An initiative to implement a triage and referral system to make exercise and rehabilitation referrals standard of care in oncology.

BACKGROUND: Clinical guidelines suggest that patients should be referred to exercise while undergoing cancer treatment. Oncology clinicians report being supportive of exercise referrals but not having the time to make referrals. Toward the goal of making exercise referrals standard of care, we implemented and evaluated a novel clinical workflow. METHODS: For this QI project, a rehabilitation navigator was inserted in chemotherapy infusion clinics. Patients were offered a validated electronic triage survey. Exercise or rehabilitation recommendations were communicated to patients during a brief counseling visit by the rehabilitation navigator. The implementation approach was guided by the EPIS framework. Acceptability and feasibility were assessed. RESULTS: Initial meetings with nursing and cancer center leadership ensured buy-in (exploration). The education of medical assistants contributed to the adoption of the triage process (preparation). Audit and feedback ensured leadership was aware of medical assistants' performance (implementation). 100% of medical assistants participated in implementing the triage tool. A total of 587 patients visited the infusion clinics during the 6-month period when this QI project was conducted. Of these, 501 (85.3%) were offered the triage survey and 391 (78%) completed the survey (acceptability). A total of 176 (45%) of triaged patients accepted a referral to exercise or rehabilitation interventions (feasibility). CONCLUSIONS: Implementation of a validated triage tool by medical assistants and brief counseling by a rehabilitation navigator resulted in 45% of infusion patients accepting a referral to exercise or rehabilitation. The triage process showed promise for making exercise referrals standard of care for patients undergoing cancer treatment.

SIS8, a putative mitogen-activated protein kinase kinase kinase, regulates sugar-resistant seedling development in Arabidopsis.

Sugar signaling pathways have been evolutionarily conserved among eukaryotes and are postulated to help regulate plant growth, development and responses to environmental cues. Forward genetic screens have identified sugar signaling or response mutants. Here we report the identification and characterization of Arabidopsis thaliana sugar insensitive8 (sis8) mutants, which display a sugar-resistant seedling development phenotype. Unlike many other sugar insensitive mutants, sis8 mutants exhibit wild-type responses to the inhibitory effects of abscisic acid and paclobutrazol (an inhibitor of gibberellin biosynthesis) on seed germination. Positional cloning of the SIS8 gene revealed that it encodes a putative mitogen-activated protein kinase kinase kinase (MAPKKK; At1g73660). SIS8mRNA is expressed ubiquitously among Arabidopsis organs. A UDP-glucosyltransferase, UGT72E1 (At3g50740), was identified as an interacting partner of SIS8 based on a yeast two-hybrid screen and in planta bimolecular fluorescence complementation. Both SIS8-yellow fluorescent protein (YFP) and UGT72E1-YFP fusion proteins localize to the nucleus when transiently expressed in tobacco leaf cells. T-DNA insertions in At3g50740 cause a sugar-insensitive phenotype. These results indicate that SIS8, a putative MAPKKK, is a regulator of sugar response in Arabidopsis and interacts with a UDP-glucosyltransferase in the nucleus.

The ORCA2 transcription factor plays a key role in regulation of the terpenoid indole alkaloid pathway.

BACKGROUND: The terpenoid indole alkaloid (TIA) pathway leads to the production of pharmaceutically important drugs, such as the anticancer compounds vinblastine and vincristine. Unfortunately, these drugs are produced in trace amounts, causing them to be very costly. To increase production of these drugs, an improved understanding of the TIA regulatory pathway is needed. Towards this end, transgenic Catharanthus roseus hairy roots that overexpress the ORCA2 TIA transcriptional activator were generated and characterized. RESULTS: Transcriptional profiling experiments revealed that overexpression of ORCA2 results in altered expression of key genes from the indole and terpenoid pathways, which produce precursors for the TIA pathway, and from the TIA pathway itself. In addition, metabolite-profiling experiments revealed that overexpression of ORCA2 significantly affects the levels of several TIA metabolites. ORCA2 overexpression also causes significant increases in transcript levels of several TIA regulators, including TIA transcriptional repressors. CONCLUSIONS: Results presented here indicate that ORCA2 plays a critical role in regulation of TIA metabolism. ORCA2 regulates expression of key genes from both feeder pathways, as well as the genes (STR and SGD) encoding the enzymes that catalyze the first two steps in TIA biosynthesis. ORCA2 may play an especially important role in regulation of the downstream branches of the TIA pathway, as it regulates four out of five genes characterized from this part of the pathway. Regulation of TIA transcriptional repressors by ORCA2 may provide a mechanism whereby increases in TIA metabolite levels in response to external stimuli are transient and limited in magnitude.

Regulation and distribution of squirrel monkey chorionic gonadotropin and secretogranin II in the pituitary.

Secretogranin II (SgII) is a member of the granin family of proteins found in neuroendocrine and endocrine cells. The expression and storage of SgII in the pituitary gland of Old World primates and rodents have been linked with those of luteinizing hormone (LH). However, New World primates including squirrel monkeys do not express LH in the pituitary gland, but rather CG is expressed. If CG takes on the luteotropic role of LH in New World primates, SgII may be associated with the expression and storage of CG in the pituitary gland. The goal of this study was to evaluate the regulation and distribution of CG and SgII in the squirrel monkey. A DNA fragment containing approximately 750 bp of squirrel monkey SgII promoter was isolated from genomic DNA and found to contain a cyclic-AMP response element that is also present in the human SgII promoter and important for GnRH responsiveness. The squirrel monkey and human SgII promoters were similarly activated by GnRH in luciferase reporter gene assays in LßT2 cells. Double immunofluorescence microscopy demonstrated close association of SgII and CG in gonadotrophs of squirrel monkey pituitary gland. These results suggest that CG and SgII have a similar intercellular distribution and are coregulated in squirrel monkey pituitary gland.

Molecular cloning of pituitary glycoprotein alpha-subunit and follicle stimulating hormone and chorionic gonadotropin beta-subunits from New World squirrel monkey and owl monkey.

The goal of this study was to characterize the gonadotropins expressed in pituitary glands of the New World squirrel monkey (Saimiri sp.) and owl monkey (Aotus sp.). The various subunits were amplified from total RNA from squirrel monkey and owl monkey pituitary glands by reverse transcription-polymerase chain reaction and the deduced amino acid sequences compared to those of other species. Mature squirrel monkey and owl monkey glycoprotein hormone alpha-polypeptides (96 amino acids in length) were determined to be 80% homologous to the human sequence. The sequences of mature beta subunits of follicle stimulating hormone (FSHbeta) from squirrel monkey and owl monkey (111 amino acids in length) are 92% homologous to human FSHbeta. New World primate glycoprotein hormone alpha-polypeptides and FSHbeta subunits showed conservation of all cysteine residues and consensus N-linked glycosylation sites. Attempts to amplify the beta-subunit of luteinizing hormone from squirrel monkey and owl monkey pituitary glands were unsuccessful. Rather, the beta-subunit of chorionic gonadotropin (CG) was amplified from pituitaries of both New World primates. Squirrel monkey and owl monkey CGbeta are 143 and 144 amino acids in length and 77% homologous with human CGbeta. The greatest divergence is in the C terminus, where all four sites for O-linked glycosylation in human CGbeta, responsible for delayed metabolic clearance, are predicted to be absent in New World primate CGbetas. It is likely that CG secreted from pituitary of New World primates exhibits a relatively short half-life compared to human CG.

Sequencing and analysis of 10,967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis reveals post-tetraploidization transcriptome remodeling.

Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection Initiative. Here we present 10,967 full ORF verified cDNA clones (8049 from X. laevis and 2918 from X. tropicalis) as a community resource. Because the genome of X. laevis, but not X. tropicalis, has undergone allotetraploidization, comparison of coding sequences from these two clawed (pipid) frogs provides a unique angle for exploring the molecular evolution of duplicate genes. Within our clone set, we have identified 445 gene trios, each comprised of an allotetraploidization-derived X. laevis gene pair and their shared X. tropicalis ortholog. Pairwise dN/dS, comparisons within trios show strong evidence for purifying selection acting on all three members. However, dN/dS ratios between X. laevis gene pairs are elevated relative to their X. tropicalis ortholog. This difference is highly significant and indicates an overall relaxation of selective pressures on duplicated gene pairs. We have found that the paralogs that have been lost since the tetraploidization event are enriched for several molecular functions, but have found no such enrichment in the extant paralogs. Approximately 14% of the paralogous pairs analyzed here also show differential expression indicative of subfunctionalization.

Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots over-expressing the alpha or the alpha and beta subunits of anthranilate synthase.

Among the pharmacologically important terpenoid indole alkaloids produced by Catharanthus roseus are the anti-cancer drugs vinblastine and vincristine. These two drugs are produced in small yields within the plant, which makes them expensive to produce commercially. Metabolic engineering has focused on increasing flux through this pathway by various means such as elicitation, precursor feeding, and introduction of genes encoding specific metabolic enzymes into the plant. Recently in our lab, a feedback-resistant anthranilate synthase alpha subunit was over-expressed in C. roseus hairy roots under the control of a glucocorticoid inducible promoter system. Upon induction we observed a large increase in the indole precursors, tryptophan, and tryptamine. The current work explores the effects of over-expressing the anthranilate synthase alpha or alpha and beta subunits in combination with feeding with the terpenoid precursors 1-deoxy-D-xylulose, loganin, and secologanin. In feeding 1-deoxy-D-xylulose to the hairy root line expressing the anthranilate synthase alpha subunit, we observed an increase of 125% in hörhammericine levels in the induced samples, while loganin feeding increased catharanthine by 45% in the induced samples. Loganin feeding to the hairy root line expressing anthranilate synthase alpha and beta subunits increases catharanthine by 26%, ajmalicine by 84%, lochnericine by 119%, and tabersonine by 225% in the induced samples. These results suggest that the terpenoid precursors to the terpenoid indole alkaloids are important factors in terpenoid indole alkaloid production.

Transient effects of overexpressing anthranilate synthase alpha and beta subunits in Catharanthus roseus hairy roots.

Catharanthus roseus produces two economically valuable anticancer drugs, vinblastine and vincristine. These drugs are members of the terpenoid indole alkaloids and accumulate in small quantities within the plant; thus these two drugs are expensive to produce. Metabolic engineering efforts have focused on increasing the alkaloids in this pathway through various means such as elicitation, precursor feeding, and gene overexpression. Recently we successfully expressed Arabidopsis genes encoding a feedback-insensitive anthranilate synthase alpha subunit under the control of the glucocorticoid-inducible promoter system and the anthranilate synthase beta subunit under the control of a constitutive promoter in C. roseus hairy roots. In this work we look at the transient behaviors of terpenoid indole alkaloids over a 72 h induction period in late exponential growth phase cultures. Upon induction, the tryptophan, tryptamine, and ajmalicine pools accumulated over 72 h. In contrast, the lochnericine, hörhammericine, and tabersonine pools decreased and leveled out over the 72 h induction period. Visible changes within the individual compounds usually took from 4 to 12 h.