Expression Elements Derived From Plant Sequences Provide Effective Gene Expression Regulation and New Opportunities for Plant Biotechnology Traits.

Plant biotechnology traits provide a means to increase crop yields, manage weeds and pests, and sustainably contribute to addressing the needs of a growing population. One of the key challenges in developing new traits for plant biotechnology is the availability of expression elements for efficacious and predictable transgene regulation. Recent advances in genomics, transcriptomics, and computational tools have enabled the generation of new expression elements in a variety of model organisms. In this study, new expression element sequences were computationally generated for use in crops, starting from native Arabidopsis and maize sequences. These elements include promoters, 5' untranslated regions (5' UTRs), introns, and 3' UTRs. The expression elements were demonstrated to drive effective transgene expression in stably transformed soybean plants across multiple tissues types and developmental stages. The expressed transcripts were characterized to demonstrate the molecular function of these expression elements. The data show that the promoters precisely initiate transcripts, the introns are effectively spliced, and the 3' UTRs enable predictable processing of transcript 3' ends. Overall, our results indicate that these new expression elements can recapitulate key functional properties of natural sequences and provide opportunities for optimizing the expression of genes in future plant biotechnology traits.

NT-3 promotes proprioceptive axon regeneration when combined with activation of the mTor intrinsic growth pathway but not with reduction of myelin extrinsic inhibitors.

Although previous studies have identified several strategies to stimulate regeneration of CNS axons, extensive regeneration and functional recovery have remained a major challenge, particularly for large diameter myelinated axons. Within the CNS, myelin is thought to inhibit axon regeneration, while modulating activity of the mTOR pathway promotes regeneration of injured axons. In this study, we examined NT-3 mediated regeneration of sensory axons through the dorsal root entry zone in a triple knockout of myelin inhibitory proteins or after activation of mTOR using a constitutively active (ca) Rheb in DRG neurons to determine the influence of environmental inhibitory or activation of intrinsic growth pathways could enhance NT-3-mediate regeneration. Loss of myelin inhibitory proteins showed modest enhancement of sensory axon regeneration. In mTOR studies, we found a dramatic age related decrease in the mTOR activation as determined by phosphorylation of the downstream marker S6 ribosomal subunit. Expression of caRheb within adult DRG neurons in vitro increased S6 phosphorylation and doubled the overall length of neurite outgrowth, which was reversed in the presence of rapamycin. In adult female rats, combined expression of caRheb in DRG neurons and NT-3 within the spinal cord increased regeneration of sensory axons almost 3 fold when compared to NT-3 alone. Proprioceptive assessment using a grid runway indicates functionally significant regeneration of large-diameter myelinated sensory afferents. Our results indicate that caRheb-induced increase in mTOR activation enhances neurotrophin-3 induced regeneration of large-diameter myelinated axons.

Quantitative assessment of neurite outgrowth over growth promoting or inhibitory substrates.

The use of sensory neurons and assessment of neurite outgrowth in vitro is an important part of understanding neuronal development and plasticity. Cultures of rat dorsal root ganglion (DRG) neurons provide quantitative results very quickly and, when grown on growth promoting or inhibitory substrates, can be utilized to study axonal growth, neurotrophic dependence, structure and function of growth cones. Since we are interested in axon regeneration and targeting, we have sought to promote neurite outgrowth by refining the techniques of growing DRG neurons in culture. This chapter describes detailed methods for the dissection and purification of DRG neurons and quantitative assessment of neurite on promoting or inhibitory substrates.

Haemorrhagic low-grade fibromyxoid sarcoma: MR findings in two young women.

Low-grade fibromyxoid sarcoma (LGFMS) is a rare soft-tissue sarcoma characterised by a deceptively bland histological appearance and a paradoxically aggressive behaviour. LGFMS usually presents in young-to-middle-aged adults as a painless, slow-growing mass with the potential for local recurrence and metastasis despite low-grade histology. Several case reports have described variable MR findings of LGFMS without haemorrhage or necrosis. We report here on the MR findings in two young women with haemorrhagic LGFMS in the thigh.

Effects of acute renal failure induced by uranyl nitrate on the pharmacokinetics of liquiritigenin and its two glucuronides, M1 and M2, in rats.

OBJECTIVES: Liver disease and acute renal failure (ARF) are closely associated. The pharmacokinetics of liquiritigenin (LQ), a candidate therapy for inflammatory liver disease, and its metabolites M1 and M2 were evaluated in rats with ARF induced by uranyl nitrate (U-ARF rats). METHODS: LQ was administered intravenously (20 mg/kg) or orally (50 mg/kg) in U-ARF and control rats, and uridine diphosphate-glucuronosyltransferases (UGT) activity and uridine 5'-diphosphoglucuronic acid (UDPGA) concentrations were determined in the liver and intestine. KEY FINDINGS: After intravenous LQ administration, U-ARF rats displayed significantly slower LQ renal clearance but no significant changes in the LQ area under the plasma concentration-time curve (AUC) compared with controls. This was because of similar hepatic UGT activity and UDPGA levels between two groups, which resulted in comparable non-renal clearance, as well as the limited contribution of LQ renal clearance to total LQ clearance. However, the AUC and AUC(M) /AUC(LQ) ratios of M1 and M2 were significantly increased in U-ARF rats because of decreased urinary excretion of M1 and M2. Similar results were observed following oral administration because of the comparable LQ intestinal metabolism in both groups and decreased urinary excretion of M1 and M2 in U-ARF rats. CONCLUSIONS: U-ARF rats displayed decreased urinary excretion of LQ glucuronides, resulting in significantly greater AUC and metabolite ratios of M1 and M2 following LQ administration.

Liquiritigenin pharmacokinetics in a rat model of diabetes mellitus induced by streptozotocin: greater formation of glucuronides in the liver, especially M2, due to increased hepatic uridine 5'-diphosphoglucuronic acid level.

Liquiritigenin (LQ) is a candidate for the treatment of inflammatory liver disease. Many studies have confirmed that hepatic disease and diabetes mellitus are closely associated. Thus, the pharmacokinetic changes of LQ and its 2 glucuronides, M1 and M2, in a rat model of diabetes mellitus induced by streptozotocin (DMIS rats) were evaluated. Liquiritigenin was administered intravenously (20 mg/kg) or orally (50 mg/kg) in DMIS and control rats. Changes in in vitro activity and in vivo uridine 5'-diphosphoglucuronic acid level in the liver and intestine of DMIS rats compared with controls were also studied. After intravenous administration of LQ in DMIS rats, no significant changes in the pharmacokinetic parameters of LQ were observed. However, the AUC(M2)/AUC(LQ) ratio was significantly greater (by 53.0%) than that of controls. After oral administration of LQ, the AUC of LQ and metabolite ratios of M1 and M2 were comparable to controls. The increase in the formation of glucuronides of LQ, especially M2, after intravenous administration of LQ was due to the increased in vivo hepatic uridine 5'-diphosphoglucuronic acid level in DMIS rats as a result of alteration in carbohydrate metabolism in diabetes. The comparable pharmacokinetics of LQ, M1, and M2 after oral administration of LQ were mainly due to the comparable intestinal metabolism of LQ between the control and DMIS rats.

Pharmacokinetic interaction between liquiritigenin (LQ) and DDB: increased glucuronidation of LQ in the liver possibly due to increased hepatic blood flow rate by DDB.

It has been reported that both liquiritigenin (LQ) and dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylenedioxybiphenyl-2,2'-dicarboxylate (DDB) have a hepatoprotective effect, and administration of both drugs together shows additive protective effect against acute liver injuries. Therefore, the pharmacokinetic interaction between LQ and DDB in rats was studied. LQ (20 and 50mg/kg for the i.v. and p.o. administration, respectively), DDB (10mg/kg for both i.v. and p.o. administration), and both drugs together were once administered intravenously or orally to rats. After the i.v. administration of both drugs together, the Cl(nr) and AUC of LQ were significantly faster (by 30.5%) and smaller (by 22.5%), respectively, than those of without DDB due to the faster hepatic blood flow rate by DDB. After the p.o. administration of both drugs together, the AUC of LQ was comparable to that of without DDB due to negligible effect of DDB on intestinal metabolism of LQ. The pharmacokinetic parameters of DDB after both i.v. and p.o. administration were not altered by LQ, indicating that LQ did not considerably affect the pharmacokinetics of DDB in rats.

Alfacalcidol increases cancellous bone in low turnover, fatty marrow sites in aged, orchidectomized rats.

The objectives of this study were to determine the responses of cancellous bone in the distal tibial metaphysis (DTM), a low turnover, fatty (yellow) marrow site, to sham-aged, orchidectomy (ORX) and alfacalcidol treatment in sham-aged and ORX rats. Eighteen-month-old male sham and ORX rats were treated with 0.1 and 0.2 microg/kg alfacalcidol 5 days/wk p.o. for 12 weeks, double fluorescent labeled, and the DTM were processed for bone histomorphometry analyses. The current study found the DTM in sham-aged male rats were resistant to age-related and ORX-induced cancellous bone loss and alfacalcidol-induced bone gain, findings that differ from that in the proximal tibial metaphysis (PTM) and lumbar vertebral body (LVB), two high turnover, red marrow bone sites. However, alfacalcidol treatment increased DTM bone mass in ORX rats where bone turnover was elevated by androgen deficiency. These results in concert with the previously positive findings in red marrow bone sites following alfacalcidol treatment suggest that alfacalcidol is more effective in increasing cancellous bone mass in the skeletal sites with higher bone turnover.

Greater efficacy of alfacalcidol in the red than in the yellow marrow skeletal sites in adult female rats.

The present study compared the bone anabolic effects of graded doses of alfacalcidol in proximal femurs (hematopoietic, red marrow skeletal site) and distal tibiae (fatty, yellow marrow skeletal site). One group of 8.5-month-old female Sprague-Dawley rats were killed at baseline and 4 groups were treated 5 days on/2 days off/week for 12 weeks with 0, 0.025, 0.05 and 0.1 microg alfacalcidol/kg by oral gavage. The proximal femur, bone site with hematopoietic marrow, as well as the distal tibia bone site with fatty marrow, were processed undecalcified for quantitative bone histomorphometry. In the red marrow site of the proximal femoral metaphysis (PFM), 0.1 microg alfacalcidol/kg induced increased cancellous bone mass, improved architecture (decreased trabecular separation, increased connectivity), and stimulated local bone formation of bone 'boutons' (localized bone formation) on trabecular surfaces. There was an imbalance in bone resorption and formation, in which the magnitude of depressed bone resorption greater than depressed bone formation resulted in a positive bone balance. In addition, bone 'bouton' formation contributed to an increase in bone mass. In contrast, the yellow marrow site of the distal tibial metaphysis (DTM), the 0.1 microg alfacalcidol/kg dose induced a non-significant increased cancellous bone mass. The treatment decreased bone resorption equal to the magnitude of decreased bone formation. No bone 'bouton' formation was observed. These findings indicate that the highest dose of 0.1 microg alfacalcidol/kg for 12 weeks increased bone mass (anabolic effect) at the skeletal site with hematopoietic marrow of the proximal femoral metaphysis, but the increased bone mass was greatly attenuated at the fatty marrow site of the distal tibial metaphysis. In addition, the magnitude of the bone gain induced by alfacalcidol treatment in red marrow cancellous bone sites of the proximal femoral metaphysis was half that of the lumbar vertebral body. The latter data were from a previous report from the same animal and protocol. These findings indicated that alfacalcidol as an osteoporosis therapy is less efficacious as a positive bone balance agent that increased trabecular bone mass in a non-vertebral skeletal site where bone marrow is less hematopoietic.

Cell identity mediates the response of Arabidopsis roots to abiotic stress.

Little is known about the way developmental cues affect how cells interpret their environment. We characterized the transcriptional response to high salinity of different cell layers and developmental stages of the Arabidopsis root and found that transcriptional responses are highly constrained by developmental parameters. These transcriptional changes lead to the differential regulation of specific biological functions in subsets of cell layers, several of which correspond to observable physiological changes. We showed that known stress pathways primarily control semiubiquitous responses and used mutants that disrupt epidermal patterning to reveal cell-layer-specific and inter-cell-layer effects. By performing a similar analysis using iron deprivation, we identified common cell-type-specific stress responses and revealed the crucial role the environment plays in defining the transcriptional outcome of cell-fate decisions.

Continuous PGE2 leads to net bone loss while intermittent PGE2 leads to net bone gain in lumbar vertebral bodies of adult female rats.

The present study examined the effects of continuous and intermittent PGE2 administration on the cancellous and cortical bone of lumbar vertebral bodies (LVB) in female rats. Six-month-old Sprague-Dawley female rats were divided into 6 groups with 2 control groups and 1 or 3 mg PGE2/kg given either continuously or intermittently for 21 days. Histomorphometric analyses were performed on the cancellous and cortical bone of the fourth and fifth LVBs. Continuous PGE2 exposure led to bone catabolism while intermittent administration led to bone anabolism. Both routes of administration stimulated bone remodeling, but the continuous PGE2 stimulated more than the intermittent route to expose more basic multicellular units (BMUs) to the negative bone balance. The continuous PGE2 caused cancellous bone loss by stimulating bone resorption greater than formation (i.e., negative bone balance) and shortening the formation period. It caused more cortical bone loss than gain, the magnitude of the negative endocortical bone balance and increased intracortical porosity bone loss was greater than for periosteal bone gain. The anabolic effects of intermittent PGE2 resulted from cancellous bone gain by positive bone balance from stimulated bone formation and shortened resorption period; while cortical bone gain occurred from endocortical bone gain exceeding the decrease in periosteal bone and increased intracortical bone loss. Lastly, a scheme to take advantage of the marked PGE2 stimulation of lumbar periosteal apposition in strengthening bone by converting it to an anabolic agent was proposed.

Continuous infusion of PGE2 is catabolic with a negative bone balance on both cancellous and cortical bone in rats.

It is well documented that intermittent PGE(2) treatment increases both trabecular and cortical bone mass. However, the effects of continuous PGE(2) administration remain undocumented. The aim of the study was to investigate the effects of continuous prostaglandin E(2) (PGE(2)) on different bone sites in skeletally mature rats. Six-month-old Sprague Dawley rats were treated with PGE(2) at 1 or 3 mg/kg/d continuously via infusion pump for 21 days. Two other groups of rats received PGE(2) at the same doses by intermittent (daily) subcutaneous injections and served as positive controls. Histomorphometry was performed on cancellous bone of the proximal tibial metaphysis and cortical bone of the tibial shaft. As expected, intermittent PGE(2) treatment increased both cancellous and cortical bone mass by stimulating bone formation at the cancellous, periosteal and endocortical bone surfaces. In contrast, continuous PGE(2) treatment decreased cancellous bone mass with bone resorption exceeding bone formation. In addition, continuous PGE(2) treatment increased endocortical and intracortical bone remodeling, inducing bone loss which was partially offset by stimulating periosteal expansion. We conclude that continuous PGE(2) treatment induces overall catabolic effects on both cancellous and cortical bone envelopes, which differs from intermittent PGE(2) treatment that is anabolic. Lastly, we speculate that superior bone mass may be achieved by co-treatment of continuous PGE(2) in combination with an anti-catabolic agent.

Cancellous bone minimodeling-based formation: a Frost, Takahashi legacy.

The current dogma is that in adult human beings, remodeling creates nearly all the new cancellous bone tissue. However, Frost, Takahashi and colleagues hypothesized that minimodeling can go on in trabeculae throughout life. The current perspective will review the available reports on minimodeling-based formation to determine whether there is any support for his hypothesis. One: describe the methodology employed to characterize remodeling and minimodeling formation sites or packets, which restrict the analyses of these packets to a known age of the specimen. Two: present quantitative minimodeling data on cancellous bone of aging rats and transiliac bone biopsy of adult humans. Three: describe the occurrence and quantitation of mixed remodeling-minimodeling formation sites that could be misinterpreted as minimodeling sites. Fourth: present irrefutable evidence that bone anabolic agents initiate minimodeling-based formation sites. Fifth: discuss the mechanism of minimodeling-based formation may be the resumption of osteoblastic activity by bone lining cells to increase cancellous bone mass and trabecular connectivity. The findings of minimodeling is a rare activity in normal individuals, but may occur in a select population, and bone anabolic agents can initiate minimodeling-based formation are in support of Frost's hypothesis that minimodeling can continue throughout human life. Thus, another Frost, Takahashi legacy lives on.

Rolipram, a phosphodiesterase 4 inhibitor, prevented cancellous and cortical bone loss by inhibiting endosteal bone resorption and maintaining the elevated periosteal bone formation in adult ovariectomized rats.

Cyclic AMP (cAMP) is a continually produced nucleotide inactivated by hydrolysis to 5'AMP via phosphodiesterase (PDE) enzymes. Rolipram is a selective PDE4 inhibitor reported to have anti-inflammatory effects and used in the treatment of asthma and chronic obstructive pulmonary disease (COPD). The current study was designed to determine whether Rolipram could prevent and restore bone loss in ovariectomized (OVX) rats. Six-month-old Sprague Dawley rats underwent either sham-operated or bilateral ovariectomy, and were left untreated for 60 days to develop osteopenia. Then they were treated with vehicle, 6 mg/kg PGE(2), 3 microg/kg Alendronate or 0.1-1.0 mg/kg Rolipram for 60 days. At sacrifice, the right tibiae were processed for quantitative bone histomorphometric measurements. The right femurs were measured by dual energy A-ray absorptiometry and the 5th lumbar vertebrae were subjected to micro-computed tomography to access bone mass and architecture changes. Our results indicated that OVX induced negative bone balance in all five bone sites we tested, with bone resorption exceeding bone formation. Rolipram at 0.1-0.6 mg/kg dose levels prevented while at 1 mg/kg restored ovariectomy-induced cancellous and cortical bone loss in the tibia, femur and lumbar vertebra. Dynamic bone histomorphometry suggested that these beneficial effects were achieved by partially maintaining the elevated bone formation at the trabecular bone surface and increasing bone formation at the periosteal bone surface of the cortex. Furthermore, it reduced bone turnover at the trabecular and the endocortical bone surfaces. The prevention of further bone loss effects were comparable to those of an anti-resorption agent (Alendronate) but were not as great as those of an anabolic agent (PGE(2)). In addition, Rolipram treatment increased body and muscle weights compared to the vehicle-treated OVX rats. In conclusion, our study in an osteopenic rat model suggested that a selective PDE4 inhibitor may be used for the treatment of established osteoporosis.

An evolutionarily conserved mechanism delimiting SHR movement defines a single layer of endodermis in plants.

Intercellular protein movement plays a critical role in animal and plant development. SHORTROOT (SHR) is a moving transcription factor essential for endodermis specification in the Arabidopsis root. Unlike diffusible animal morphogens, which form a gradient across multiple cell layers, SHR movement is limited to essentially one cell layer. However, the molecular mechanism is unknown. We show that SCARECROW (SCR) blocks SHR movement by sequestering it into the nucleus through protein-protein interaction and a safeguard mechanism that relies on a SHR/SCR-dependent positive feedback loop for SCR transcription. Our studies with SHR and SCR homologs from rice suggest that this mechanism is evolutionarily conserved, providing a plausible explanation why nearly all plants have a single layer of endodermis.

Simvastatin did not prevent nor restore ovariectomy-induced bone loss in adult rats.

Current published results on whether statins have beneficial effects on bone metabolism have been conflicting so far. In order to further investigate if statins were promising candidates for the treatment for osteoporosis, we conducted a study in which rats were ovariectomized (OVX) at 6 months of age, allowed to lose bone for 60 days and followed by oral administration of simvastatin at the dose levels of 0.3-10 mg/kg/d for 60 days. PGE2 (6 mg/kg) was used as a positive control. Study endpoints included bone histomorphometry on the proximal tibial metaphysis (PTM) and the tibial diaphysis (TX), dual-energy X-ray absorptiometry on the right femur and micro computed tomography (ICT) on the 5th lumbar vertebra (LV). After 120 days of OVX, cancellous bone lost by 80% in the PTM and 18% in the LV accompanied by increased bone formation and resorption. Simvastatin at all dose levels did not affect bone volume, bone formation rate and bone erosion surface when compared to 120 day ovariectomized animals at all bone sites studied. By contrast, PGE2 restored cancellous and cortical bone area to sham control levels. In conclusion, this study demonstrated that unlike PGE2, oral administration of simvastatin did not have effects on cancellous or cortical bone formation and resorption; and consequently was not able to prevent further bone loss or restore bone mass in the osteopenic, OVX rats.

Harold M. Frost, M.D., D.Sc. (hon) -- one man's association.

The author described how and when he first met Harold M. Frost, M.D., that began a journey from the Henry Ford Hospital to the University of Utah and Sun Valley Hard TissueWorkshops that sequentially developed the technology of dynamic cancellous bone histomorphometry, the ever-evolving mechanostat hypothesis and the Utah Paradigm for Bone Physiology.

Ancestry of beagles in lifespan studies of radionuclide toxicity at the University of Utah.

Analysis of the ancestry of the 1,262 lifespan beagles (LSB) entered into lifespan studies at the Radiobiology Division, University of Utah, beginning in 1952 and ending in 1980, indicated that about 97% of ancestor citations in the various pedigrees were of only 10 breeding animals (breeders) among breeders within the beagle colony. In turn, just 18 AKC-registered "champion" beagles from outside of this colony (founders) accounted for about 98% of all ancestor citations among founders for the LSB. We conclude from this study that the animals used in the lifespan radionuclide experiments can be considered to be somewhat genetically interrelated.

Conservation and diversification of SCARECROW in maize.

The SCARECROW (SCR) gene in Arabidopsis is required for asymmetric cell divisions responsible for ground tissue formation in the root and shoot. Previously, we reported that Zea mays SCARECROW (ZmSCR) is the likely maize ortholog of SCR. Here we describe conserved and divergent aspects of ZmSCR. Its ability to complement the Arabidopsis scr mutant phenotype suggests conservation of function, yet its expression pattern during embryogenesis and in the shoot system indicates divergence. ZmSCR expression was detected early during embryogenesis and localized to the endodermal lineage in the root, showing a gradual regionalization of expression. Expression of ZmSCR appeared to be analogous to that of SCR during leaf formation. However, its absence from the maize shoot meristem and its early expression pattern during embryogenesis suggest a diversification of ZmSCR in the patterning processes in maize. To further investigate the evolutionary relationship of SCR and ZmSCR, we performed a phylogenetic analysis using Arabidopsis, rice and maize SCARECROW-LIKE genes (SCLs). We found SCL23 to be the most closely related to SCR in both eudicots and monocots, suggesting that a gene duplication resulting in SCR and SCL23 predates the divergence of dicots and monocots.