A Simple,Quantitative Method Using Alginate Gel to Determine Rat Colonic Tumor Volume In Vivo

Many studies of the response of colonic tumors to therapeutics use tumor multiplicity as the endpoint to determine the effectiveness of the agent. These studies can be greatly enhanced by accurate measurements of tumor volume. Here we present a quantitative method to easily and accurately determine colonic tumor volume. This approach uses a biocompatible alginate to create a negative mold of a tumor-bearing colon; this mold is then used to make positive casts of dental stone that replicate the shape of each original tumor. The weight of the dental stone cast correlates highly with the weight of the dissected tumors. After refinement of the technique, overall error in tumor volume was 16.9% ± 7.9% and includes error from both the alginate and dental stone procedures. Because this technique is limited to molding of tumors in the colon, we utilized the Apc^Pirc/+ rat, which has a propensity for developing colonic tumors that reflect the location of the majority of human intestinal tumors. We have successfully used the described method to determine tumor volumes ranging from 4 to 196 mm³. Alginate molding combined with dental stone casting is a facile method for determining tumor volume in vivo without costly equipment or knowledge of analytic software. This broadly accessible method creates the opportunity to objectively study colonic tumors over time in living animals in conjunction with other experiments and without transferring animals from the facility where they are maintained.Colon cancer is the third leading cause of cancer in men and women, with more than 100,000 new cases diagnosed each year in the United States alone. This disease is not limited to humans—cancers of the colon and rectum also affect companion species, such as dogs, albeit less frequently than in humans.²⁰ Colorectal cancers generally develop from precancerous polyps, which can be detected and removed during colonoscopy screening before they become invasive cancers. However, not all precancers will become cancerous,²³ and a better understanding of early tumor growth dynamics in models of the disease can simultaneously increase the rate of detection of polyps destined to become cancerous and decrease the rate of unnecessary removal of benign polyps.Sizing of tumors creates an additional dimension beyond studies examining tumor multiplicity alone. Terminal sizing of tumors uses an eyepiece reticule under a dissection microscope to measure the maximal diameter of each tumor. However, this method likely misrepresents tumor volume for several reasons. First, tumors often are not symmetrical in shape, thereby limiting the interpretation of even multiple linear measurements. When volume calculations rely on the use of a formula, the irregular shape of solid tumors may require the testing of many different formulas to find the optimal one for that particular measurement and model.⁸ Second, if tumor sizing occurs after fixation, the original shape of the tumor can be affected. However, when tumor sizing occurs before fixation, the added time to size the tumors may result in degradation of the intestinal tissue, limiting further analysis. An alternate method of tumor sizing involves using the surrogate of tumor weight, the current ‘gold standard,’ for terminal studies. Tumor weight correlates closely with tumor size, although tumor density may vary depending on the tumor type. In addition, this technique is limited to use at the terminal time point. Methods that determine true tumor volume are powerful; those that can be applied in vivo to study the tumor longitudinally are even more compelling.It recently has been recognized that not all early colonic tumors grow; some remain static for years whereas a few spontaneously regress.²³ Importantly, the early growth profile of a tumor may correlate with its eventual fate.²³ This aspect of tumor biology is a newly emerging area that deserves deeper study. The current gold standard for determining longitudinal tumor volume is CT, given that tumor weight is available only through terminal experiments. In mice, microCT colonography can be used to detect a 16% change in tumor volume with 95% confidence in living animals.⁵ However, the cost of CT equipment limits this technology to shared facilities, and the pathogen status of these facilities may preclude returning animals to the place where they were original housed, limiting the opportunities for longitudinal study. Importantly, many institutions do not have access to microCT technology, and even if available, 3D renderings must be recreated to determine tumor volume, a process requiring specialized software and detailed computing knowledge. Furthermore, CT exposes animal subjects to radiation, which may interfere with the tumor biology. Although MRI can be used to determine tumor volume accurately in the absence of ionizing radiation, specialized scanners and software are required, and enemas or intravenous treatments are needed to visualize tumors clearly.²⁶Another imaging modality uses the surface area of signal due to proteins expressing a fluorescent marker, such as red fluorescent protein, as a surrogate for tumor volume.¹⁷ However, tumor volume measured by fluorescent surface area¹² may not accurately represent tumor volume in irregularly shaped tumors. In addition, this method necessitates a surgical procedure to orthotopically transplant fluorophore-expressing cells, raising questions of immune interactions between the recipient animal and the donor cells or to the surgery itself. If nude or immunocompromised animals are used in the procedure, the ability to study the immune aspect of tumor biology is reduced or eliminated.Alternatively, tumor volume can be estimated from endoscopic images. The study of tumors by colonoscopy has become routine for both mouse^6,10 and rat^1,15 models of the disease. In contrast to terminal assessments, colonoscopy allows tumors to be visualized in vivo over time, capturing the dynamics of tumor growth. Documentation of this aspect of tumor biology can greatly enrich studies evaluating chemopreventive or therapeutic agents.^6,15 Quantitative methods for determining tumor volume take this benefit a step further, allowing the investigation of the effects of background strain, therapeutic agents, environmental factors, or other modifiers of tumor growth pattern. One method to estimate tumor size uses the fraction of luminal cross-section occluded by tumor.² However, the colonic lumen expands as the animal grows, and its size often increases to accommodate the growing tumor, to prevent intestinal blockage. Optical methods to extrapolate tumor sizes from 2D images obtained in vivo during colonoscopy are achieved by inserting a flexible metal rod of known dimensions into the working channel of the endoscope.¹⁰ However, because colonic tumors can differ in shape (some are flat whereas others are pedunculated), area measurements may not translate accurately to tumor volume.To overcome these limitations and to add another tool to the growing cancer-research toolbox, we have developed a method using a biologically inert alginate to create negative molds of colonic tumors. These molds are filled with dental stone to achieve a positive cast of each tumor. A conversion factor then is used to calculate the volume of the original tumor from the dry weight of the dental stone cast. This procedure, which requires no specialized or expensive equipment and no complicated analytical methods, can be performed within the facility where the rats are housed and takes less than 15 min, including the 8 to 12 min during which the alginate sets. Therefore, our new method offers possibilities to study the dynamics of tumor growth in virtually any animal facility, regardless of the health status of subject animals or equipment availability.     

Measuring cortisol in fish scales to study stress in wild tropical tuna

Environmental Biology of Fishes - Cortisol is recognized as a physiological indicator of stress in fish. However, this hormone is typically measured in plasma samples. In this study, cortisol...     

Ringed seal (Pusa hispida) seasonal movements,diving, and haul‐out behavior in the Beaufort,Chukchi, and Bering Seas (2011–2017)

Continued Arctic warming and sea‐ice loss will have important implications for the conservation of ringed seals, a highly ice‐dependent species. A better understanding of their spatial ecology will help characterize emerging ecological trends and inform management decisions. We deployed satellite transmitters on ringed seals in the summers of 2011, 2014, and 2016 near Utqia?vik (formerly Barrow), Alaska, to monitor their movements, diving, and haul‐out behavior. We present analyses of tracking and dive data provided by 17 seals that were tracked until at least January of the following year. Seals mostly ranged north of Utqia?vik in the Beaufort and Chukchi Seas during summer before moving into the southern Chukchi and Bering Seas during winter. In all seasons, ringed seals occupied a diversity of habitats and spatial distributions, from near shore and localized, to far offshore and wide‐ranging in drifting sea ice. Continental shelf waters were occupied for >96% of tracking days, during which repetitive diving (suggestive of foraging) primarily to the seafloor was the most frequent activity. From mid‐summer to early fall, 12 seals made ~1‐week forays off‐shelf to the deep Arctic Basin, most reaching the retreating pack‐ice, where they spent most of their time hauled out. Diel activity patterns suggested greater allocation of foraging efforts to midday hours. Haul‐out patterns were complementary, occurring mostly at night until April‐May when midday hours were preferred. Ringed seals captured in 2011—concurrent with an unusual mortality event that affected all ice‐seal species—differed morphologically and behaviorally from seals captured in other years. Speculations about the physiology of molting and its role in energetics, habitat use, and behavior are discussed; along with possible evidence of purported ringed seal ecotypes.     

Exercise decreases speedball self-administration

Aims

Epidemiological studies report that individuals who exercise are less likely to abuse drugs. Preclinical studies report that exercise, in the form of treadmill or wheel running, reliably decreases the self-administration of psychomotor stimulants and opioids. To date, preclinical studies have only examined the effects of exercise on responding maintained by individual drugs and not by combinations of multiple drugs. This limits the translational appeal of these studies because polydrug abuse is common among substance abusing populations. The purpose of this study was to examine the effects of exercise on the self-administration of speedball, a combination of cocaine and heroin that is frequently encountered in intravenous drug abusing populations.

Main methods

Female rats were obtained at weaning and assigned to sedentary or exercising conditions. Sedentary rats were housed in standard cages that permitted no exercise beyond normal cage ambulation; exercising rats were housed in similar cages with an activity wheel. After 6 weeks, rats were implanted with intravenous catheters and trained to self-administer cocaine, heroin, and dose combinations of cocaine and heroin (i.e., speedball) on a progressive ratio schedule of reinforcement.

Key findings

Doses of speedball maintained greater levels of responding than corresponding doses of cocaine and heroin alone. Importantly, responding maintained by cocaine, heroin, and speedball was lower in exercising rats than sedentary rats.

Significance

These data indicate that exercise decreases the self-administration of speedball and suggest that exercise may reduce the abuse of drug combinations that have traditionally been resistant to treatment.     

The cumulative impact of annual coral bleaching can turn some coral species winners into losers

Mass coral bleaching events caused by elevated seawater temperatures result in extensive coral loss throughout the tropics, and are projected to increase in frequency and severity. If bleaching becomes an annual event later in this century, more than 90% of coral reefs worldwide may be at risk of long‐term degradation. While corals can recover from single isolated bleaching and can acclimate to recurring bleaching events that are separated by multiple years, it is currently unknown if and how they will survive and possibly acclimatize to annual coral bleaching. Here, we demonstrate for the first time that annual coral bleaching can dramatically alter thermal tolerance in Caribbean corals. We found that high coral energy reserves and changes in the dominant algal endosymbiont type (Symbiodinium spp.) facilitated rapid acclimation in Porites divaricata, whereas low energy reserves and a lack of algal phenotypic plasticity significantly increased susceptibility in Porites astreoides to bleaching the following year. Phenotypic plasticity in the dominant endosymbiont type of Orbicella faveolata did not prevent repeat bleaching, but may have facilitated rapid recovery. Thus, coral holobiont response to an isolated single bleaching event is not an accurate predictor of its response to bleaching the following year. Rather, the cumulative impact of annual coral bleaching can turn some coral species ‘winners’ into ‘losers’, and can also facilitate acclimation and turn some coral species ‘losers’ into ‘winners’. Overall, these findings indicate that cumulative impact of annual coral bleaching could result in some species becoming increasingly susceptible to bleaching and face a long‐term decline, while phenotypically plastic coral species will acclimatize and persist. Thus, annual coral bleaching and recovery could contribute to the selective loss of coral diversity as well as the overall decline of coral reefs in the Caribbean.     

Mapping DNA damage‐dependent genetic interactions in yeast via party mating and barcode fusion genetics

Condition‐dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State‐of‐the‐art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double‐mutant strains, does not scale readily to multi‐condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG‐GI), by which double‐mutant strains generated via en masse “party” mating can also be monitored en masse for growth to detect genetic interactions. By using site‐specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG‐GI enables multiplexed quantitative tracking of double mutants via next‐generation sequencing. We applied BFG‐GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4‐nitroquinoline 1‐oxide (4NQO), bleomycin, zeocin, and three other DNA‐damaging environments. BFG‐GI recapitulated known genetic interactions and yielded new condition‐dependent genetic interactions. We validated and further explored a subnetwork of condition‐dependent genetic interactions involving MAG1, SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53.     

Slit2 Inactivates GSK3β to Signal Neurite Outgrowth Inhibition

Slit molecules comprise one of the four canonical families of axon guidance cues that steer the growth cone in the developing nervous system. Apart from their role in axon pathfinding, emerging lines of evidence suggest that a wide range of cellular processes are regulated by Slit, ranging from branch formation and fasciculation during neurite outgrowth to tumor progression and to angiogenesis. However, the molecular and cellular mechanisms downstream of Slit remain largely unknown, in part, because of a lack of a readily manipulatable system that produces easily identifiable traits in response to Slit. The present study demonstrates the feasibility of using the cell line CAD as an assay system to dissect the signaling pathways triggered by Slit. Here, we show that CAD cells express receptors for Slit (Robo1 and Robo2) and that CAD cells respond to nanomolar concentrations of Slit2 by markedly decelerating the rate of process extension. Using this system, we reveal that Slit2 inactivates GSK3β and that inhibition of GSK3β is required for Slit2 to inhibit process outgrowth. Furthermore, we show that Slit2 induces GSK3β phosphorylation and inhibits neurite outgrowth in adult dorsal root ganglion neurons, validating Slit2 signaling in primary neurons. Given that CAD cells can be conveniently manipulated using standard molecular biological methods and that the process extension phenotype regulated by Slit2 can be readily traced and quantified, the use of a cell line CAD will facilitate the identification of downstream effectors and elucidation of signaling cascade triggered by Slit.