, 95% CI 0.993-1.035), when compared to non-migraineurs. CONCLUSIONS:Greater BA curvature was associated with MA. Given aura typically originates from the occipital cortex, understanding the physiopathology of this association may provide clues to migraine's underlying mechanisms and relationship with stroke.

PURPOSE OF REVIEW/OBJECTIVE:Diagnostic delay is an increasingly recognized issue in epilepsy. At the same time, there is a clear disparity between public awareness of epilepsy and that of other public health issues. A contributing factor for this seems to be a lack of studies testing interventions designed to improve seizure recognition. In this review, we summarize the main findings from recent studies investigating diagnostic delay in epilepsy, highlighting causes, consequences, and potential interventions in future research that may improve quality of care in this population. RECENT FINDINGS/RESULTS:Building on prior evidence, diagnostic delay in patients with new-onset focal epilepsy has been identified as an important problem for patients with epilepsy. Such delay in diagnosis can lead to delayed treatment and potentially preventable morbidity and mortality including motor vehicle accidents. Nonmotor seizure semiology appears to be a major contributor for delay; such seizures are largely unrecognized when patients present to emergency departments for care. Improving recognition and diagnosis of recurrent nonmotor seizures in emergency departments represents a significant opportunity for improving time to diagnosis, particularly when patients present following a first lifetime motor seizure and meet diagnostic criteria for epilepsy. Diagnostic delay in epilepsy is a significant public health issue and recent studies have highlighted potential areas for intervention.

Introduction: Inappropriate activation of Wnt/beta-catenin signaling plays a role in some cancers. beta-catenin (beta-cat) levels in the cell can be regulated by a cadherin-mediated sequestration into membrane-bound and free cytosolic pools, with the later translocating to the nucleus and driving TCF-mediated transcriptional activity following Wnt signal transduction. While sequencing has shown that MM lacks the mutations that typically lead to constitutive beta-cat activation seen in other cancers, we and others have demonstrated that Wnt/beta-catenin signaling is nonetheless activated in MM and can regulate MM growth. The mechanism driving beta-cat stabilization and activation in MM is unclear. E- and N-cadherin (N-cad) expression is elevated in MM compared to plasma cells from healthy donors. We hypothesized that that cadherins can regulate Wnt/beta-catenin signaling in MM.
Material(s) and Method(s): We detected different forms of beta-cat expression in a panel of human MM cell lines (HMCLs) and CD138 PC from MM patients by several approaches. Cadherin gain- or loss-of-function MM models were produced by expressing wild-type N-cad in MMS1 and ARP1 (lack endogenous N-cadherin expression) using a lentiviral system to create stable cell lines (N-Cad/MMS1 and N-cad/ARP1) and empty vector control (EV/MMS1, and EV-ARP1). We knocked down N-cadherin in the JJN3 cell line expressing high level of endogenous N-cadherin using shRNA specific for N-cad (shNcad/JJN3) or scrambled control shRNA (shCont/JJN3) by lentiviral-mediated transfection. We used a TCF reporter system to evaluate beta-cat transcriptional activity as previously described.
Result(s): We surveyed 25 HMCLs and CD138-selected plasma cells from 72 newly diagnosed MM for active beta-cat with an antibody that specifically recognizes the unphosphorylated active form of beta-cat. Higher levels of cytosolic and/or nuclear beta-cat protein were seen in 13 of 25 (52%) HMCLs and 36 of 72 (50%) primary MM PC. Correlation of beta-cat protein levels with global mRNA expression levels in primary PC revealed significant correlation with only one gene, CDH2 (N-cad). Remarkably, those primary MM with high beta-cat levels but low CDH2 levels expressed high levels of E-cadherin/CHD1 mRNA. This posed the question of whether CDH2 is a direct target of TCF/beta-cat transcriptional activity or whether high levels of CDH2 lead to increased levels of beta-cat protein via sequestration. Both CDH2 mRNA and protein were correlated with beta-cat protein but not beta-cat mRNA in 23/25 HMCLs. Co-immunoprecipitation revealed that N-cad and beta-cat complexes could be identified in HMCLs and primary MM. Consistent with N-cad-mediated stabilization of beta-cat both total and unphosphorylated beta-cat levels and TCF activity were significantly elevated in N-cad/MMS1 and N-Cad/ARP1 cells relative to controls. In contrast, shRNA mediated knockdown of N-cad led to a loss of both N-cad and beta-cat protein levels and TCF-dependent transcription activity relative to controls. These findings provide evidence that beta-cat/TCF signaling can be regulated by N-cad in MM. CDH2 mRNA is significantly elevated in the MS and HY subgroups of MM. To search for a potential mechanism of CDH2 transcriptional regulation in MS MM, we compared TCF activity and beta-cat protein levels in MS versus non-MS HMCLs. TCF activity and active beta-cat were elevated in MS versus non-MS forms of MM and B-cell lymphoma lacking N-cadherin. To determine if MMSET is required to up-regulate N-cad expression, we depleted the full-length MMSET protein in KMS11 cells. The results revealed a dramatic loss of total and unphosphorylated beta-cat protein, but not mRNA, and loss of both CDH2 mRNA and protein relative to controls. These data suggest that MMSET can regulate the transcription of the CDH2 gene. MMS1 and ARP1 cells stably expressing N-cad exhibited enhanced adhesion to bone marrow stromal cells and decreased sensitivity to bortezomib (Bzb). In contrast, blocking N-cadherin-mediated adhesion by CDH2 shRNA increased sensitivity to Bzb. These results suggests that N-cad/beta-cat complexes can contribute to adhesion-mediated drug resistance in MM.
Conclusion(s): Taken together, these findings establish that beta-cat is stabilized by N-cadherin-, and likely E-cadherin-, adhesins junction formation in MM. This in turn leads to an increased pool of beta-cat that can drive TCF transcriptional activation as well participate in cadherin-mediated cell adhesion and drug resistance. Disclosures: Davies: Amgen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Walker: Bristol Myers Squibb: Research Funding; Sanofi: Speakers Bureau.
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BACKGROUND:Open surgery has traditionally been preferred for the management of bifurcation middle cerebral artery (MCA) aneurysms. Flow diverting stents present a novel endovascular strategy for aneurysm treatment. OBJECTIVE:To add to the limited literature describing the outcomes and complications in the use of flow diverters for the treatment of these complex aneurysms. METHODS:This is a multicenter retrospective review of MCA bifurcation aneurysms undergoing flow diversion. We assessed post-treatment radiological outcomes and both thromboembolic and hemorrhagic complications. RESULTS:We reviewed the outcomes of 54 aneurysms treated with flow diversion. Four (7.4%) of the aneurysms had a history of rupture (3 remote and 1 acute). Fourteen (25.9%) of the aneurysms already underwent either open surgery or coiling prior to flow diversion. A total of 36 out of the 45 aneurysms (80%) with available follow-up data had adequate aneurysm occlusion with a median follow-up time of 12 mo. There were no hemorrhagic complications but 16.7% (9/54) had thromboembolic complications. CONCLUSION/CONCLUSIONS:Flow diverting stents may be a viable option for the endovascular treatment of complex bifurcation MCA aneurysms. However, compared to published series on the open surgical treatment of this subset of aneurysms, flow diversion has inferior outcomes and are associated with a higher rate of complications.