Mechanisms underlying divergent relationships between Ca2+ and YAP/TAZ signalling.

Yes-associated protein (YAP) and its homologue TAZ are transducers of several biochemical and biomechanical signals, integrating multiplexed inputs from the microenvironment into higher level cellular functions such as proliferation, differentiation and migration. Emerging evidence suggests that Ca2+ is a key second messenger that connects microenvironmental input signals and YAP/TAZ regulation. However, studies that directly modulate Ca2+ have reported contradictory YAP/TAZ responses: in some studies, a reduction in Ca2+ influx increases the activity of YAP/TAZ, while in others, an increase in Ca2+ influx activates YAP/TAZ. Importantly, Ca2+ and YAP/TAZ exhibit distinct spatiotemporal dynamics, making it difficult to unravel their connections from a purely experimental approach. In this study, we developed a network model of Ca2+ -mediated YAP/TAZ signalling to investigate how temporal dynamics and crosstalk of signalling pathways interacting with Ca2+ can alter the YAP/TAZ response, as observed in experiments. By including six signalling modules (e.g. GPCR, IP3-Ca2+ , kinases, RhoA, F-actin and Hippo-YAP/TAZ) that interact with Ca2+ , we investigated both transient and steady-state cell response to angiotensin II and thapsigargin stimuli. The model predicts that stimuli, Ca2+ transients and frequency-dependent relationships between Ca2+ and YAP/TAZ are primarily mediated by cPKC, DAG, CaMKII and F-actin. Simulation results illustrate the role of Ca2+ dynamics and CaMKII bistable response in switching the direction of changes in Ca2+ -induced YAP/TAZ activity. A frequency-dependent YAP/TAZ response revealed the competition between upstream regulators of LATS1/2, leading to the YAP/TAZ non-monotonic response to periodic GPCR stimulation. This study provides new insights into underlying mechanisms responsible for the controversial Ca2+ -YAP/TAZ relationship observed in experiments. KEY POINTS: YAP/TAZ integrates biochemical and biomechanical inputs to regulate cellular functions, and Ca2+ acts as a key second messenger linking cellular inputs to YAP/TAZ. Studies have reported contradictory Ca2+ -YAP/TAZ relationships for different cell types and stimuli. A network model of Ca2+ -mediated YAP/TAZ signalling was developed to investigate the underlying mechanisms of divergent Ca2+ -YAP/TAZ relationships. The model predicts context-dependent Ca2+ transient, CaMKII bistable response and frequency-dependent activation of LATS1/2 upstream regulators as mechanisms governing the Ca2+ -YAP/TAZ relationship. This study provides new insights into the underlying mechanisms of the controversial Ca2+ -YAP/TAZ relationship to better understand the dynamics of cellular functions controlled by YAP/TAZ activity.

Centennial Review: History and husbandry recommendations for raising Pekin ducks in research or commercial production.

By some accounts, ducks were domesticated between 400 and 10,000 yr ago and have been a growing portion of the poultry industry for decades. Ducks specifically, and waterfowl in general, have unique health, housing, nutrition and welfare concerns compared to their galliform counterparts. Although there have been many research publications in regards to health, nutrition, behavior, and welfare of ducks there have been very few reviews to provide an overview of these numerous studies, and only one text has attempted to review all aspects of the duck industry, from breeders to meat ducks. This review covers incubation, hatching, housing, welfare, nutrition, and euthanasia and highlights the needs for additional research at all levels of duck production. The purpose of this review is to provide guidelines to raise and house ducks for research as specifically related to industry practices.

A feed restriction milieu for Pekin meat ducks that may improve gait characteristics but also affects gut leakiness.

In a previous study, we demonstrated that a 15% feed restriction (FR) during the first 2 wk after hatch could improve gait in Pekin meat ducks, but did result in reduced breast mass. We hypothesized that feed restriction after day 5 following muscle satellite cell development would allow the full growth of the breast meat. To accomplish this goal, 300 1-day-old ducklings (Maple Leaf Farms Inc.) were randomly allocated to 1 of the 3 groups (n = 4 pens, 25 ducks per pen): (1) Control group fed to ad libitum; (2) 85% daily feed intake from days 1 to 14 (FR 85% 1-14); 85% daily feed intake from days 5 to 14 (FR 85% 5-14). All ducks were vaccinated with inactivated Salmonella enteritidis on day 14 and boosted on day 26. The FR 85% 5-14 did show similar production standards to controls, and improved gait characteristics (P < 0.05). To determine if the partial feed restriction would have an impact on intestinal epithelial tight junction integrity, we treated ducks on days 7, 14, 21, 28, and 35 with 8.32 mg/kg FITC-d in water per os and blood samples were obtained via the tibial vein 1 h later. Serum samples were analyzed for presence and quantification of FITC-d. Feed restriction elicited a significant increase in FITC-d permeability at all points of evaluation. Anti-S. enteritidis specific IgY responses were assessed by ELISA from serum collected at 14 D, 28 D, and 35 D. Although all ducks showed an increase humoral immune response to the S. enteritidis, both feed restricted groups showed reduced IgY production compared to ad lib controls. Our data suggest that although the FR 5-14 feed restriction paradigm may reduce gait abnormalities without affecting production rates, some challenges exist due to increased gut leakiness or decreased acquired immune activity. Future studies will look at altering the feed restriction milieu to ameliorate these challenges.

Macromolecular crowding tunes 3D collagen architecture and cell morphogenesis.

Collagen I is the primary extracellular matrix component of most solid tumors and influences metastatic progression. Collagen matrix engineering techniques are useful for understanding how this complex biomaterial regulates cancer cell behavior and for improving in vitro cancer models. Here, we establish an approach to tune collagen fibril architecture using PEG as an inert molecular crowding agent during gelation and cell embedding. We find that crowding produces matrices with tighter fibril networks that are less susceptible to proteinase mediated degradation, but does not significantly alter matrix stiffness. The resulting matrices have the effect of preventing cell spreading, confining cells, and reducing cell contractility. Matrix degradability and fibril length are identified as strong predictors of cell confinement. Further, the degree of confinement predicts whether breast cancer cells will ultimately undergo individual or collective behaviors. Highly confined breast cancer cells undergo morphogenesis to form either invasive networks reminiscent of aggressive tumors or gland and lobule structures reminiscent of normal breast epithelia. This morphological transition is accompanied by expression of cell-cell adhesion genes, including PECAM1 and ICAM1. Our study suggests that cell confinement, mediated by matrix architecture, is a design feature that tunes the transcriptional and morphogenic state of breast cancer cells.

Low light intensity in Pekin duck breeder barns has a greater impact on the fertility of drakes than hens.

The Pekin duck is an excellent model for the study of seasonal reproduction. To more completely understand the lighting requirements for maximal fertility in duck breeder houses, we housed adult (45 week old) drakes and hens in the Hope College aviary as 5 drakes and 25 hens. Light conditions in each floor pen were normalized based upon quantal energy and divided into the following categories: (1) to simulate summer, 14.5 h 65 lux with 9.5 h 1 lux; (2) to simulate winter, 8 h 65 lux with 16 h 1 lux; (3) winter augmented, 8 h 65 lux with 16 h at 15 lux. The experiment was repeated with rotation of light treatments among 3 pens until a final N = 6 was obtained. Daily, total number of eggs laid was tallied, and a daily average of eggs laid was calculated throughout the study. Weekly, eggs were weighed and the perivitilline membrane was assayed for the number of sperm holes as an indirect measure of drake fertility. As expected, winter conditions caused a reduction in the percent of eggs laid and a reduction in the number of fertilized eggs compared to the summer light conditions. The augmented winter light conditions prevented the loss in the percent eggs laid and fertilized eggs. Surprisingly, even after 4 wk of the study, the winter conditions did not cause a complete loss of fertility in the Pekin ducks. At the end of the study, no differences in the relative expression in brain deep brain photoreceptors or gonadotropin inhibitory hormone mRNAs were observed among any light treatment. Although a minimum (1 lux) of light can support some fertility, our findings suggest commercial Pekin duck barns may benefit from increasing the augmented light to 15 lux to maintain optimal fertility during winter months. Furthermore, our data suggest that drakes may be more sensitive to environmental light conditions than hens.

3D collagen architecture induces a conserved migratory and transcriptional response linked to vasculogenic mimicry.

The topographical organization of collagen within the tumor microenvironment has been implicated in modulating cancer cell migration and independently predicts progression to metastasis. Here, we show that collagen matrices with small pores and short fibers, but not Matrigel, trigger a conserved transcriptional response and subsequent motility switch in cancer cells resulting in the formation of multicellular network structures. The response is not mediated by hypoxia, matrix stiffness, or bulk matrix density, but rather by matrix architecture-induced ß1-integrin upregulation. The transcriptional module associated with network formation is enriched for migration and vasculogenesis-associated genes that predict survival in patient data across nine distinct tumor types. Evidence of this gene module at the protein level is found in patient tumor slices displaying a vasculogenic mimicry (VM) phenotype. Our findings link a collagen-induced migration program to VM and suggest that this process may be broadly relevant to metastatic progression in solid human cancers.

Increased water contamination and grow-out Pekin duck mortality when raised with water troughs compared to pin-metered water lines using a United States management system.

Controversy has developed as to whether or not pin-metered water lines or water troughs are more appropriate for Pekin ducks. We hypothesized that water troughs would show improved duck body conditions and environmental quality compared to pin-metered water lines. To test this hypothesis, we housed ducks in 2 barns, one with water lines and one with water troughs. Water troughs were constructed to meet RSPCA guidelines for number and density of ducks and with recently described verandas. Ducks were divided into 4 pens per barn (n=1,000 ducks/pen). The study was then repeated (n=8 pens per water source) in a cross-over design so the barns each contained the opposite water source to the first experiment. We scored the ducks' body condition using an established scoring rubric and analyzed using SAS Proc GLM-Mix as binomial data. Ducks housed with water troughs showed higher (thus worse condition; P<0.001) scores for eyes, nostrils, feather quality, feather cleanliness, and foot pads. We also compared water condition, water quality, and duck mortality using a Student t test for both water sources each week. We found that the water troughs showed higher iron (P<0.001), nitrites (P<0.001), pH (P<0.01), and bacterial growth (P<0.001). The bacterial growth was shown to have higher (P<0.001)E. coli, coliforms, and Staphylococcusin the water troughs. Water lines typically showed no bacterial growth in culture-based assays. Ducks housed with water troughs used greater (P=0.001) volumes of water compared to ducks housed with water lines. Ducks with water troughs also showed a greater percent (P=0.008) mortality at all ages compared to ducks with water lines. These data suggest that water troughs may not be beneficial for duck welfare and could adversely impact both environment and duck or human health.

Effects of different wavelengths of light on the biology, behavior, and production of grow-out Pekin ducks.

Previous research has shown that red light conditions may improve growth and decrease aggressive behaviors in chickens and turkeys; however, more recent studies suggest that blue-green light may improve production of broilers over red light. To date, no research has been conducted to examine whether different wavelengths of light have an impact on production in the Pekin duck. To determine this, we raised Pekin ducks under aviary conditions that were similar to standard commercial barns. The ducks were kept in 3 different pens: red light (approximately 625 nm), blue light (approximately 425 nm), and white light. Light sources in each pen were standardized to produce a peak energy at 1.6 × 10³ µM photons/m²/s at the level of the ducks' heads. Ducks were given ad libitum access to water and commercial duck diet, and were housed on pine shavings at a density of 0.43 m²/duck. Ducks were evaluated weekly for BW and condition and a subjective measure of the duck's anxiety levels was determined. We found that ducks housed under blue light had significantly (P < 0.01) reduced BW at every age until the end of the study (processing age; 35 d). Unlike ducks housed under red or white light, ducks housed in the blue pen showed a higher level of anxiety; while evaluators were in the pen a majority of them began panting, they were much less inquisitive than other ducks, they took longer to exhibit normal social behavior once evaluation was completed, and they frequently "swarmed" when no people were present. There were no differences in any measurements between the red and white-lighted pens. These data suggest that unlike the chicken, blue lights may be inappropriate for raising Pekin ducks in a commercial setting.

Effect of variable water intake as mediated by dietary potassium carbonate supplementation on rumen dynamics in lactating dairy cows.

Water is a critical nutrient for dairy cows, with intake varying with environment, production, and diet. However, little work has evaluated the effects of water intake on rumen parameters. Using dietary potassium carbonate (K2CO3) as a K supplement to increase water intake, the objective of this study was to evaluate the effect of K2CO3 supplementation on water intake and on rumen parameters of lactating dairy cows. Nine ruminally cannulated, late-lactation Holstein cows (207±12d in milk) were randomly assigned to 1 of 3 treatments in a replicated 3×3 Latin square design with 18-d periods. Dietary treatments (on a dry matter basis) were no added K2CO3 (baseline dietary K levels of 1.67% dietary K), 0.75% added dietary K, and 1.5% added dietary K. Cows were offered treatment diets for a 14-d adaption period followed by a 4-d collection period. Ruminal total, liquid, and dry matter digesta weights were determined by total rumen evacuations conducted 2h after feeding on d 4 of the collection period. Rumen fluid samples were collected to determine pH, volatile fatty acids, and NH3 concentrations, and Co-EDTA was used to determine fractional liquid passage rate. Milk samples were collected twice daily during the collection period. Milk, milk fat, and protein yields showed quadratic responses with greatest yields for the 0.75% added dietary K treatment. Dry matter intake showed a quadratic response with 21.8kg/d for the 0.75% added dietary K treatment and 20.4 and 20.5kg/d for control and the 1.5% added dietary K treatment, respectively. Water intake increased linearly with increasing K2CO3 supplementation (102.4, 118.4, and 129.3L/d) as did ruminal fractional liquid passage rate in the earlier hours after feeding (0.118, 0.135, and 0.141 per hour). Total and wet weights of rumen contents declined linearly and dry weight tended to decline linearly as dietary K2CO3 increased, suggesting that the increasing water intake and fractional liquid passage rate with increasing K2CO3 increased the overall ruminal turnover rate. Ruminal ammonia concentrations declined linearly and pH increased linearly as K supplementation increased. As a molar percentage of total volatile fatty acids, acetate increased linearly as dietary K increased, though propionate declined. Increasing dietary K2CO3 and total K in the diets of lactating dairy cows increased water consumption and modified ruminal measures in ways suggesting that both liquid and total ruminal turnover were increased as both water and K intake increased.

Influence of plastic slatted floors compared with pine shaving litter on Pekin Duck condition during the summer months.

The management and well-being of commercial Pekin ducks has been studied in the European Union where straw is the predominant litter source. In the United States, however, the most prevalent litter is wood shavings, with a recent trend toward using plastic slatted flooring. A previous study in the United States evaluated the relationship between flooring type (litter, slats) and duck condition during winter months and found very few differences between the 2 in terms of overall duck condition. The purpose of the current study was to reevaluate the 2 flooring systems during the summer months to determine if seasonal differences would interact with flooring type to have an impact on duck condition. Eighteen commercial barns that produce Pekin ducks for Maple Leaf Farms Inc. (Leesburg, IN), located in northern Indiana and southern Wisconsin (n = 9 litter; n = 9 raised slatted floor), were used for this study. Twenty ducks were randomly selected from 5 predetermined areas within each house (n = 100 total) and scored for eye condition, nostril and feather cleanliness, and feather and foot pad quality at 7, 21, and 32 d of age. Environmental data, including carbon monoxide, ammonia, RH, and temperature, were also obtained at each collection day. The only statistical differences in body condition occurred at 7 d; there were more ducks with clear eyes and eye rings on the litter flooring, whereas average nostril scores were better on the plastic slatted floors. Live weight, weight gain per day, flock mortality, and condemnations at the plant were collected, and the only statistical difference was a higher gain per day for ducks reared on slatted floors compared with litter (P < 0.05). There were no differences between flooring systems in the environmental parameters measured within the barns. In summary, there were very few differences between the litter and slatted flooring systems, indicating that there may not be clear advantages for one particular flooring system over the other from the point of view of duck well-being and production.

Influence of raised plastic floors compared with pine shaving litter on environment and Pekin duck condition.

Commercial poultry production management practices have been under increased public scrutiny driven by concerns for food safety and animal welfare. Within the United States, wood shavings and raised plastic floors are common flooring systems used in duck production. It is intuitive that each flooring type would present different management challenges influencing physical characteristics of growing ducks. This study evaluated the relationship between flooring type and duck condition during the winter. Random samples of 20 ducks from 5 predetermined areas (n = 100) were examined in commercial duck houses (n = 9, litter; n = 11, raised plastic slats). Ducks were assessed at 7, 21, and 32 d of age for eye, nostril, and feather cleanliness, feather and foot pad quality, and gait. The data were analyzed to determine the proportion of ducks with a given score. In both housing types, the proportion of 0 scores for foot pad quality improved during the production cycle (P < 0.0001). Feather hygiene declined with age in ducks reared on litter flooring, whereas ducks reared on slatted flooring had cleaner feathers at d 32 (P < 0.011). With the exception of foot pad scores, the majority of ducks had no detectable problems for any single trait. The only main effect due to flooring pertained to feather quality with the proportion of ducks having a 0 or 1 score greater in litter flooring systems than slats (P < 0.05). Overall, the condition of ducks reared, regardless of flooring system, was considered to be good.

Effect of snack food by-product inclusion on production of laying hens.

The increased interest in becoming green for consumers and companies is driving groups to develop innovative ways to become more efficient and reduce their waste. Foods past their expiration dates are large sources of waste and are causing food-manufacturing companies to develop waste disposal strategies. Integrating by-products from these companies into animal diets, specifically that of laying hens, could be significantly more cost effective for both the human food manufacturers and the agricultural producers. The study's objective is to evaluate laying hen diets containing snack food by-product, consisting mostly of expired potato chips, and the effect on hen performance. In total, 192 White Leghorn laying hens (45 wk old) were selected from the Michigan State University Poultry Farm. Hens were housed in conventional cages (3 birds/cage) and received 1 of 4 diets for 5 wk: 1) industry control corn-soybean meal, 2) control with 3% by-product, 3) control with 6% by-product, and 4) control with 9% by-product. Diets were formulated to be isocaloric, isonitrogenous, and balanced for sodium. Feed intake was measured for 3 consecutive days each week, and no overall differences between treatments were observed. However, during the first week, feed intake was significantly higher in birds fed the 6% and 9% diets compared with those fed control (P < 0.05). Birds fed the 6% had a higher feed intake than that of the control again during the fourth week (P < 0.01). Egg production, egg weight, and specific gravity were measured weekly. Hen BW was measured on d 1, 14, 28, and 35. Egg production, egg weight, specific gravity, and BW were not significantly affected by the addition of snack food by-products to the diet. In conclusion, the addition of expired snack food by-product into poultry diets does not significantly affect laying hen egg production and has the potential to be used as an alternative feed stuff in the future.

Retinal topography in the optic tract of adult goldfish.

The present study was aimed at determining how transformations in fiber order establish a retinal topography in the optic tract of adult Carassius auratus. Horseradish peroxidase was applied to the optic nerve or retina, and the pathways of labeled axons originating from retinal annuli or wedges were analysed in reconstructed serial-sections and wholeamounts of the optic pathway. The age-related fiber order of the optic tract involves a rotation of the optic pathway that begins near the chiasm, continues through the optic tract as it wraps around the brain, and extends through the brachia. The relative order of laminae, in which each lamina is composed of age-related axons, is maintained in the optic pathway. The laminae add systematically onto the optic tract in a mediolateral direction with the oldest lamina forming the medial margin. Retinal sector order in the optic tract is established by the rearrangement of axons from each lamina. These rearrangements begin at the chiasm and, in part, involve transposition of axons originating from the ventrotemporal and dorsonasal sectors of the retina. The transformations achieve a fiber order in the optic tract that is appropriate for entry into the tectum. It is proposed that the final retinal topography of the optic tract is determined by the combined influences of selective affinities along the neural axis and substrate guidance mechanisms, the latter being mediated largely by the oldest axons of the fasciculus medialis tractus opticus.

Topography of retinodiencephalic projections in adult channel catfish.

In order to determine the topography of retinal axons in diencephalic nuclei of Ictalurus punctatus, horseradish peroxidase was applied to the optic papillae. The topography was found to be similar in all nuclei in that the terminal endings of ventral retinal axons innervated rostrodorsally whereas dorsal retinal axons innervated caudoventrally. In general, the retinal topography in the diencephalon corresponded with the topography of retinal axons in the optic tracts. These data suggest a relation between retinal axon ordering within a target and the pathway by which axons enter the target.

Retinal origin of the fasciculus medialis tractus optici in adult goldfish.

In teleosts, the fasciculus medialis tractus optici departs the optic tract and follows an aberrant path towards the midbrain tectum. To determine the retinal origin of the fascicle, horseradish peroxidase was injected into localized lesions of either the optic nerve or the retina. The fasciculus medialis axons of adult goldfish derive selectively from ganglion cells residing in the dorsotemporal portion of the central retina. The fasciculus medialis appears to be an integral part of the chronotopic ordering of fibers in the optic tract.

Influence of litter size on antepartum serum relaxin and progesterone immunoactivity levels and on birth in the rat.

In the rat, the antepartum elevation of serum relaxin levels consists of two phases separated by a 24-h interval. The second phase, which occurs between 36 and 24 h before birth, is temporally closely associated with functional luteolysis. Relaxin levels then decline throughout the last approximately 24 h of pregnancy. We have postulated that the two phases in the antepartum elevation of serum relaxin levels may be indicative of an increasingly effective endogenous circadian luteolytic process. There is limited evidence that both luteolysis and birth are delayed in rats with small litters. The present study investigated in detail the relationship between litter size and the timing of both functional luteolysis and birth in rats. The number of conceptuses (C) in Sprague-Dawley-derived rats was surgically adjusted on day 8 of pregnancy (day 8) so that rats bore one, two, three, five, or a full complement (FC) of eight or more C. Rats were maintained under a photoperiod regimen of 14 h of light and 10 h of darkness (lights on from 2100-1100 h) beginning on day 8 and observed for birth at 10-min intervals from 2100 h on day 22. Serum levels of both relaxin and progesterone were determined in blood samples obtained at 4-h intervals from 2400 h on day 19 until birth. Ninety-five percent of the rats that had five or more C gave birth during the light phase on day 23, which was designated the normal birth interval. However, only 20% of the rats with three C or less, gave birth during the normal birth interval, and 47% gave birth about 24 h later during the light phase on day 24, which was designated the late birth interval. The 24-h delay in birth of rats with small litters which delivered during the late birth interval appears to be attributable to a delay in functional luteolysis; the antepartum decline in serum relaxin and progesterone levels occurred about 24 h later in these rats than in rats that delivered during the normal birth interval. It is concluded that the C may be associated with the luteolytic process and thereby influence the time of birth in rats. Additionally, the results of this study are consistent with our hypothesis that there is an endogenous circadian luteolytic process in rats during the antepartum period.

Topography of retinal axons in the diencephalon of goldfish.

The projections of horseradish peroxidase-filled axons from each quadrant of the retina were studied to determine whether retinal projections of goldfish are topographically organized in diencephalic target nuclei. A distinct topography of the dorsal, nasal, ventral and temporal retina exists in the lateral geniculate nucleus and in the dorsolateral optic nucleus of the thalamus. The projections of retinal quadrants show minimal spatial overlap in each of these nuclei. The suprachiasmatic nucleus of the hypothalamus is extensively innervated by ventral retinal fibers, whereas the nucleus is sparsely innervated by fibers from the other three retinal quadrants. A rudimentary topography also exists in the pretectum where the dorsal pretectal area receives projections primarily from the ventral retina and the ventral pretectal area receives projections mostly from the dorsal retina. These data show that retinal projections to some diencephalic nuclei are topographically organized.