Methods in Molecular Biology

Developmental processes are inherently dynamic and understanding them requires quantitative measurements of gene and protein expression levels in space and time. While live imaging is a powerful approach for obtaining such data, it is still a challenge to apply it over long periods of time to large tissues, such as the embryonic spinal cord in mouse and chick. Nevertheless, dynamics of gene expression and signaling activity patterns in this organ can be studied by collecting tissue sections at different developmental stages. In combination with immunohistochemistry, this allows for measuring the levels of multiple developmental regulators in a quantitative manner with high spatiotemporal resolution. The mean protein expression levels over time, as well as embryo-to-embryo variability can be analyzed. A key aspect of the approach is the ability to compare protein levels across different samples. This requires a number of considerations in sample preparation, imaging and data analysis. Here we present a protocol for obtaining time course data of dorsoventral expression patterns from mouse and chick neural tube in the first 3 days of neural tube development. The described workflow starts from embryo dissection and ends with a processed dataset. Software scripts for data analysis are included. The protocol is adaptable and instructions that allow the user to modify different steps are provided. Thus, the procedure can be altered for analysis of time-lapse images and applied to systems other than the neural tube

Differential Impact of Tumor Suppressor Pathways on DNA Damage Response and Therapy-Induced Transformation in a Mouse Primary Cell Model

The RB and p53 tumor suppressors are mediators of DNA damage response, and compound inactivation of RB and p53 is a common occurrence in human cancers. Surprisingly, their cooperation in DNA damage signaling in relation to tumorigenesis and therapeutic response remains enigmatic. In the context of individuals with heritable retinoblastoma, there is a predilection for secondary tumor development, which has been associated with the use of radiation-therapy to treat the primary tumor. Furthermore, while germline mutations of the p53 gene are critical drivers for cancer predisposition syndromes, it is postulated that extrinsic stresses play a major role in promoting varying tumor spectrums and disease severities. In light of these studies, we examined the tumor suppressor functions of these proteins when challenged by exposure to therapeutic stress. To examine the cooperation of RB and p53 in tumorigenesis, and in response to therapy-induced DNA damage, a combination of genetic deletion and dominant negative strategies was employed. Results indicate that loss/inactivation of RB and p53 is not sufficient for cellular transformation. However, these proteins played distinct roles in response to therapy-induced DNA damage and subsequent tumorigenesis. Specifically, RB status was critical for cellular response to damage and senescence, irrespective of p53 function. Loss of RB resulted in a dramatic evolution of gene expression as a result of alterations in epigenetic programming. Critically, the observed changes in gene expression have been specifically associated with tumorigenesis, and RB-deficient, recurred cells displayed oncogenic characteristics, as well as increased resistance to subsequent challenge with discrete therapeutic agents. Taken together, these findings indicate that tumor suppressor functions of RB and p53 are particularly manifest when challenged by cellular stress. In the face of such challenge, RB is a critical suppressor of tumorigenesis beyond p53, and RB-deficiency could promote significant cellular evolution, ultimately contributing to a more aggressive disease

Systematic Review of Potential Health Risks Posed by Pharmaceutical, Occupational and Consumer Exposures to Metallic and Nanoscale Aluminum, Aluminum Oxides, Aluminum Hydroxide and Its Soluble Salts

Aluminum (Al) is a ubiquitous substance encountered both naturally (as the third most abundant element) and intentionally (used in water, foods, pharmaceuticals, and vaccines); it is also present in ambient and occupational airborne particulates. Existing data underscore the importance of Al physical and chemical forms in relation to its uptake, accumulation, and systemic bioavailability. The present review represents a systematic examination of the peer-reviewed literature on the adverse health effects of Al materials published since a previous critical evaluation compiled by Krewski et al. (2007). Challenges encountered in carrying out the present review reflected the experimental use of different physical and chemical Al forms, different routes of administration, and different target organs in relation to the magnitude, frequency, and duration of exposure. Wide variations in diet can result in Al intakes that are often higher than the World Health Organization provisional tolerable weekly intake (PTWI), which is based on studies with Al citrate. Comparing daily dietary Al exposures on the basis of “total Al”assumes that gastrointestinal bioavailability for all dietary Al forms is equivalent to that for Al citrate, an approach that requires validation. Current occupational exposure limits (OELs) for identical Al substances vary as much as 15-fold. The toxicity of different Al forms depends in large measure on their physical behavior and relative solubility in water. The toxicity of soluble Al forms depends upon the delivered dose of Al+ 3 to target tissues. Trivalent Al reacts with water to produce bidentate superoxide coordination spheres [Al(O2)(H2O4)+ 2 and Al(H2O)6 + 3] that after complexation with O2•−, generate Al superoxides [Al(O2•)](H2O5)]+ 2. Semireduced AlO2• radicals deplete mitochondrial Fe and promote generation of H2O2, O2 • − and OH•. Thus, it is the Al+ 3-induced formation of oxygen radicals that accounts for the oxidative damage that leads to intrinsic apoptosis. In contrast, the toxicity of the insoluble Al oxides depends primarily on their behavior as particulates. Aluminum has been held responsible for human morbidity and mortality, but there is no consistent and convincing evidence to associate the Al found in food and drinking water at the doses and chemical forms presently consumed by people living in North America and Western Europe with increased risk for Alzheimer\u27s disease (AD). Neither is there clear evidence to show use of Al-containing underarm antiperspirants or cosmetics increases the risk of AD or breast cancer. Metallic Al, its oxides, and common Al salts have not been shown to be either genotoxic or carcinogenic. Aluminum exposures during neonatal and pediatric parenteral nutrition (PN) can impair bone mineralization and delay neurological development. Adverse effects to vaccines with Al adjuvants have occurred; however, recent controlled trials found that the immunologic response to certain vaccines with Al adjuvants was no greater, and in some cases less than, that after identical vaccination without Al adjuvants. The scientific literature on the adverse health effects of Al is extensive. Health risk assessments for Al must take into account individual co-factors (e.g., age, renal function, diet, gastric pH). Conclusions from the current review point to the need for refinement of the PTWI, reduction of Al contamination in PN solutions, justification for routine addition of Al to vaccines, and harmonization of OELs for Al substances

Retinoblastoma pathway deregulatory mechanisms determine clinical outcome in high-grade serous ovarian carcinoma

Alterations in the retinoblastoma pathway are frequent in ovarian/tubal high-grade serous cancers, but the mechanism of deregulation and the impact on patient outcome are poorly understood. A cohort of 334 high-grade serous carcinomas was studied by immunohistochemical analysis of RB1, p16, cyclin D1, cyclin E1, and Ki67. Additional detailed analyses including RB1 allelic deletion (n=42), mutation (n=75), methylation (n=31), and SNP array analyses (n=75) were performed on cases with clinical parameters, including age, debulking status, treatment, and clinical outcome. p16/RB1 expression results yielded three distinct clinically relevant subgroups upon multivariable analysis controlling for stage, debulking status, and treatment types: p16 homogeneous/RB1+ with the shortest progression-free survival (median 15 months (95% CI: 13-18); P=0.016) compared with the p16 heterogeneous/RB1+ subgroup (median 22 months (95% CI: 16-32)) and the p16 homogeneous/RB1- subgroup (median 20 months (95% CI: 15-24)). Patients in the p16 homo/RB1- subgroup showed a significant increase in overall survival (>60 months; P=0.013), which suggests an increase in sensitivity to cytotoxic agents. Analyses of Rb pathway mechanistic differences among these groups revealed frequent RB1 genomic alterations such as RB1 allelic loss and/or large spanning deletions (83%) in the p16 homo/RB1- subgroups, also indicating that RB1 deletions are frequent in high-grade serous carcinoma. CCNE1 gene gains/amplifications were frequent in the p16 homogeneous/RB1+ subgroup (68%) and cyclin D1 protein overexpression was predominantly characteristic of the p16 heterogeneous/RB1+ subgroup. These subcategories occur early in tumor progression and are seen with similar frequency in the cancer precursor lesion, serous tubal intra-epithelial carcinoma. Overall, this study uniquely identifies multiple non-synonymous mechanisms of retinoblastoma pathway deregulation that correlate with significantly different clinical outcomes. Furthermore, deregulations identified in precursor lesions suggest a key role of this pathway in serous tumor development. Recognition of these categories may identify patients with increased sensitivity to chemotherapy and new opportunities for novel therapeutics

Anti-Aβ Oligomer IgG and Surface Sialic Acid in Intravenous Immunoglobulin: Measurement and Correlation with Clinical Outcomes in Alzheimer’s Disease Treatment

<div><p>The fraction of IgG antibodies with anti-oligomeric Aβ affinity and surface sialic acid was compared between Octagam and Gammagard intravenous immunoglobulin (IVIG) using two complementary surface plasmon resonance methods. These comparisons were performed to identify if an elevated fraction existed in Gammagard, which reported small putative benefits in a recent Phase III clinical trial for Alzheimer’s Disease. The fraction of anti-oligomeric Aβ IgG was found to be higher in Octagam, for which no cognitive benefits were reported. The fraction and location of surface-accessible sialic acid in the Fab domain was found to be similar between Gammagard and Octagam. These findings indicate that anti-oligomeric Aβ IgG and total surface sialic acid alone cannot account for reported clinical differences in the two IVIG products. A combined analysis of sialic acid in anti-oligomeric Aβ IgG did reveal a notable finding that this subgroup exhibited a high degree of surface sialic acid lacking the conventional α2,6 linkage. These results demonstrate that the IVIG antibodies used to engage oligomeric Aβ in both Gammagard and Octagam clinical trials did not possess α2,6-linked surface sialic acid at the time of administration. Anti-oligomeric Aβ IgG with α2,6 linkages remains untested as an AD treatment.</p></div