A Quantitative Diffuse Reflectance Imaging (QDRI) System for Comprehensive Surveillance of the Morphological Landscape in Breast Tumor Margins

In an ongoing effort to address the clear clinical unmet needs surrounding breast conserving surgery (BCS), our group has developed a next-generation multiplexed optical-fiber-based tool to assess breast tumor margin status during initial surgeries. Specifically detailed in this work is the performance and clinical validation of a research-grade intra-operative tool for margin assessment based on diffuse optical spectroscopy. Previous work published by our group has illustrated the proof-of-concept generations of this device; here we incorporate a highly optimized quantitative diffuse reflectance imaging (QDRI) system utilizing a wide-field (imaging area = 17cm²) 49-channel multiplexed fiber optic probe, a custom raster-scanning imaging platform, a custom dual-channel white LED source, and an astronomy grade imaging CCD and spectrograph. The system signal to noise ratio (SNR) was found to be greater than 40dB for all channels. Optical property estimation error was found to be less than 10%, on average, over a wide range of absorption (μ_a = 0–8.9cm^-1) and scattering (μ_s’ = 7.0–9.7cm^-1) coefficients. Very low inter-channel and CCD crosstalk was observed (2% max) when used on turbid media (including breast tissue). A raster-scanning mechanism was developed to achieve sub-pixel resolution and was found to be optimally performed at an upsample factor of 8, affording 0.75mm spatially resolved diffuse reflectance images (λ = 450–600nm) of an entire margin (area = 17cm²) in 13.8 minutes (1.23cm²/min). Moreover, controlled pressure application at the probe-tissue interface afforded by the imaging platform reduces repeated scan variability, providing <1% variation across repeated scans of clinical specimens. We demonstrate the clinical utility of this device through a pilot 20-patient study of high-resolution optical parameter maps of the ratio of the β-carotene concentration to the reduced scattering coefficient. An empirical cumulative distribution function (eCDF) analysis is used to reduce optical property maps to quantitative distributions representing the morphological landscape of breast tumor margins. The optimizations presented in this work provide an avenue to rapidly survey large tissue areas on intra-operative time scales with improved sensitivity to regions of focal disease that may otherwise be overlooked.     

Quantitative analysis of anandamide and related acylethanolamides in human seminal plasma by ultra performance liquid chromatography tandem mass spectrometry

The endocannabinoids anandamide, palmitoylethanolamide and oleoylethanolamide have been detected in human seminal plasma and are bioactive lipids implicated in regulation of sperm motility, capacitation and acrosome reaction. Several methods exist for endocannabinoid quantification but none have been validated for measurement in human seminal plasma. We describe sensitive, robust, reproducible solid phase and isotope-dilution UHPLC-ESI-MS/MS methods for the extraction and quantification of anandamide, palmitoylethanolamide and oleoylethanolamide in human seminal plasma. Precision and accuracy were evaluated using pooled seminal plasma over a 4 day period. For all analytes, the inter- and intraday precision (CV%) was between 6.6-17.7% and 6.3-12.5%, respectively. Analyses were linear over the range 0.237-19nM for anandamide and oleoylethanolamide and 0.9-76nM for PEA. Limits of detection (signal-to-noise >3) were 50, 100 and 100fmol/mL and limits of quantification (signal-to-noise >10) were 100, 200 and 200fmol/mL, respectively for anandamide, palmitoylethanolamide and oleoylethanolamide. Anandamide and oleoylethanolamide were stable at -80°C for up to 4 weeks, but palmitoylethanolamide declined significantly. We assessed seminal plasma from 40 human donors with normozoospermia and found mean (inter-quartile range) concentrations of 0.21nM (0.09-0.27), 1.785nM (0.48-2.32) and 15.54nM (7.05-16.31) for anandamide, oleoylethanolamide and palmitoylethanolamide, respectively. Consequently, this UHPLC-ESI-MS/MS method represents a rapid, reliable and reproducible technique for the analysis of these endocannabinoids in fresh seminal plasma.     

The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons

To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-G(q/11)alpha-binding, catalytically-inactive (D110A,K220R)G protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCepsilon, but not Ca2+/diacylglycerol-sensitive eGFP-PKCbetaII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.     

Conditional deletion of focal adhesion kinase leads to defects in ventricular septation and outflow tract alignment

To examine a role for focal adhesion kinase (FAK) in cardiac morphogenesis, we generated a line of mice with a conditional deletion of FAK in nkx2-5-expressing cells (herein termed FAKnk mice). FAKnk mice died shortly after birth, likely resulting from a profound subaortic ventricular septal defect and associated malalignment of the outflow tract. Additional less penetrant phenotypes included persistent truncus arteriosus and thickened valve leaflets. Thus, conditional inactivation of FAK in nkx2-5-expressing cells leads to the most common congenital heart defect that is also a subset of abnormalities associated with tetralogy of Fallot and the DiGeorge syndrome. No significant differences in proliferation or apoptosis between control and FAKnk hearts were observed. However, decreased myocardialization was observed for the conal ridges of the proximal outflow tract in FAKnk hearts. Interestingly, chemotaxis was significantly attenuated in isolated FAK-null cardiomyocytes in comparison to genetic controls, and these effects were concomitant with reduced tyrosine phosphorylation of Crk-associated substrate (CAS). Thus, it is possible that ventricular septation and appropriate outflow tract alignment is dependent, at least in part, upon FAK-dependent CAS activation and subsequent induction of polarized myocyte movement into the conal ridges. Future studies will be necessary to determine the precise contributions of the additional nkx2-5-derived lineages to the phenotypes observed.     

Relationships to skating performance in competitive hockey players

The purpose of this study was to identify off-ice variables that would correlate to on-ice skating sprint performance and cornering ability. Previous literature has not reported any off-ice testing variables that strongly correlate to on-ice cornering ability in ice hockey players. Thirty-six male hockey players aged 15-22 years (mean +/- SD: 16.3 +/- 1.7 years; weight = 70.8 +/- 10.4 kg; height = 175.6 +/- 4.1 cm) with an average of 10.3 +/- 3.0 years hockey playing experience (most at AA and AAA levels) participated in the study. The on-ice tests included a 35-m sprint and the cornering S test. The off-ice tests included the following: 30-m sprint, vertical jump, broad jump, 3 hop jump, Edgren side shuffle, Hexagon agility, side support, push-ups, and 15-second modified Wingate. The on-ice sprint test and cornering S test were strongly correlated (r = 0.70; p < 0.001). While many off-ice tests correlated with on-ice skating, measures of horizontal leg power (off-ice sprint and 3 hop jump) were the best predictors of on-ice skating performance, once weight and playing level were accounted for. These 4 variables accounted for a total of 78% (p < 0.0001) of the variance in on-ice sprint performance. No off-ice test accounted for unique variance in S-cornering performance beyond weight, playing level, and skating sprint performance. These data indicate that coaches should include horizontal power tests of off-ice sprint and 3 hop jump to adequately assess skating ability. To improve on-ice skating performance and cornering ability, coaches should also focus on the development of horizontal power through specific off-ice training, although future research will determine whether off-ice improvements in horizontal power directly transfer to improvements in on-ice skating.     

Effect of starting stance on initial sprint performance

The effect of different starting stances from a standing position on short sprint times and the subsequent variability in times was investigated in this study. A dual-beam timing light system was used to measure 5- and 10-m times for 3 different standing starts commonly found in the sporting environment: parallel (feet parallel to the start line), split (lead left foot on start line, right leg back), and false (initial parallel start, right leg drops back to split start when movement initiated). The parallel start was found to be significantly (alpha < 0.05) slower than the other 2 stances for both the 5- ( approximately 8.3%) and the 10-m (approximately 5.9%) distances. Within the trial, variation of the different starting stances was equally consistent; however, there was less variability for the 10-m distance (CV = 1.16-1.67%) than the 5-m distance (CV = 1.43-2.15%) for each start for both men and women. The split and false start seem to offer the best option as a movement strategy for minimizing short-distance sprint times. However, the benefits of these 2 starts are less clear if total movement time is the variable of interest.     

Restoration of reproductive potential after expiration or removal of melengestrol acetate contraceptive implants in Tigers (Panthera tigris)

The need for contraception in the successful management of captive wild animals is becoming increasingly apparent. Because concerns exist regarding the reversibility of the contraceptive implant melengestrol acetate (MGA), reproductive data for 94 female Amur (Panthera tigris altaica) and Sumatran tigers (Panthera tigris sumatrae) were analyzed using survival analyses to evaluate return to reproductive status after implant removal or assumed expiration. Females placed in potential breeding situations after MGA implants were surgically removed showed a 62% return to reproduction by 5.25 years, whereas females with implants that were assumed to have expired showed only a 30% return to reproduction by 6 years. Implanted females did not reproduce as successfully as non‐implanted control females, which showed an 85% probability of reproducing after placement in a new breeding situation by 2.66 years. Parturition increased the probability of reproducing in non‐implanted females, but not in implanted females. Litter size, stillbirths, and offspring survival were not significantly different between non‐implanted, implant‐removed and implant‐expired female tigers. Ten female tigers reproduced both before and after implant placement, and the differences in litter size, stillbirths, and offspring survival were not significant, nor were they significantly different from non‐implanted females. Prior parturition, age when implant was removed, and duration of implantation did not affect the probability of reproducing for females after implant removal. These results show substantial reversibility of MGA implants, leading to 62% successful reproduction after implant removal. The reasons for lower successful reproduction in animals previously treated with the contraceptive compared to non‐implanted females are not known, but a greater delay in reversibility was seen when implants were left in place and only presumed expired. Zoo Biol 26:275–288, 2007. © 2007 Wiley‐Liss, Inc.     

Plasticity in thermal tolerance has limited potential to buffer ectotherms from global warming

Global warming is increasing the overheating risk for many organisms, though the potential for plasticity in thermal tolerance to mitigate this risk is largely unknown. In part, this shortcoming stems from a lack of knowledge about global and taxonomic patterns of variation in tolerance plasticity. To address this critical issue, we test leading hypotheses for broad-scale variation in ectotherm tolerance plasticity using a dataset that includes vertebrate and invertebrate taxa from terrestrial, freshwater and marine habitats. Contrary to expectation, plasticity in heat tolerance was unrelated to latitude or thermal seasonality. However, plasticity in cold tolerance is associated with thermal seasonality in some habitat types. In addition, aquatic taxa have approximately twice the plasticity of terrestrial taxa. Based on the observed patterns of variation in tolerance plasticity, we propose that limited potential for behavioural plasticity (i.e. behavioural thermoregulation) favours the evolution of greater plasticity in physiological traits, consistent with the ‘Bogert effect’. Finally, we find that all ectotherms have relatively low acclimation in thermal tolerance and demonstrate that overheating risk will be minimally reduced by acclimation in even the most plastic groups. Our analysis indicates that behavioural and evolutionary mechanisms will be critical in allowing ectotherms to buffer themselves from extreme temperatures.