Differential role of RIP1 in Smac mimetic-mediated chemosensitization of neuroblastoma cells

We explored the potential of Smac mimetics, which antagonize Inhibitor of Apoptosis (IAP) proteins, for chemosensitization of neuroblastoma (NB). Here, we report that Smac mimetics, e.g. BV6, prime NB cells for chemotherapeutics including the topoisomerase II inhibitor doxorubicin (DOX) and vinca alkaloids such as Vincristine (VCR), Vinblastine (VBL) and Vinorelbine (VNR). Additionally, BV6 acts in concert with DOX or VCR to suppress long-term clonogenic growth. While BV6 causes rapid downregulation of cellular IAP (cIAP)1 protein and nuclear factor-kappaB (NF-κB) activation, DOX/BV6- or VCR/BV6-induced apoptosis occurs independently of NF-κB or TNFα signaling, since overexpression of dominant-negative IκBα superrepressor or the Tumor Necrosis Factor (TNF)α-blocking antibody Enbrel fail to block cell death. Mechanistic studies reveal that Receptor-interacting protein (RIP)1 is required for DOX/BV6-, but not for VCR/BV6-induced apoptosis, since transient or stable knockdown of RIP1 or the pharmacological RIP1 inhibitor necrostatin-1 significantly reduce apoptosis. By comparison, VCR/BV6-mediated apoptosis critically depends on the mitochondrial pathway. VCR/BV6 cotreatment causes phosphorylation of BCL-2 during mitotic arrest, enhanced activation of BAX and BAK and loss of mitochondrial membrane potential (MMP). Additionally, overexpression of BCL-2 profoundly suppresses VCR/BV6-induced apoptosis. Thus, BV6 sensitizes NB cells to chemotherapy-induced apoptosis via distinct initial signaling mechanisms depending on the chemotherapeutic drug. These findings provide novel mechanistic insights into Smac mimetic-mediated chemosensitization of NB.     

The FNTB promoter polymorphism rs11623866 as a potential predictive biomarker for lonafarnib treatment of ovarian cancer patients

Aim

Despite promising preclinical findings regarding clinical utility of farnesyltransferase inhibitors (FTI), such as lonafarnib, success of clinical trials is limited. A multicentre AGO-OVAR-15 phase II trial reported an unfavourable effect of lonafarnib on the outcome of patients with advanced ovarian cancer. This study was performed as a genetic subgroup analysis of the AGO-OVAR-15 trial, and investigated the utility of the promoter polymorphism rs11623866 of the farnesyltransferase ß-subunit gene (FNTB) in predicting the clinical effectiveness of lonafarnib.

Methods

The influence of rs11623866 (c.-609G > C) on FNTB promoter activity was investigated by electrophoretic-mobility-shift assay, luciferase-reporter assay and RT-qPCR. A total of 57 out of 105 patients from the AGO-OVAR-15 trial, treated with carboplatin and paclitaxel ± lonafarnib, was genotyped for rs11623866 by restriction fragment length polymorphism analysis. Genotype-dependent survival analysis was performed by Kaplan–Meier analysis.

Results

The presence of the G allele was associated with increased FNTB promoter activity compared with the C allele. An unfavourable effect of lonafarnib was limited to patients carrying a GG genotype (HR_PFS 6.2, 95%CI = 2.01, 19.41, P = 0.002; HR_OS 9.6, 95%CI = 1.89, 48.54, P = 0.006). Median progression free survival (PFS) for patients with the GG genotype in the lonafarnib treated arm was 10 months, whereas median PFS without FTI-treatment was 40 months. Median overall survival (OS) in the lonafarnib-treated group was 19 months, whereas median OS was not reached in the untreated group.

Conclusions

Discrepancies between preclinical success and clinical failure may be due to the patients'' genetic variability of FNTB. Therefore, our results may encourage retrospective evaluation of FNTB polymorphisms in previous FTI studies, especially those reporting positive FTI response.     

Trunk exercise training improves muscle size,strength, and function in older adults: A randomized controlled trial

The aim of this study was to assess the effectiveness of a multimodal exercise program to increase trunk muscle morphology and strength in older individuals, and their associated changes in functional ability. Using a single‐blinded parallel‐group randomized controlled trial design, 64 older adults (≥60 years) were randomly allocated to a 12‐week exercise program comprising walking and balance exercises with or without trunk strengthening/motor control exercises; followed by a 6‐week walking‐only program (detraining; 32 per group). Trunk muscle morphology (ultrasound imaging), strength (isokinetic dynamometer), and functional ability and balance (6‐Minute Walk Test; 30 second Chair Stand Test; Sitting and Rising Test; Berg Balance Scale, Multi‐Directional Reach Test; Timed Up and Go; Four Step Square Test) were the primary outcome measures. Sixty‐four older adults (mean [SD]; age: 69.8 [7.5] years; 59.4% female) were randomized into two exercise groups. Trunk training relative to walking‐balance training increased (mean difference [95% CI]) the size of the rectus abdominis (2.08 [1.29, 2.89] cm²), lumbar multifidus (L4/L5:0.39 [0.16, 0.61] cm; L5/S1:0.31 [0.07, 0.55] cm), and the lateral abdominal musculature (0.63 [0.40, 0.85] cm); and increased trunk flexion (29.8 [4.40, 55.31] N), extension (37.71 [15.17, 60.25] N), and lateral flexion (52.30 [36.57, 68.02] N) strength. Trunk training relative to walking‐balance training improved 30‐second Chair Stand Test (5.90 [3.39, 8.42] repetitions), Sitting and Rising Test (1.23 [0.24, 2.23] points), Forward Reach Test (4.20 [1.89, 6.51] cm), Backward Reach Test (2.42 [0.33, 4.52] cm), and Timed Up and Go Test (?0.76 [?1.40, ?0.13] seconds). Detraining led to some declines but all outcomes remained significantly improved when compared to pre‐training. These findings support the inclusion of trunk strengthening/motor control exercises as part of a multimodal exercise program for older adults.     

Comparison of novel risk factors for cardiovascular disease between hemodialysis patients with and without protein-energy wasting

Purpose

The present study was designed to compare novel risk factors for cardiovascular diseases (CVD) between hemodialysis (HD) patients with or without protein-energy wasting (PEW) for determining novel risk factors for CVD in HD patients with PEW.

Methods

In this cross-sectional study, 291 HD patients were randomly selected from among 2,302 adult HD patients in Tehran hemodialysis centers. The presence of PEW in HD patients was determined by subjective global assessment. In addition, 4 mL blood was obtained before dialysis and analyzed for serum concentrations of novel risk factors for CVD, including C-reactive protein (CRP), soluble intercellular adhesion molecule type 1 (sICAM-1), soluble vascular cell adhesion molecule type 1 (sVCAM-1), sE-selectin, malondialdehyde (MDA), nitric oxide (NO), endothelin-1 and lipoprotein (a) [Lp (a)].

Results

Serum CRP and sICAM-1 were significantly higher in HD patients with PEW as compared to those without PEW (P < 0.01), whereas there were no significant differences in serum sVCAM-1, sE-selectin, MDA, NO, endothelin-1 and Lp (a) between the two groups. Serum CRP and sICAM-1 were significantly higher in HD patients with PEW type IIa and IIb than in those with PEW type I (P < 0.01).

Conclusion

The present study indicates that serum CRP and sICAM-1, as two CVD risk factors, increase in HD patients with PEW as compared to those without PEW and these increases occur in HD patients with PEW type IIa and IIb who have inflammation.     

A Novel Protocol to Differentiate Induced Pluripotent Stem Cells by Neuronal microRNAs to Provide a Suitable Cellular Model

Neurodegenerative diseases are one of the most challenging subjects in medicine. Investigation of their underlying genetic or epigenetic factors is hampered by lack of suitable models. Patient‐specific induced pluripotent stem cells (iPS cells) represent a valuable approach to provide a proper model for poorly understood mechanisms of neuronal diseases and the related drug screenings. miR‐124 and miR‐128 are the two brain‐enriched miRNAs with different time‐points of expression during neuronal development. Herein, we transduced human iPS cells with miR‐124 and miR‐128 harboring lentiviruses sequentially. The transduced plasmids contained GFP and puromycin antibiotic‐resistant genes for easier selection and identification. Morphological assessment and immunocytochemistry (overexpressions of beta‐tubulin and neuron‐specific enolase) confirmed that induced hiPS cells by miR‐124 and miR‐128 represent similar characteristics to those of mature neurons. In addition, the upregulation of neuron‐specific enolase, beta‐tubulin, Map2, GFAP, and BDNF was detected by quantitative real‐time PCR. In conclusion, it seems that our novel protocol remarks the combinatorial effect of miR‐124 and miR‐128 on neural differentiation in the absence of any extrinsic factor. Moreover, such cellular models could be used in personalized drug screening and applied for more effective therapies.     

Self-care intervention to reduce oral candidiasis recurrences in HIV-seropositive persons: a pilot study

OBJECTIVES: This single-blind randomized controlled pilot study evaluated the efficacy of a behavioral intervention program, PRO-SELF: Candidiasis, to reduce time to recurrence of oral candidiasis over 6 months in susceptible HIV-seropositive persons. The intervention involved instruction by dentists on improving oral hygiene, minimizing sugar intake, and self-diagnosing candidiasis. METHODS: Participants were adults with oral candidiasis responsive to antifungals who presented to the UCSF Stomatology Clinic between 1997 and 2000. At 2-3 weeks of follow-up visits, a dentist "examiner", masked to group assignment, quizzed participants as to the presence of candidiasis, and assessed candidiasis status. A second, unmasked dentist "instructor" then delivered the program to intervention participants. Participants recorded dietary and oral hygiene practices in 24-h recall diaries: intervention participants at each visit and controls at initial and final visits. RESULTS: At randomization, CD4+ cell counts (cells/mm(3)) were 298 +/- 188 among 18 intervention participants and 396 +/- 228 among 17 controls. The candidiasis recurrence rates at 6 months were 78% among intervention compared with 88% among control participants (hazard ratio 0.72; 95% CI 0.35-1.50). Performing oral hygiene after meals/snacks showed the largest relative improvement: intervention-control difference in proportion of meals/snacks affected was 24% (95% CI -1 to 48%). Self-diagnoses of candidiasis were inaccurate, possibly because of mild episodes. CONCLUSIONS: The results weakly indicate that regular instruction from healthcare professionals helps patients delay candidiasis recurrence by improving oral hygiene. Among HIV-seropositive persons, those with poor oral hygiene, and high-sugar diets are most likely to benefit.     

Structural correlates of a functional streptokinase antigenic epitope: serine 138 is an essential residue for antibody binding

We determined the pattern of cross-reactivity of a panel of anti-streptokinase (SK) monoclonal antibodies (mAbs) with SK variants in order to map the antigenic and functional epitope of SK. Comparison of the pattern of cross-reactivity of the anti-SK mAb A4.3 with SK variants and sequence alignments of SK variants and native (n) SK suggested that mutation of Ser 138 to Lys results in loss of binding of mAb A4.3 to SK variants. However, this mutation does not affect formation of activator complex by these proteins. The epitope specificity of the mAb A4.3 was further confirmed by mutating Ser 138 to Lys in n SK. Monoclonal Ab A4.3 did not bind to mutant SK (Ser138Lys). Activator activity of mutant SK (Ser138Lys) was indistinguishable from that of n SK and recombinant n SK. Since addition of A4.3 mAb to an equimolar mixture of SK and human plasminogen inhibits activator complex formation, the sequences spanning position 138 are likely important for formation of streptokinase-plasminogen activator complex or processing of the plasminogen substrate.