Gene expression profiles from formalin fixed paraffin embedded breast cancer tissue are largely comparable to fresh frozen matched tissue

Background and Methods

Formalin Fixed Paraffin Embedded (FFPE) samples represent a valuable resource for cancer research. However, the discovery and development of new cancer biomarkers often requires fresh frozen (FF) samples. Recently, the Whole Genome (WG) DASL (cDNA-mediated Annealing, Selection, extension and Ligation) assay was specifically developed to profile FFPE tissue. However, a thorough comparison of data generated from FFPE RNA and Fresh Frozen (FF) RNA using this platform is lacking. To this end we profiled, in duplicate, 20 FFPE tissues and 20 matched FF tissues and evaluated the concordance of the DASL results from FFPE and matched FF material.

Methodology and Principal Findings

We show that after proper normalization, all FFPE and FF pairs exhibit a high level of similarity (Pearson correlation >0.7), significantly larger than the similarity between non-paired samples. Interestingly, the probes showing the highest correlation had a higher percentage G/C content and were enriched for cell cycle genes. Predictions of gene expression signatures developed on frozen material (Intrinsic subtype, Genomic Grade Index, 70 gene signature) showed a high level of concordance between FFPE and FF matched pairs. Interestingly, predictions based on a 60 gene DASL list (best match with the 70 gene signature) showed very high concordance with the MammaPrint® results.

Conclusions and Significance

We demonstrate that data generated from FFPE material with the DASL assay, if properly processed, are comparable to data extracted from the FF counterpart. Specifically, gene expression profiles for a known set of prognostic genes for a specific disease are highly comparable between two conditions. This opens up the possibility of using both FFPE and FF material in gene expressions analyses, leading to a vast increase in the potential resources available for cancer research.     

Decreased spontaneous eye blink rates in chronic cannabis users: evidence for striatal cannabinoid-dopamine interactions

Chronic cannabis use has been shown to block long-term depression of GABA-glutamate synapses in the striatum, which is likely to reduce the extent to which endogenous cannabinoids modulate GABA- and glutamate-related neuronal activity. The current study aimed at investigating the effect of this process on striatal dopamine levels by studying the spontaneous eye blink rate (EBR), a clinical marker of dopamine level in the striatum. 25 adult regular cannabis users and 25 non-user controls matched for age, gender, race, and IQ were compared. Results show a significant reduction in EBR in chronic users as compared to non-users, suggesting an indirect detrimental effect of chronic cannabis use on striatal dopaminergic functioning. Additionally, EBR correlated negatively with years of cannabis exposure, monthly peak cannabis consumption, and lifetime cannabis consumption, pointing to a relationship between the degree of impairment of striatal dopaminergic transmission and cannabis consumption history.     

Changes in the distribution of ZO-1, occludin,and claudins in the rat uterine epithelium during the estrous cycle

During the estrous cycle, the endometrium epithelium experiences marked cellular structural changes. For fertilization to proceed, maintenance of an adequate uterine environment by ovarian hormones is essential. Epithelial cells lining the uterine lumen are associated with each other by tight junctions (TJs), which regulate the passage of ions and molecules through the paracellular pathway. The aim of the present study was to assess by confocal immunofluorescence the distribution pattern of the TJ proteins ZO-1, occludin, and claudins 1–7 in the rat uterus during the estrous cycle. Our results reveal that on proestrus, the day when mating takes place, ZO-1, occludin, and claudins 1 and 5 are located in the TJs, while claudins 3 and 7 display a basolateral distribution. In contrast, on metestrus day, when no sexual mating occurs and the uterine lumen is devoid of secretions, none of these proteins were detected in the TJ region, and only a diffuse cytosolic staining was observed for some of the proteins. On estrus and diestrus days, an intermediate situation was encountered, since ZO-1 localized in the TJs, whereas occludin was no longer detectable in the TJs. The distribution of claudins during these stages varied from the lowermost portion of the basolateral membrane to its apex. In conclusion, the results show that the protein composition of TJs present in the luminal epithelial cells of the uterus changes during the different days of the estrous cycle, and suggest that the expression of TJ proteins participates in providing an adequate environment for a successful fertilization.This work was supported by grants PAPIIT (IN210902, IX228504) and PAIP (6190-08) from the National Autonomous University of Mexico (UNAM), and by grants G34511-M and 37846-N from the Mexican National Council on Science and Technology (CONACYT).     

Computational studies of shape memory alloy behavior in biomedical applications

BACKGROUND: Nowadays, shape memory alloys (SMAs) and in particular Ni-Ti alloys are commonly used in bioengineering applications as they join important qualities as resistance to corrosion, biocompatibility, fatigue resistance, MR compatibility, kink resistance with two unique thermo-mechanical behaviors: the shape memory effect and the pseudoelastic effect. They allow Ni-Ti devices to undergo large mechanically induced deformations and then to recover the original shape by thermal loading or simply by mechanical unloading. METHOD OF APPROACH: A numerical model is developed to catch the most significant SMA macroscopic thermo-mechanical properties and is implemented into a commercial finite element code to simulate the behavior of biomedical devices. RESULTS: The comparison between experimental and numerical response of an intravascular coronary stent allows to verify the model suitability to describe pseudo-elasticity. The numerical study of a spinal vertebrae spacer where the effects of different geometries and material characteristic temperatures are investigated, allows to verify the model suitability to describe shape memory effect. CONCLUSION: the results presented show the importance of computational studies in designing and optimizing new biomedical devices.     

Rickettsia africae in Amblyomma variegatum and domestic ruminants on eight Caribbean islands

We used PCRs with omp A primers to determine if spotted fever group rickettsiae occurred in Amblyomma variegatum from 6 Caribbean islands. Positive amplicons were obtained from ticks from the U.S. Virgin Islands (9/18; 50%), Dominica (39/171; 30%), Montserrat (2/5; 40%), Nevis (17/34; 50%), St. Kitts (46/227; 20%), and St. Lucia (1/14; 7%). Sequences for a convenience sample of reaction products obtained from A. variegatum on St. Kitts (7), American Virgin Islands (4), Montserrat (2), and St. Lucia (1) were 100% homologous with that of Rickettsia africae , the agent of African tick-bite fever. To determine if transmission of R. africae occurred, we used Rickettsia rickettsii antigen in IFA tests and found positive titers (≥ 1/80) with sera from cattle, goats, and sheep from Dominica (24/95 [25%], 2/136 [2%], 0/58 [0%]), Nevis (12/45 [27%], 5/157 [3%], 0/90 [0%]), St. Kitts (2/43 [5%], 1/25 [4%), 1/35 [3%]), and St. Lucia (6/184 [3%] cattle), respectively. No seropositive animals were found in Grenada (0/4, 0/98/, 0/86), Montserrat (0/12, 0/26, 0/52), or Puerto Rico (0/80 cattle). Our study indicates that R. africae and African tick-bite fever are widespread in the Caribbean.     

Hepatocyte growth factor installs a survival platform for colorectal cancer cell invasive growth and overcomes p38 MAPK-mediated apoptosis

Hepatocyte growth factor (HGF) induces invasive growth, a biological program that confers tumor cells the capability to invade and metastasize by integrating cell proliferation, motility, morphogenesis, and survival. We here demonstrate that HGFR activation promotes survival of colorectal carcinoma (CRC) cells exposed to conditions that mimic those met during tumor progression, i.e. nutrient deprivation or substrate detachment, and following chemotherapeutic treatment. In all these conditions, a sustained activation of p38 MAPK delivers a main death signal that is overcome by cell treatment with HGF. HGF-driven survival requires the engagement of the PI3K/Akt/mTOR/p70S6K and ERK MAPK transduction pathways. Abrogation of p38 MAPK activity prevents CRC cell apoptosis also when these transduction pathways are inhibited, and treatment with HGF further increases survival. Engagement of these signaling cascades is also needed for HGF to induce CRC cell scattering, morphogenesis, motility and invasion. Activation of p38 MAPK signaling is therefore a main apoptotic switch for CRC cells in the stressful conditions encountered during tumor progression. Conversely, HGF orchestrates several biochemical pathways, which allow cell survival in these same conditions and promote the biological responses required for tumor invasive growth. Both p38 MAPK and HGF/HGFR signaling constitute potential molecular targets for inhibiting colorectal carcinogenesis.     

Geldanamycins trigger a novel Ron degradative pathway, hampering oncogenic signaling

Ron, the tyrosine kinase receptor for macrophage-stimulating protein is responsible for proliferation and migration of cells from different tissues. Ron can acquire oncogenic potential by single point mutations in the kinase domain, and dysregulated Ron signaling has been involved in the development of different human cancers. We have previously shown that ligand-activated Ron recruits the negative regulator c-Cbl, which mediates its ubiquitylation and degradation. Here we report that Ron is ubiquitylated also by the U-box E3 ligase C-terminal Hsc70-interacting protein (CHIP), recruited via chaperone intermediates Hsp90 and Hsc70. Gene silencing shows that CHIP activity is necessary to mediate Ron degradation upon cell treatment with Hsp90 inhibitors geldanamycins. The oncogenic Ron(M1254T) receptor escapes from c-Cbl negative regulation but retains a strong association with CHIP. This constitutively active mutant of Ron displays increased sensitivity to geldanamycins, enhanced physical interaction with Hsp90, and more rapid degradation rate. Cell growth and migration, as well as the transforming potential evoked by Ron(M1254T), are abrogated upon Hsp90 inhibition. These data highlight a novel mechanism for Ron degradation and propose Hsp90 antagonists like geldanamycins as suitable pharmacological agents for therapy of cancers where altered Ron signaling is involved.     

Changes in intranuclear chromatin architecture induce bipolar nuclear localization of histone variant H1T2 in male haploid spermatids

Spermiogenesis entails a major biochemical and morphological restructuring of the germ cell packing the DNA into the condensed spermatid nucleus. H1T2 is a histone H1 variant selectively and transiently expressed in male haploid germ cells during spermiogenesis that specifically localizes to a chromatin domain at the apical pole under the acrosome. We explored the mechanisms determining polar localization of H1T2 in spermatids. In acrosome-deficient round spermatids of hrb -/- and gopc -/- mice, H1T2 localization is not altered, indicating that proper acrosome development is not required for specifying nuclear polarity. In contrast, in late round spermatids from trf2 -/- or hmgb2 -/- mice, a bipolar H1T2 localization was observed revealing that polarity is modified by loss of proteins specifying chromatin architecture. Our results show that intranuclear chromatin organization is critical for correct polar localization of H1T2 and that H1T2 can be a useful molecular marker revealing chromatin disorganization in spermatids.