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Supplementary Figure 1 from Phosphorylated Caveolin-1 Regulates Rho/ROCK-Dependent Focal Adhesion Dynamics and Tumor Cell Migration and Invasion

Rho/ROCK signaling and caveolin-1 (Cav1) are implicated in tumor cell migration and metastasis; however, the underlying molecular mechanisms remain poorly defined. Cav1 was found here to be an independent predictor of decreased survival in breast and rectal cancer and significantly associated with the presence of distant metastasis for colon cancer patients. Rho/ROCK signaling promotes tumor cell migration by regulating focal adhesion (FA) dynamics through tyrosine (Y14) phosphorylation of Cav1. Phosphorylated Cav1 is localized to protrusive domains of tumor cells and Cav1 tyrosine phosphorylation is dependent on Src kinase and Rho/ROCK signaling. Increased levels of phosphorylated Cav1 were associated with elevated GTP-RhoA levels in metastatic tumor cells of various tissue origins. Stable expression and knockdown studies of Cav1 in tumor cells showed that phosphorylated Cav1 expression stimulates Rho activation, stabilizes FAK association with FAs, and promotes cell migration and invasion in a ROCK-dependent and Src-dependent manner. Tyrosine-phosphorylated Cav1, therefore, functions as an effector of Rho/ROCK signaling in the regulation of FA turnover and, thereby, tumor cell migration and invasion. These studies define a feedback loop between Rho/ROCK, Src, and phosphorylated Cav1 in tumor cell protrusions, identifying a novel function for Cav1 in tumor metastasis that may contribute to the poor prognosis of some Cav1-expressing tumors. [Cancer Res 2008;68(20):8210–20]

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Policy-makers are increasingly turning to behavioural science for insights about how to improve citizens’ decisions and outcomes(1). Typically, different scientists test different intervention ideas in different samples using different outcomes over different time intervals(2). The lack of comparability of such individual investigations limits their potential to inform policy. Here, to address this limitation and accelerate the pace of discovery, we introduce the megastudy–a massive field experiment in which the effects of many different interventions are compared in the same population on the same objectively measured outcome for the same duration. In a megastudy targeting physical exercise among 61,293 members of an American fitness chain, 30 scientists from 15 different US universities worked in small independent teams to design a total of 54 different four-week digital programmes (or interventions) encouraging exercise. We show that 45% of these interventions significantly increased weekly gym visits by 9% to 27%; the top-performing intervention offered microrewards for returning to the gym after a missed workout. Only 8% of interventions induced behaviour change that was significant and measurable after the four-week intervention. Conditioning on the 45% of interventions that increased exercise during the intervention, we detected carry-over effects that were proportionally similar to those measured in previous research(3–6). Forecasts by impartial judges failed to predict which interventions would be most effective, underscoring the value of testing many ideas at once and, therefore, the potential for megastudies to improve the evidentiary value of behavioural science.

DNA double-strand breaks (DSBs) are a particularly lethal form of DNA damage that must be repaired to restore genomic integrity. Canonical nonhomologous end joining (NHEJ), is a widely conserved pathway that detects and directly ligates the broken ends to repair the DSB. These events globally require the two proteins that form the Ku ring complex, Ku70 and Ku80, and the terminal ligase LIG4. While the NHEJ pathway in vertebrates is elaborated by more than a dozen factors of varying conservation and is similarly complex in other eukaryotes, the entire known NHEJ toolkit in Caenorhabditis elegans consists only of the core components CKU-70, CKU-80, and LIG-4. Here, we report the discovery of the first accessory NHEJ factor in C. elegans. Our analysis of the DNA damage response in young larvae revealed that the canonical wild-type N2 strain consisted of two lines that exhibited a differential phenotypic response to ionizing radiation (IR). Following the mapping of the causative locus to a candidate on chromosome V and clustered regularly interspaced short palindromic repeats-Cas9 mutagenesis, we show that disruption of the nhj-1 sequence induces IR sensitivity in the N2 line that previously exhibited IR resistance. Using genetic and cytological analyses, we demonstrate that nhj-1 functions in the NHEJ pathway to repair DSBs. Double mutants of nhj-1 and lig-4 or cku-80 do not exhibit additive IR sensitivity, and the post-IR somatic and fertility phenotypes of nhj-1 mimic those of the other NHEJ factors. Furthermore, in com-1 mutants that permit repair of meiotic DSBs by NHEJ instead of restricting their repair to the homologous recombination pathway, loss of nhj-1 mimics the consequences of loss of lig-4. Diakinesis-stage nuclei in nhj-1; com-1 and nhj-1; lig-4 mutant germlines exhibit increased numbers of DAPI-staining bodies, consistent with increased chromosome fragmentation in the absence of NHEJ-mediated meiotic DSB repair. Finally, we show that NHJ-1 and LIG-4 localize to somatic nuclei in larvae, but are excluded from the germline progenitor cells, consistent with NHEJ being the dominant DNA repair pathway in the soma. nhj-1 shares no sequence homology with other known eukaryotic NHEJ factors and is taxonomically restricted to the Rhabditid family, underscoring the evolutionary plasticity of even highly conserved pathways.

The objectives of this study were 1) to determine if supplementation of zilpaterol hydrochloride (ZH) altered select organ weights, histology, and cardiac anatomical features at harvest and 2) to determine if administration of a corticotropin-releasing hormone (CRH) and vasopressin (VP) challenge following 20 d of ZH supplementation altered the blood chemistry profile in cattle. Crossbred heifers (n = 20; 556 ± 7 kg BW) were randomized into 2 treatment groups: 1) control (CON), without ZH, and 2) zilpaterol (ZIL; ZH at 8.33 mg/kg [DM basis] for 20 d). On d 20 of supplementation, heifers were fitted with indwelling jugular catheters. On d 24, starting at 0800 h and continuing until 1600 h, blood samples were collected at 60-min intervals. At 1000 h, heifers received an i.v. bolus of CRH (0.3 µg/kg BW) and VP (1.0 µg/kg BW) to activate the stress axis. Serum was separated and stored at −80°C until analyzed for a large-animal chemistry panel. Following the CRH/VP challenge, heifers were harvested on d 25, 26, and 27 (5, 6, and 7 d after ZH supplementation); BW, HCW, select organ weights, and histology were measured, and a total heart necropsy was performed. A treatment effect (P ≤ 0.02) was observed for Ca, K, creatinine, alkaline phosphatase, and sorbitol dehydrogenase. Zilpaterol-fed heifers had decreased (P ≤ 0.02) concentrations of Ca and K and increased concentrations (P < 0.01) of creatinine (P = 0.02) during the CRH/VP challenge when compared to control heifers. Control heifers had greater (P ≤ 0.05) alkaline phosphatase and sorbitol dehydrogenase concentrations when compared with ZIL heifers. A treatment × time interaction (P = 0.02) was observed for P; concentrations were similar between treatments from −2 to 6 h postchallenge, and 7 h postchallenge CON heifers had decreased P. Liver (P = 0.06) and kidney (P = 0.08) weights as a percentage of BW tended (P ≤ 0.08) to be reduced in ZIL heifers. Gross liver weights tended (P = 0.08) to be lower in ZIL heifers. Other organ (heart, lung, adrenals) to BW ratios remained similar (P ≥ 0.41). These data suggest that there are some variations observed between treatments in terms of response to ZH supplementation and the CRH/VP challenge; however, in the environmental conditions of this study, limited variation in blood metabolic responses and organ weights suggests that the supplementation of ZH did not detrimentally alter the physiology of cattle.

Purpose of the study: This study was conducted to define the role of Dachshund in prostate cancer, through assessing human prostate cancer samples and through genetic deletion in the mouse. Prostate cancer (PCa), the second leading cause of death in American men, is a genetically heterogeneous disease, likely representing distinct genetic drivers, with terminal events caused primarily by metastasis. Substratification of PCa into genetic subtypes, forms the basis of rational therapy for PCa. A better molecular understanding of the disease is necessary in order to develop novel targeted therapies of metastatic PCa. Known genetic drivers to tumor initiation include PTEN and NKX3.1 deletions, rearrangements of the TMPRSS2 gene to the oncogenic ETS transcription factor, ERG, and genetic predisposing factors include germline DNA-repair gene mutations. The DACH1 gene, initially cloned as an inhibitor of Elipse, the hyperactive epidermal growth factor (EGFR) in Drosophila, was found to be reduced in abundance in several malignancies including breast and prostate cancer. Results: In order to determine whether the DACH1 gene is deleted or mutated in prostate cancer we interrogated the genomic sequencing analysis of over 490 patients from 5 population cohorts. Homozygous deletion of DACH1 was identified in 18% (N=61), 11% (N=136), 10% (N=492), 7% (N=103) and 3% (N=150) of prostate cancer in 5 distinct cohorts. The prevalence of DACH1 gene deep deletions was higher in the metastasis than in the primary tumors. The Transgenic Adenocarcinoma Mouse Prostate (TRAMP) transgenic, Dach1fl/fl, and Probasin-Cre, ROSA26mT/mG transgenic mice were used to generate a prostate epithelial cell specific Dach1 gene knockout mouse (Probasin-Cre-Dach1fl/fl ROSA26mT/mG-TRAMP) lines. Prostate specific deletion of the murine Dach1 gene enhanced progression of prostatic intraepithelial neoplasia (PIN), associated with increased prostate epithelial cell proliferation, epithelial mesenchymal transition (EMT), DNA damage and inflammation. Conclusions: DACH1 gene deletion may define a distinct subclass of prostate cancer that may benefit from PARP inhibitors, and platinum-based chemotherapy. Citation Format: Xuanmao Jiao, Gabriele Di Sante, Zhiping Li, Agnese Di Rocco, Min Wang, Adam Ertel, Peter A. McCue, Andrew P. South, Carlos Cordon-Cardo, Matthew P. Stokes, Marco Marra, Steven J. Jones, Andrew Kossenkov, Richard G. Pestell. DACH1 gene deletion extends portraits of human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1730.

Forty-eight crossbred rabbits were used in three replications of a 2 x 4 factorial arrangement to investigate the short-term responses of tissue accretion, calpains and calpastatin activity, and nucleic acid and protein concentrations to beta-adrenergic agonist (BAA) feeding. Rabbits were fed a 17% CP diet with or without 7 ppm of L644,969 and slaughtered after 1, 4, 8, or 16 d of treatment. Empty body dressing percentage and biceps femoris weight (as a percentage of empty body weight [EBW]) were significantly higher in the treated rabbits than in the controls after 16 d of treatment. Heart and liver weights (as a percentage of EBW) were higher (P < .05) after 1 d and liver weight (as a percentage of EBW) was lower (P < .05) after 16 d in treated vs controls. Except for an elevation of skeletal muscle m-calpain after 16 d, BAA-supplementation did not affect the calpain-calpastatin system. Muscle RNA concentrations and RNA:DNA ratios were higher (P < .05) in treated rabbits after 1 d and remained higher thereafter. Protein:RNA ratios were lower (P < .01) in treated than in control rabbits after 4 d and remained lower throughout the trial. Muscle DNA content was lower after 4 d and higher after 16 d; RNA content was higher after 4, 8, and 16 d; and protein content was higher after 16 d in treated vs control rabbits. Liver nucleic acid and protein concentrations were not affected by BAA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

For select men with low risk prostate cancer active surveillance is more often being considered a management strategy. In a multicenter retrospective study we evaluated the actuarial rates and predictors of remaining on active surveillance, the incidence of cancer progression and the pathological findings of delayed radical prostatectomy.A cohort of 262 men from 4 institutions met the inclusion criteria of age 75 years or younger, prostate specific antigen 10 ng/ml or less, clinical stage T1-T2a, biopsy Gleason sum 6 or less, 3 or less positive cores at diagnostic biopsy, repeat biopsy before active surveillance and no treatment for 6 months following the repeat biopsy. Active surveillance started on the date of the second biopsy. Actuarial rates of remaining on active surveillance were calculated and univariate Cox regression was used to assess predictors of discontinuing active surveillance.With a median followup of 29 months 43 patients ultimately received active treatment. The 2 and 5-year probabilities of remaining on active surveillance were 91% and 75%, respectively. Patients with cancer on the second biopsy (HR 2.23, 95% CI 1.23-4.06, p = 0.007) and a higher number of cancerous cores from the 2 biopsies combined (p = 0.002) were more likely to undergo treatment. Age, prostate specific antigen, clinical stage, prostate volume and number of total biopsy cores sampled were not predictive of outcome. Skeletal metastases developed in 1 patient 38 months after starting active surveillance. Of the 43 patients undergoing delayed treatment 41 (95%) are without disease progression at a median of 23 months following treatment.With a median followup of 29 months active surveillance for select patients appears to be safe and associated with a low risk of systemic progression. Cancer at restaging biopsy and a higher total number of cancerous cores are associated with a lower likelihood of remaining on active surveillance. A restaging biopsy should be strongly considered to finalize eligibility for active surveillance.

To examine the effects of cimaterol (CIM) and its withdrawal on meat tenderness and carcass composition, 21-d-old broiler chickens (n = 288) were randomly assigned to one of nine treatments. For Treatments 1 through 6, birds were fed a control diet or a diet containing 1 ppm CIM until slaughter at 35, 42, or 49 d of age. Treatments 7 and 8 consisted of birds fed the CIM diet for 14 d and then withdrawn from CIM for either 7 or 14 d before slaughter (42 or 49 d of age). In Treatment 9, birds were fed the CIM diet to 42 d of age, then withdrawn from CIM for 7 d. Breast muscle (BM) weight, leg muscle (LM) weight, whole body weight, and BM and LM cathepsin B and L activities were obtained on 12 birds/treatment. Body, LM and BM composition and BM shear values were obtained on 12 additional birds/treatment. Eight birds/treatment were used to balance the number of birds per pen. Leg muscle weight, as a percentage of whole body weight, was elevated in CIM-fed birds at all ages, and BM percentage was greater at 35 d of age (P less than .05). Leg muscle fat percentage was reduced at 35 and 42 d of age (P less than .05), and LM protein was elevated at 42 and 49 d of age (P less than .05) in CIM-fed birds. Percentage of protein in the BM of CIM-fed birds was elevated at 35 and 42 d of age. Protein content of the whole body was also increased at 35 d of age. Shear values were higher in 42- and 49-d-old CIM-fed birds.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of this study was to determine the effects of different implanting schemes on serum testosterone and cortisol concentrations, and carcass traits of bulls and steers implanted with trenbolone acetate (TBA) and zeranol (Z). Twenty Polled Hereford bulls were randomly assigned to one of three treatments after birth. Five calves served as nonimplanted control bulls (NIB). Nine bulls were implanted (IB) with 140 mg of TBA and 36 mg of Z at about 1 mo of age and reimplanted with both compounds 10 wk later. When IB calves were about 21 wk of age, the TBA implant was removed and calves were reimplanted with Z every 10 wk until slaughter. Six calves were castrated at 3 wk of age and implanted (IS) with TBA and Z every 10 wk until slaughter. Blood samples from each animal were obtained at 14-d intervals beginning at 14 wk of age and serum cortisol (C) and testosterone (T) concentrations were determined. The NIB had higher C levels than IB or IS (P less than .05) during the preweaning period. During the finishing period, there were no differences in C concentrations between NIB and IB; however, IS had lower levels (P less than .05) than both bull treatments. Serum T concentrations began to increase about 12 wk later (42 vs 30 wk, respectively) in IB compared with NIB. Testicular size was smaller (P less than .05) in IB than in NIB. No differences (P greater than .05) were observed in carcass characteristics. Taste-panel scores were not different among treatments. In conclusion, implanting schemes using TBA and Z lowered serum levels of C and delayed puberty in bulls; however, they did not alter carcass characteristics or eating quality.

The objective of this study was to determine the effect of feed restriction and repletion on myofibrillar protein turnover in cattle. Crossbred steer calves (n = 12) about 310 d of age were assigned randomly to a diet of corn and silage that was 1) provided ad libitum for 146 d (ALC) or 2) restricted so steers gained .2 kg/d for 80 d but received ad libitum access to feed thereafter for 66 d (RFC). At 27, 55, 97, 118 and 146 d a 24-h urine sample and a blood sample were obtained. Urine was analyzed for N tau-methylhistidine (N tau-MH), creatinine (C), urea nitrogen (UN) and total nitrogen (TN). Serum samples were analyzed for hydroxyproline (HYP), C and albumin (A). Body weights were lower (P less than .05) in RFC at 55, 97, 118 and 146 d. Excretion of N tau-MH was lower (P less than .05) in the RFC at 27 and 55 d but higher at 118 d. Urinary C excretion was higher in ALC at the last four sample times. Urinary UN and TN excretion were lower (P less than .05) in RFC at 55, 97 and 118 d; urinary UN also was lower (P less than .05) at d 146. Serum A was higher (P less than .05) in ALC at 55 and 118 d, respectively. Serum HYP was higher (P less than .05) in RFC at 27 and 55 d. Calculated myofibrillar protein breakdown rates (FBR) and fractional synthesis rates (FSR) were higher (P less than .05) in RFC at the last two sampling periods; FSR was lower for the RFC at the first sampling period.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate the impact of cimaterol (CIM) on muscle protein turnover, carcass and muscle composition, muscle cathepsin B + L activity and meat tenderness, 21-d-old broiler chickens (n = 88) were assigned to dietary treatments of either 0 or 1 ppm CIM. Fractional synthesis rates (FSR), fractional accretion rates (FAR), proximate composition and collagen content were determined in the breast muscle (BM); pectoralis major) and a group of leg muscles (LM; gastrocnemius and peroneous longus) from groups of six birds per treatment within each of two time periods (age = 38 or 56 d). Whole body composition, serum hydroxyproline content and BM cathepsin B + L activity also were measured. Fractional degradation rates (FDR) were calculated as the difference between FSR and FAR. Feeding CIM increased (P less than .01) whole body protein content. Weights of LM and percentage of body weight as BM and LM were increased (P less than .05) when CIM was included in the diet. Although FSR was not significantly reduced by CIM feeding it decreased (P less than .05) with increasing age. Due to decreases in FAR, FDR thereby was reduced by CIM 31.5% and 11.9% in BM and 38.2% and 37.4% in LM at 38 d and 56 d of age, respectively. Cathepsin B + L activities also were reduced 33.6% (P less than .01) and shear forces were increased by 41% (P less than .05) by CIM feeding. For chickens fed CIM, the correlation between cathepsin B + L activity and shear force was -.63 (P less than .01). Feeding CIM improved carcass leanness and muscling due to reductions in FDR and proteolytic enzyme activity. Feeding CIM also reduced meat tenderness.