Evaluation of glyphosate resistance in Arabidopsis thaliana expressing an altered target site EPSPS.

BACKGROUND: Glyphosate-resistant goosegrass has recently evolved and is homozygous for the double mutant of EPSPS (T102 I, P106 S or TIPS). These same mutations combined with EPSPS overexpression, have been used to create transgenic glyphosate-resistant crops. Arabidopsis thaliana (Wt EPSPS Ki ∼ 0.5 µM) was engineered to express a variant AtEPSPS-T102 I, P106 A (TIPA Ki = 150 µM) to determine the resistance magnitude for a more potent variant EPSPS that might evolve in weeds. RESULTS: Transgenic A. thaliana plants, homozygous for one, two or four copies of AtEPSPS-TIPA, had resistance (IC50 values, R/S) as measured by seed production ranging from 4.3- to 16-fold. Plants treated in reproductive stage were male sterile with a range of R/S from 10.1- to 40.6-fold. A significant hormesis (∼ 63% gain in fresh weight) was observed for all genotypes when treated at the initiation of reproductive stage with 0.013 kg ha-1 . AtEPSPS-TIPA enzyme activity was proportional to copy number and correlated with resistance magnitude. CONCLUSIONS: A. thaliana, as a model weed expressing one copy of AtEPSPS-TIPA (300-fold more resistant), had only 4.3-fold resistance to glyphosate for seed production. Resistance behaved as a single dominant allele. Vegetative tissue resistance was 4.7-fold greater than reproductive tissue resistance and was linear with gene copy number. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Endogenous tassel-specific small RNAs-mediated RNA interference enables a novel glyphosate-inducible male sterility system for commercial production of hybrid seed in Zea mays L.

Hybrid crops produce higher yields than their inbred parents due to heterosis. For high purity of hybrid seeds, it is critical to eliminate self-pollination. Manual or mechanical removal of male parts (such as detasseling in maize) is labor-intensive, fuel and time-consuming, and can cause physical damage to female plants, resulting in significant seed yield reductions. Many male-sterility systems either require a maintainer for male-sterile line propagation or are often affected by environmental factors. Roundup® Hybridization System (RHS) utilizes glyphosate to induce male sterility, which effectively eliminates the need for maintainer lines and removal of male parts for commercial hybrid seed production. The first-generation RHS (RHS1) is based on low expression of a glyphosate-insensitive 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) in pollen. This report presents the second-generation RHS (RHS2) technology built on RNA interference (RNAi) combined with CP4 EPSPS. It utilizes maize endogenous male tissue-specific small interfering RNAs (mts-siRNAs) to trigger cleavage of the CP4 EPSPS mRNA specifically in tassels, resulting in glyphosate-sensitive male cells due to lack of the CP4 EPSPS protein. Male sterility is then induced by glyphosate application at the stages critical for pollen development, and the male-sterile plants are used as the female parent to produce hybrid seed. The endogenous mts-siRNAs are conserved across maize germplasms, and the inducible male sterility was replicated in representative germplasms through introgression of a CP4 EPSPS transgene containing the mts-siRNA target sequence. This technology combines the relative simplicity and convenience of a systemic herbicide spray methodology with targeted protein expression to create an inducible male sterility system for industrial production of row crop hybrid seeds in an environmentally-independent manner.

The impact of methicillin resistance in Staphylococcus aureus bacteremia on patient outcomes: mortality, length of stay, and hospital charges.

OBJECTIVE: To evaluate the impact of methicillin resistance in Staphylococcus aureus on mortality, length of hospitalization, and hospital charges. DESIGN: A cohort study of patients admitted to the hospital between July 1, 1997, and June 1, 2000, who had clinically significant S. aureus bloodstream infections. SETTING: A 630-bed, urban, tertiary-care teaching hospital in Boston, Massachusetts. PATIENTS: Three hundred forty-eight patients with S. aureus bacteremia were studied; 96 patients had methicillin-resistant S. aureus (MRSA). Patients with methicillin-susceptible S. aureus (MSSA) and MRSA were similar regarding gender, percentage of nosocomial acquisition, length of hospitalization, ICU admission, and surgery before S. aureus bacteremia. They differed regarding age, comorbidities, and illness severity score. RESULTS: Similar numbers of MRSA and MSSA patients died (22.9% vs 19.8%; P = .53). Both the median length of hospitalization after S. aureus bacteremia for patients who survived and the median hospital charges after S. aureus bacteremia were significantly increased in MRSA patients (7 vs 9 days, P = .045; 19,212 dollars vs 26,424 dollars, P = .008). After multivariable analysis, compared with MSSA bacteremia, MRSA bacteremia remained associated with increased length of hospitalization (1.29 fold; P = .016) and hospital charges (1.36 fold; P = .017). MRSA bacteremia had a median attributable length of stay of 2 days and a median attributable hospital charge of 6916 dollars. CONCLUSION: Methicillin resistance in S. aureus bacteremia is associated with significant increases in length of hospitalization and hospital charges.

Decreased response of plasma cortisol to intravenous metyrapone in ocular hypertension and primary open-angle glaucoma.

PURPOSE: To determine if there is a difference in response to metyrapone, which blocks the conversion of 11-deoxycortisol to cortisol in the adrenal gland between normals and ocular hypertensives (OH) plus primary open-angle glaucomas (POAG) without pigmented angles. METHODS: Intravenous metyrapone was given to 20 normals and 15 ocular hypertensives plus primary open-angle glaucomas without pigmented angles. Blood samples were obtained at 4, 6, and 8 hours after administration of intravenous metyrapone for analyses of 11-deoxycortisol and cortisol. RESULTS: The ocular hypertensives plus primary open-angle glaucoma group showed significantly lower cortisol values compared with the normal group at 6 hours (P = 0.002) but not at 4 or 8 hours. There were no significant differences between the two groups for 11-deoxycortisol values at 4, 6, or 8 hours. The percent decrease of plasma cortisol from baseline was significantly greater for the ocular hypertensives plus open-angle glaucoma group compared with the normals at 4 hours (P = 0.010) and 6 hours (P = 0.0004). Significant negative correlations were observed for the total group of subjects between levels of plasma cortisol at 6 hours and intraocular pressure, worse eye (P = 0.029), percent area of cupping, worse eye (P = 0.045), pallor, worse eye (P = 0.001), and visual field loss, worse eye (P = 0.048), so that the less the plasma cortisol, the greater the abnormality of the glaucomatous parameters. Multivariate analyses with the 6-hour plasma cortisol level as the dependent variable showed that the only significant (P = 0.0004) independent variable was the percent area of pallor, worse eye, associated with a smaller level of plasma cortisol at 6 hours. Similarly, the multiple regression models using the percent change from baseline of the 6-hour plasma cortisol value showed a significant association of larger percent decreases of plasma cortisol in the ocular hypertensives plus open-angle glaucoma compared with the normals. CONCLUSIONS: The ocular hypertensives plus primary open-angle glaucoma subjects show greater adrenal inhibition to metyrapone in the synthesis of cortisol from 11-deoxycortisol compared with normals. This observation suggests an adrenal abnormality in the ocular hypertensive plus primary open-angle glaucoma subjects.

Dose response of dexamethasone for the enhanced ocular hypotensive response to epinephrine in rabbits with prior dexamethasone treatment.

We have previously shown that administration of epinephrine to the rabbit eye with dexamethasone pretreatment resulted in a significant decrease in intraocular pressure (IOP), compared to the use of epinephrine alone. Furthermore, this decrease in IOP is dose-dependent on epinephrine with the largest response of decrease of IOP occurring at the lower doses of epinephrine. We now extend this study to determine whether the decrease in IOP is dose-dependent on dexamethasone. Rabbit eyes were pretreated with five applications of topical dexamethasone or saline, administered at fifteen-minute intervals. Eyes were then treated with 0.005% epinephrine (0.01% epinephrine bitartrate). IOP was monitored for the next four hours. Different groups of rabbits received varying concentrations of dexamethasone base, from 0.0004% to 0.07% (dexamethasone phosphate), and a dose response curve was obtained. When compared to eyes treated with epinephrine alone, eyes pretreated with dexamethasone showed a significantly greater decrease in pressure at free base dexamethasone concentrations of 0.07%, 0.007% and 0.0007%, with the greatest difference at the 0.007% concentration (mean = 6.8 mm Hg). Similarly, the duration taken for the IOP to return to baseline levels was prolonged in the groups receiving dexamethasone pretreatment. The synergism between dexamethasone and epinephrine in lowering IOP may be a useful alternative in the treatment of ocular hypertension and glaucoma especially using a combination of a low dose of epinephrine with a low dose of dexamethasone.

Maternal and breast pump factors associated with breast pump problems and injuries.

BACKGROUND: Benefits of using a breast pump are well documented, but pump-related problems and injuries and the associated risk factors have not been reported. OBJECTIVES: This study aimed to describe breast pump-related problems and injuries and identify factors associated with these problems and injuries. METHODS: Data were from the Infant Feeding Practices Study II; mothers were recruited from a nationally distributed consumer opinion panel. Mothers were asked about breast pump use, problems, and injuries at infant ages 2, 5, and 7 months. Survival analysis was used to identify factors associated with pump-related problems and injuries. RESULTS: The sample included 1844 mothers. About 62% and 15% of mothers reported pump-related problems and injuries, respectively. The most commonly reported problem was that the pump did not extract enough milk and the most commonly reported injury was sore nipples. Using a battery-operated pump and intending to breastfeed less than 12 months were associated with higher risks of pump-related problems and injury. Learning from a friend to use the pump was associated with lower risk of pump-related problems, and using a manual pump and renting a pump were associated with a higher risk of problems. CONCLUSION: Our results suggest that problems and injuries associated with breast pump use can happen to mothers of all socioeconomic characteristics. Breastfeeding mothers may reduce their risks of problems and injury by not using battery-operated pumps and may reduce breast pump problems by not using manual pumps and by learning breast pump skills from a person rather than following written or video instructions.

Improving hybrid seed production in corn with glyphosate-mediated male sterility.

BACKGROUND: Hybrid corn varieties exhibit benefits associated with heterosis and account for most of the corn acreage in the USA. Hybrid seed corn is produced by crossing a female parent which is male-sterile and therefore incapable of self-pollination with a male parent as the pollen donor. The majority of hybrid seed corn is produced by mechanical detasseling which involves physically removing the tassel, a process that is laborious and costly. RESULTS: Glyphosate-resistant corn was developed via expression of a glyphosate insensitive 5-enolpyruvyl-shikimate 3-phosphate synthase enzyme (CP4-EPSPS). Experimentation with molecular expression elements resulted in selective reduction of CP4-EPSPS expression in male reproductive tissues. The resulting plant demonstrated sterile tassel following glyphosate application with little to no injury to the rest of the plant. Using (14)C-glyphosate as a marker, we also examined the translocation of glyphosate to the tassel via spray application in a track sprayer to simulate field application. The results allowed optimization of spray parameters such as dose, spray timing and target to maximize tassel delivery of glyphosate for efficient sterilization. CONCLUSION: The Roundup hybridization system (RHS) is a novel process for hybrid seed production based on glyphosate-mediated male sterility. RHS replaces mechanical detasseling with glyphosate spray and greatly simplifies the process of hybrid seed corn production.

Emergency department visits for medical device-associated adverse events among children.

OBJECTIVES: The purposes of this study were to provide national estimates of emergency department (ED) visits for medical device-associated adverse events (MDAEs) in the pediatric population and to characterize these events further. METHODS: ED medical record reports from the National Electronic Injury Surveillance System All Injury Program database from January 1, 2004, through December 21, 2005, were reviewed. MDAEs among pediatric patients were identified, and data were abstracted. National estimates for pediatric MDAEs were determined according to medical specialty, device category and class, injury diagnosis, and patient characteristics and outcome. RESULTS: The total estimated number of pediatric MDAEs during the 24-month period was 144,799 (95% confidence interval: 113,051-183,903), involving devices from 13 medical specialties. Contact lenses accounted for most MDAEs (23%), followed by hypodermic needles (8%). The distribution of MDAEs according to medical specialty varied according to age subgroup. The most-prevalent types of injuries included contusions/abrasions, foreign-body intrusions, punctures, lacerations, and infections. The most-frequently affected body parts were the eyeball, pubic region, finger, face, and ear. The majority of pediatric MDAEs involved class II (moderate-risk) devices. The incidence of pediatric MDAEs decreased with increasing age from early to late childhood and then spiked after 10 years of age. More girls than boys were affected at older ages (16-21 years) and more boys than girls at younger ages (< or =10 years). Hospitalizations were more likely to involve invasive or implanted devices. CONCLUSIONS: This study provides national estimates of pediatric MDAEs resulting in ED visits and highlights the need to develop interventions to prevent pediatric device-related injuries.

Dicamba monooxygenase: structural insights into a dynamic Rieske oxygenase that catalyzes an exocyclic monooxygenation.

Dicamba (2-methoxy-3,6-dichlorobenzoic acid) O-demethylase (DMO) is the terminal Rieske oxygenase of a three-component system that includes a ferredoxin and a reductase. It catalyzes the NADH-dependent oxidative demethylation of the broad leaf herbicide dicamba. DMO represents the first crystal structure of a Rieske non-heme iron oxygenase that performs an exocyclic monooxygenation, incorporating O(2) into a side-chain moiety and not a ring system. The structure reveals a 3-fold symmetric trimer (alpha(3)) in the crystallographic asymmetric unit with similar arrangement of neighboring inter-subunit Rieske domain and non-heme iron site enabling electron transport consistent with other structurally characterized Rieske oxygenases. While the Rieske domain is similar, differences are observed in the catalytic domain, which is smaller in sequence length than those described previously, yet possessing an active-site cavity of larger volume when compared to oxygenases with larger substrates. Consistent with the amphipathic substrate, the active site is designed to interact with both the carboxylate and aromatic ring with both key polar and hydrophobic interactions observed. DMO structures were solved with and without substrate (dicamba), product (3,6-dichlorosalicylic acid), and either cobalt or iron in the non-heme iron site. The substitution of cobalt for iron revealed an uncommon mode of non-heme iron binding trapped by the non-catalytic Co(2+), which, we postulate, may be transiently present in the native enzyme during the catalytic cycle. Thus, we present four DMO structures with resolutions ranging from 1.95 to 2.2 A, which, in sum, provide a snapshot of a dynamic enzyme where metal binding and substrate binding are coupled to observed structural changes in the non-heme iron and catalytic sites.

Transgenic studies on the involvement of cytokinin and gibberellin in male development.

Numerous plant hormones interact during plant growth and development. Elucidating the role of these various hormones on particular tissue types or developmental stages has been difficult with exogenous applications or constitutive expression studies. Therefore, we used tissue-specific promoters expressing CKX1 and gai, genes involved in oxidative cytokinin degradation and gibberellin (GA) signal transduction, respectively, to study the roles of cytokinin and GA in male organ development. Accumulation of CKX1 in reproductive tissues of transgenic maize (Zea mays) resulted in male-sterile plants. The male development of these plants was restored by applications of kinetin and thidiazuron. Similarly, expression of gai specifically in anthers and pollen of tobacco (Nicotiana tabacum) and Arabidopsis resulted in the abortion of these respective tissues. The gai-induced male-sterile phenotype exhibited by the transgenic plants was reversible by exogenous applications of kinetin. Our results provide molecular evidence of the involvement of cytokinin and GA in male development and support the hypothesis that the male development is controlled in concert by multiple hormones. These studies also suggest a potential method for generating maintainable male sterility in plants by using existing agrochemicals that would reduce the expense of seed production for existing hybrid crops and provide a method to produce hybrid varieties of traditionally non-hybrid crops.

RNA-binding protein-mediated translational repression of transgene expression in plants.

We have demonstrated that RNA-binding proteins from coliphages and yeast can function as translational repressors in plants. RNA sequences called translational operators were inserted at a cap-proximal position in the 5'-UTR of mRNAs of two reporter genes, gus or aroA:CP4. Translation of the reporter mRNAs was efficiently repressed when the RNA binding protein that specifically binds to its cognate operator was co-expressed. The efficiency of translational repression by RNA-binding protein positively correlated with the amount of binding protein in transformed plant cells. Detailed studies on coliphage MS2 coat protein-mediated translational repression also suggested that the efficiency of translational repression was position-dependent. A translational operator situated at the cap-proximal position was more efficient in conferring repression than one that was placed cap-distal. Translational repression can be an efficient means for regulation of transgene expression, thereby broadening current approaches for transgene regulation in plants.

Variety of beta-lactamases produced by amoxicillin-clavulanate-resistant Escherichia coli isolated in the northeastern United States.

This study analyzed the enzymatic basis and molecular epidemiology of amoxicillin-clavulanate-resistant Escherichia coli isolated by the microbiology laboratory of a United States tertiary care hospital. From October 1998 to December 1999, all E. coli isolates were screened for ampicillin-sulbactam resistance. Of 283 isolates that tested resistant to ampicillin-sulbactam, 69 unique patient isolates were also resistant to amoxicillin-clavulanate by disk diffusion testing (zone diameter /= 32 micro g/ml). Two isolates were susceptible to amoxicillin-clavulanic acid by agar dilution, although they were resistant by disk diffusion testing. The distribution of beta-lactamases was as follows: the TEM type alone was found in 52 isolates, the AmpC type was found in 4 isolates (2 identified as containing CMY-2), the TEM type and CMY-2 were found in 2 isolates, and the OXA type was found in 1 isolate. Also, there was one isolate with the TEM type and the SHV type and one with the TEM type and a second, unidentified enzyme. Among the isolates with TEM-type enzymes, two extended-spectrum beta-lactamase-producing isolates were identified but two isolates with inhibitor-resistant TEM (IRT) enzymes (one with TEM-34 [IRT-6] and the other with a novel enzyme [tentatively assigned the designation TEM-122]) were more interesting.