Expression characterization of the herbicide tolerance gene Aryloxyalkanoate Dioxygenase (aad-1) controlled by seven combinations of regulatory elements.

BACKGROUND: Availability of well characterized maize regulatory elements for gene expression in a variety of tissues and developmental stages provides effective alternatives for single and multigene transgenic concepts. We studied the expression of the herbicide tolerance gene aryloxyalkanoate dioxygenase (aad-1) driven by seven different regulatory element construct designs including the ubiquitin promoters of maize and rice, the actin promoters of melon and rice, three different versions of the Sugarcane Bacilliform Badnavirus promoters in association with other regulatory elements of gene expression. RESULTS: Gene expression of aad-1 was characterized at the transcript and protein levels in a collection of maize tissues and developmental stages. Protein activity against its target herbicide was characterized by herbicide dosage response. Although differences in transcript and protein accumulation were observed among the different constructs tested, all events were tolerant to commercially relevant rates of quizalafop-P-ethyl compared to non-traited maize under greenhouse conditions. DISCUSSION: The data reported demonstrate how different regulatory elements affect transcript and protein accumulation and how these molecular characteristics translate into the level of herbicide tolerance. The level of transcript detected did not reflect the amount of protein quantified in a particular tissue since protein accumulation may be influenced not only by levels of transcript produced but also by translation rate, post-translational regulation mechanisms and protein stability. The amount of AAD-1 enzyme produced with all constructs tested showed sufficient enzymatic activity to detoxify the herbicide and prevent most herbicidal damage at field-relevant levels without having a negative effect on plant health. CONCLUSIONS: Distinctive profiles of aad-1 transcript and protein accumulation were observed when different regulatory elements were utilized in the constructs under study. The ZmUbi and the SCBV constructs showed the most consistent robust tolerance, while the melon actin construct provided the lowest level of tolerance compared to the other regulatory elements used in this study. These data provide insights into the effects of differing levels of gene expression and how these molecular characteristics translate into the level of herbicide tolerance. Furthermore, these data provide valuable information to optimize future designs of single and multiple gene constructs for maize research and crop improvement.

mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging.

BACKGROUND: The mammalian target of rapamycin (mTOR) signalling pathway has a key role in cellular regulation and several diseases. While it is thought that Rheb GTPase regulates mTOR, acting immediately upstream, while raptor is immediately downstream of mTOR, direct interactions have yet to be verified in living cells, furthermore the localisation of Rheb has been reported to have only a cytoplasmic cellular localization. RESULTS: In this study a cytoplasmic as well as a significant sub-cellular nuclear mTOR localization was shown , utilizing green and red fluorescent protein (GFP and DsRed) fusion and highly sensitive single photon counting fluorescence lifetime imaging microscopy (FLIM) of live cells. The interaction of the mTORC1 components Rheb, mTOR and raptor, tagged with EGFP/DsRed was determined using fluorescence energy transfer-FLIM. The excited-state lifetime of EGFP-mTOR of ~2400 ps was reduced by energy transfer to ~2200 ps in the cytoplasm and to 2000 ps in the nucleus when co-expressed with DsRed-Rheb, similar results being obtained for co-expressed EGFP-mTOR and DsRed-raptor. The localization and distribution of mTOR was modified by amino acid withdrawal and re-addition but not by rapamycin. CONCLUSIONS: The results illustrate the power of GFP-technology combined with FRET-FLIM imaging in the study of the interaction of signalling components in living cells, here providing evidence for a direct physical interaction between mTOR and Rheb and between mTOR and raptor in living cells for the first time.

CAPG Is Required for Ebola Virus Infection by Controlling Virus Egress from Infected Cells.

The replication of Ebola virus (EBOV) is dependent upon actin functionality, especially at cell entry through macropinocytosis and at release of virus from cells. Previously, major actin-regulatory factors involved in actin nucleation, such as Rac1 and Arp2/3, were shown important in both steps. However, downstream of nucleation, many other cell factors are needed to control actin dynamics. How these regulate EBOV infection remains largely unclear. Here, we identified the actin-regulating protein, CAPG, as important for EBOV replication. Notably, knockdown of CAPG specifically inhibited viral infectivity and yield of infectious particles. Cell-based mechanistic analysis revealed a requirement of CAPG for virus production from infected cells. Proximity ligation and split-green fluorescent protein reconstitution assays revealed strong association of CAPG with VP40 that was mediated through the S1 domain of CAPG. Overall, CAPG is a novel host factor regulating EBOV infection through connecting actin filament stabilization to viral egress from cells.

Knowledge and attitudes of syphilis and syphilis pre-exposure prophylaxis (PrEP) among men who have sex with men in Vancouver, Canada: a qualitative study.

OBJECTIVES: In British Columbia, Canada, syphilis is at record-high rates, with over 80% of cases in 2017 seen in gay, bisexual and other men who have sex with men (GBM). The syphilis epidemic is of particular concern for those living with HIV, since syphilis may lead to more serious complications in this population. We sought to explore syphilis-related knowledge and attitudes around biomedical prevention options for syphilis, with the goal of informing effective strategies to prevent syphilis. DESIGN: We conducted a qualitative study consisting of in-depth, individual interviews from December 2016 to June 2017. Our interviews focused on participants' knowledge about syphilis and perceptions regarding syphilis pre-exposure prophylaxis (PrEP). Interviews were analysed using Grounded Theory. PARTICIPANTS: Twenty-five GBM were interviewed (64% white; median age: 43 years), including men living with HIV and/or with a history of syphilis. SETTING: Vancouver, British Columbia. RESULTS: Five interrelated themes emerged. First, GBM were aware of the local syphilis epidemic. Second, syphilis-related knowledge differed according to syphilis and HIV serostatus. Third, competing ideas emerged regarding men's concerns about syphilis. While our participants expressed concern about getting syphilis, they also described the importance of sexual pleasure. Fourth, many participants said that syphilis was not perceived to be alarming; preventing HIV infection remained a primary concern for many. Finally, while syphilis PrEP was appealing to those living with HIV or a prior syphilis diagnosis, others were concerned about antibiotic resistance, cost and side effects. CONCLUSIONS: Our participants organised their safer sex strategies around HIV, not syphilis. Although syphilis-related knowledge was relatively high among GBM living with HIV and those with a prior syphilis diagnosis, this knowledge did not appear to be related with safer sexual practices, such as increased condom use. This work highlights the importance of examining other potential prevention solutions, such as syphilis PrEP.

Acute ischemia of the lower extremity in the pediatric population: a protocol for management.

Each year, a large number of infants and children undergo vascular catheterization via access in a lower extremity. The femoral system is relatively easy to cannulate, and this approach avoids use of more central access approaches that may present more serious complications. Infrequently, however, pediatric patients develop acute ischemia of the ipsilateral lower extremity. Several surgical and nonsurgical treatment options exist for the management of such problems. However, no adequate protocol exists for the management of acute lower extremity ischemia in the pediatric population. The authors present several cases of distal lower extremity ischemia as a result of femoral artery catheterization and offer a protocol for management of similar cases.

A comprehensive study of the use of a homologous promoter in antisense cotton lines exhibiting a high seed oleic acid phenotype.

As opposed to first-generation biotechnology products, such as pest-resistant crops and herbicide-resistant crops, second-generation products often utilize plant-derived, homologous or heterologous genes and/or promoters. In this study, we evaluated the ability of a promoter from a gene encoding a major storage protein in cottonseed to drive an antisense sequence of the cotton FAD2 gene to down-regulate the activity of Delta-12 desaturase enzyme in cottonseeds. The oleic acid level in the transgenic cottonseeds approximately doubled from the wild-type level of 15%, with a concomitant decrease in the level of linoleic acid. A more extensive study of one line revealed a higher degree of seed-to-seed variability in the transgenic phenotype. A thorough investigation was conducted to determine the impact of the use of a homologous promoter to drive a transgene on the activity of the endogenous promoter. The results showed that the use of the homologous alpha-globulin B promoter for transgenic purposes did not adversely affect the expression of alpha-globulin B storage protein in cottonseed. The results obtained in this investigation on the use of a homologous promoter and antisense technology will be useful in the design of strategies to alter biosynthetic pathways for nutritional quality improvements and for the production of heterologous proteins of commercial value in seeds.

Proteases as drug targets.

The effective management of AIDS with HIV protease inhibitors, or the use of angiotensin-converting enzyme inhibitors to treat hypertension, indicates that proteases do make good drug targets. On the other hand, matrix metalloproteinase (MMP) inhibitors from several companies have failed in both cancer and rheumatoid arthritis clinical trials. Mindful of the MMP inhibitor experience, this chapter explores how tractable proteases are as drug targets from a chemistry perspective. It examines the recent success of other classes of drug for the treatment of rheumatoid arthritis, and highlights the need to consider where putative targets lie on pathophysiological pathways--regardless of what kind of therapeutic entity would be required to target them. With genome research yielding many possible new drug targets, it explores the likelihood of discovering proteolytic enzymes that are causally responsible for disease processes and that might therefore make better targets, especially if they lead to the development of drugs that can be administered orally. It also considers the impact that biologics are having on drug discovery, and in particular whether biologically derived therapeutics such as antibodies are likely to significantly alter the way we view proteases as targets and the methods used to discover therapeutic inhibitors.

Minimizing tolerance and withdrawal to prolonged pediatric sedation: case report and review of the literature.

Midazolam and fentanyl infusions are commonly used for prolonged sedation and analgesia in the pediatric intensive care setting. Tolerance and withdrawal are major concerns when these infusions are used for days or weeks. Here, we review the current approaches to prolonged pediatric sedation using midazolam and fentanyl and discuss newer strategies to avoid tolerance and withdrawal syndromes. We report the case of a pediatric burn patient who developed tolerance syndrome and a movement disorder in our institution. We also review the relevant literature and methods of minimizing tolerance and withdrawal. Prolonged sedation is often necessary in treating critically ill children, and tolerance and abstinence syndrome can complicate a successful recovery. Scoring systems can be used to minimize oversedation and to titrate effectively. "Drug cycling," "wake-up protocols," and weaning regimens, possibly combined with adjuvant drugs, are being implemented successfully. Such novel approaches may decrease the incidence of tolerance and withdrawal associated with prolonged sedative and analgesic use.

First Things First: a framework for successful secondary school reform.

If youth development initiatives are going to focus on outcomes that we know are important in settings that we know can change these outcomes, the first outcomes should be educational, and the first setting should be school. School reform presents the most feasible, defensible, and informed opportunity for public policy to improve the life chances of children and youth in disadvantaged communities. This chapter introduces First Things First (FTF), a school reform framework grounded in research about how young people develop and how schools promote students' engagement and learning. The chapter explores four critical features of FTF that focus on students: (1) continuity of care; (2) increased instructional time; (3) high, clear, and fair standards; and (4) enriched opportunities for students. The critical features of FTF are implemented through three strategies: small learning communities, a family advocate system, and instructional improvement.

Cotton alpha-globulin promoter: isolation and functional characterization in transgenic cotton, Arabidopsis, and tobacco.

Globulins are the most abundant seed storage proteins in cotton and, therefore, their regulatory sequences could potentially provide a good source of seed-specific promoters. We isolated the putative promoter region of cotton alpha-globulin B gene by gene walking using the primers designed from a cotton staged embryo cDNA clone. PCR amplified fragment of 1108 bp upstream sequences was fused to gusA gene in the binary vector pBI101.3 to create the test construct. This was used to study the expression pattern of the putative promoter region in transgenic cotton, Arabidopsis, and tobacco. Histochemical GUS analysis revealed that the promoter began to express during the torpedo stage of seed development in tobacco and Arabidopsis, and during cotyledon expansion stage in cotton. The activity quickly increased until embryo maturation in all three species. Fluorometric GUS analysis showed that the promoter expression started at 12 and 15 dpa in tobacco and cotton, respectively, and increased through seed maturation. The strength of the promoter expression, as reflected by average GUS activity in the seeds from primary transgenic plants, was vastly different amongst the three species tested. In Arabidopsis, the activity was 16.7% and in tobacco it was less than 1% of the levels detected in cotton seeds. In germinating seedlings of tobacco and Arabidopsis, GUS activity diminished until it was completely absent 10 days post imbibition. In addition, absence of detectable level of GUS expression in stem, leaf, root, pollen, and floral bud of transgenic cotton confirmed that the promoter is highly seed-specific. Analysis of GUS activity at individual seed level in cotton showed a gene dose effect reflecting their homozygous or hemizygous status. Our results show that this promoter is highly tissue-specific and it can be used to control transgene expression in dicot seeds.