Caspase-3 and inhibitor of apoptosis protein(s) interactions in Saccharomyces cerevisiae and mammalian cells

AbstractUsing a heterologous yeast expression assay, we show that inhibitor of apoptosis proteins (IAPs) suppress caspase-3-mediated cytotoxicity in the order of XIAP>c-IAP2>c-IAP1>survivin. The same ordering of IAP activities was demonstrated in mammalian cells expressing an auto-activating caspase-3. The relative anti-apoptotic activities of each IAP depended on the particular death stimulus. For IAP-expressing cells treated with camptothecin, survival correlated with their intrinsic anti-caspase-3 activity. However, c-IAP1-transfected cells were disproportionately resistant to tumor necrosis factor-α, suggesting that its anti-apoptotic activities extend beyond caspase-3 or -7 inhibition. Yeast-based caspase assays provide rapid, reliable information on specificity and activity of the IAPs and aid in identifying critical targets in mammalian apoptotic pathways

Resection Cavity Contraction Effects in the Use of Radioactive Sources (1-25 versus Cs-131) for Intra-Operative Brain Implants.

Background and Objectives Intra-parenchymal brain surgical resection cavities usually contract in volume following low dose rate (LDR) brachytherapy implants. In this study, we systematically modeled and assessed dose variability resulting from such changes for I-125 versus Cs-131 radioactive sources. Methods Resection cavity contraction was modeled based on 95 consecutive patient cases, using surveillance magnetic resonance (MR) images. The model was derived for single point source geometry and then fully simulated in 3D where I-125 or Cs-131 seeds were placed on the surface of an ellipsoidal resection cavity. Dose distribution estimated via TG-43 calculations and biological effective dose (BED) calculations were compared for both I-125 and Cs-131, accounting for resection cavity contractions. Results Resection cavity volumes were found to contract with an effective half-life of approximately 3.4 months (time to reach 50% of maximum volume contraction). As a result, significant differences in dose distributions were noted between I-125 and Cs-131 radioactive sources. For example, when comparing with static volume, assuming no contraction effect, I-125 exhibited a 31.8% and 30.5% increase in D90 and D10 values (i.e., the minimal dose to 90% and 10% of the volume respectively) in the peripheral target areas over the follow-up period of 20.5 months. In contrast, Cs-131 seeds only exhibited a 1.44% and 0.64% increase in D90 and D10 values respectively. Such discrepancy is likewise similar for BED calculations. Conclusion Resection cavity contractions affects Cs-131 dose distribution significantly less than that of I-125 for permanent brain implants. Care must be taken to account for cavity contractions when prescribing accumulative doses of a radioactive source in performing the brain implant procedures

A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies.

Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening

Impact of Altered Airway Pressure on Intracranial Pressure, Perfusion, and Oxygenation: A Narrative Review.

OBJECTIVES: A narrative review of the pathophysiology linking altered airway pressure and intracranial pressure and cerebral oxygenation. DATA SOURCES: Online search of PubMed and manual review of articles (laboratory and patient studies) of the altered airway pressure on intracranial pressure, cerebral perfusion, or cerebral oxygenation. STUDY SELECTION: Randomized trials, observational and physiologic studies. DATA EXTRACTION: Our group determined by consensus which resources would best inform this review. DATA SYNTHESIS: In the normal brain, positive-pressure ventilation does not significantly alter intracranial pressure, cerebral oxygenation, or perfusion. In injured brains, the impact of airway pressure on intracranial pressure is variable and determined by several factors; a cerebral venous Starling resistor explains much of the variability. Negative-pressure ventilation can improve cerebral perfusion and oxygenation and reduce intracranial pressure in experimental models, but data are limited, and mechanisms and clinical benefit remain uncertain. CONCLUSIONS: The effects of airway pressure and ventilation on cerebral perfusion and oxygenation are increasingly understood, especially in the setting of brain injury. In the face of competing mechanisms and priorities, multimodal monitoring and individualized titration will increasingly be required to optimize care

Comparative evaluation of [(99m)tc]tilmanocept for sentinel lymph node mapping in breast cancer patients: results of two phase 3 trials.

BackgroundSentinel lymph node (SLN) surgery is used worldwide for staging breast cancer patients and helps limit axillary lymph node dissection. [(99m)Tc]Tilmanocept is a novel receptor-targeted radiopharmaceutical evaluated in 2 open-label, nonrandomized, within-patient, phase 3 trials designed to assess the lymphatic mapping performance.MethodsA total of 13 centers contributed 148 patients with breast cancer. Each patient received [(99m)Tc]tilmanocept and vital blue dye (VBD). Lymph nodes identified intraoperatively as radioactive and/or blue stained were excised and histologically examined. The primary endpoint, concordance (lower boundary set point at 90 %), was the proportion of nodes detected by VBD and [(99m)Tc]tilmanocept.ResultsA total of 13 centers contributed 148 patients who were injected with both agents. Intraoperatively, 207 of 209 nodes detected by VBD were also detected by [(99m)Tc]tilmanocept for a concordance rate of 99.04 % (p < 0.0001). [(99m)Tc]tilmanocept detected a total of 320 nodes, of which 207 (64.7 %) were detected by VBD. [(99m)Tc]Tilmanocept detected at least 1 SLN in more patients (146) than did VBD (131, p < 0.0001). In 129 of 131 patients with ≥1 blue node, all blue nodes were radioactive. Of 33 pathology-positive nodes (18.2 % patient pathology rate), [(99m)Tc]tilmanocept detected 31 of 33, whereas VBD detected only 25 of 33 (p = 0.0312). No pathology-positive SLNs were detected exclusively by VBD. No serious adverse events were attributed to [(99m)Tc]tilmanocept.Conclusion[(99m)Tc]Tilmanocept demonstrated success in detecting a SLN while meeting the primary endpoint. Interestingly, [(99m)Tc]tilmanocept was additionally noted to identify more SLNs in more patients. This localization represented a higher number of metastatic breast cancer lymph nodes than that of VBD

Identification of Novel Tumor Antigens With Patient-Derived Immune-Selected Antibodies

The identification of tumor antigens capable of eliciting an immune response in vivo may be an effective method to identify therapeutic cancer targets. We have developed a method to identify such antigens using frozen tumor-draining lymph node samples from breast cancer patients. Immune responses in tumor-draining lymph nodes were identified by immunostaining lymph node sections for B-cell markers (CD20&CD23) and Ki67 which revealed cell proliferation in germinal center zones. Antigen-dependent somatic hypermutation (SH) and clonal expansion (CE) were present in heavy chain variable (VH) domain cDNA clones obtained from these germinal centers, but not from Ki67 negative germinal centers. Recombinant VH single-domain antibodies were used to screen tumor proteins and affinity select potential tumor antigens. Neuroplastin (NPTN) was identified as a candidate breast tumor antigen using proteomic identification of affinity selected tumor proteins with a recombinant VH single chain antibody. NPTN was found to be highly expressed in approximately 20% of invasive breast carcinomas and 50% of breast carcinomas with distal metastasis using a breast cancer tissue array. Additionally, NPTN over-expression in a breast cancer cell line resulted in a significant increase in tumor growth and angiogenesis in vivo which was related to increased VEGF production in the transfected cells. These results validate NPTN as a tumor-associated antigen which could promote breast tumor growth and metastasis if aberrantly expressed. These studies also demonstrate that humoral immune responses in tumor-draining lymph nodes can provide antibody reagents useful in identifying tumor antigens with applications for biomarker screening, diagnostics and therapeutic interventions

Racial/Ethnic Discrimination and Mental Health in Mexican-Origin Youths and Their Parents: Testing the "Linked Lives" Hypothesis

PURPOSE: Using a life course perspective, the present study tested the concept of "linked lives" applied to the problem of not only how racial/ethnic discrimination may be associated with poor mental health for the target of discrimination but also how discrimination may exacerbate the discrimination-distress link for others in the target's social network-in this case, the family. METHODS: The discrimination-distress link was investigated among 269 Mexican-origin adolescents and their parents both cross-sectionally and longitudinally. It was hypothesized that parents' discrimination experiences would adversely affect their adolescent children's mental health via a moderating effect on the target adolescent discrimination-distress link. The converse was also hypothesized for the target parents. Multilevel moderation analyses were conducted to test the moderating effect of parents' discrimination experiences on the youth discrimination-distress link. We also tested the moderating effect of youths' discrimination experiences on the parent discrimination-distress link. RESULTS: Parents' discrimination experiences significantly moderated the longitudinal association between youths' discrimination stress appraisals and mental health, such that the father's discrimination experiences exacerbated the youth discrimination-depression link. Youths' discrimination stress appraisals were not a significant moderator of the cross-sectional parent discrimination-mental health association. CONCLUSIONS: Implications of these findings are discussed from a linked lives perspective, highlighting how fathers' discrimination experiences can adversely affect youths who are coping with discrimination, in terms of their mental health