Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta.

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression. The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified. Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

Comparative analysis of the genetic variability within the Q-type C2H2 zinc-finger transcription factors in the economically important cabbage, canola and Chinese cabbage genomes.

BACKGROUND: Brassica oleracea, B. rapa and B. napus encompass many economically important vegetable and oil crops; such as cabbage, broccoli, canola and Chinese cabbage. The genome sequencing of these species allows for gene discovery with an eye towards discerning the natural variability available for future breeding. The Q-type C2H2 zinc-finger protein (ZFP) transcription factors contain zinc finger motifs with a conserved QALGGH as part of the motif and they may play a critical role in the plants response to stress. While they may contain from one to five ZF domains (ZFD) this work focuses on the ZFPs that contain two zinc-fingers, which bind to the promoter of genes, and negatively regulate transcription via the EAR motif. B. oleracea and rapa are diploid and evolved into distinct species about 3.7 million years ago. B. napus is polyploid and formed by fusion of the diploids about 7500 years ago. RESULTS: This work identifies a total of 146 Q-type C2H2-ZFPs with 37 in B. oleracea, 35 in B. rapa and 74 in B. napus. The level of sequence similarity and arrangement of these genes on their chromosomes have mostly remained intact in B. napus, when compared to the chromosomes inherited from either B. rapa or oleracea. In contrast, the difference between the protein sequences of the orthologs of B. rapa and oleracea is greater and their organization on the chromosomes is much more divergent. In general, the 146 proteins are highly conserved especially within the known motifs. Differences within subgroups of ZFPs were identified. Considering that B. napus has twice the number of these proteins in its genome, RNA-Seq data was mined and the expression of 68 of the 74 genes was confirmed. CONCLUSION: Alignment of these proteins gives a snapshot of the variability that may be available naturally in Brassica species. The aim is to study how different ZFPs bind different genes or how dissimilar EAR motifs alter the negative regulation of the genes bound to the ZFP. Results from such studies could be used to enhance tolerance in future Brassica breeding programs.

The remarkable plethora of infestation-responsive Q-type C2H2 transcription factors in potato.

OBJECTIVE: Q-type C2H2 transcription factors (TF) play crucial roles in the plant response to stress, often leading to regulation of downstream genes required for tolerance to these challenges. An infestation-responsive Q-type C2H2 TF (StZFP2) is induced by wounding and infestation in potato. While mining the Solanum tuberosum Group Phureja genome for additional members of this family of proteins, five StZFP2-like genes were found on a portion of chromosome 11. The objective of this work was to differentiate these genes in tissue specificity and expression upon infestation. RESULTS: Examination of different tissues showed that young roots had the highest amounts of transcripts for five of the genes. Expression of their transcripts upon excision or infestation by Manduca sexta, showed that all six genes were induced. Overall, each gene showed variations in its response to infestation and specificity for tissue expression. The six genes encode very similar proteins but most likely play unique roles in the plant response to infestation. In contrast, only two homologs have been identified in Arabidopsis and tomato. Overexpression of similar genes has led to enhanced tolerance to, for example, salinity, drought and pathogen stress. Discovery of these new StZFP2 homologs could provide additional resources for potato breeders.

A first-in-human phase I study of MORAb-004, a monoclonal antibody to endosialin in patients with advanced solid tumors.

PURPOSE: Endosialin (TEM-1, CD248) is a protein expressed on the surface of activated mesenchymal cells, including certain subsets of tumors. Preclinical models suppressing endosialin function have shown antitumor activity. A humanized monoclonal antibody, MORAb-004, was engineered to target endosialin and is the first agent in clinical development for this mesenchymal cell target. EXPERIMENTAL DESIGN: This first-in-human, open-label, phase I study recruited patients with treatment-refractory solid tumors. MORAb-004 was administered intravenously once weekly in 4-week cycles. Objectives included determination of the safety of multiple infusions of MORAb-004, identification of the maximum tolerated dose (MTD), pharmacokinetic modeling, detection of any anti-human antibody response, and assessment of objective radiographic response to therapy. RESULTS: Thirty-six patients were treated at 10 dose levels of MORAb-004, ranging from 0.0625 to 16 mg/kg. Drug-related adverse events were primarily grade 1-2 infusion toxicities. Dose-limiting toxicity of grade 3 vomiting was observed at 16 mg/kg. Eighteen of 32 evaluable patients across all doses achieved disease stability, with minor radiographic responses observed in 4 patients (pancreatic neuroendocrine, hepatocellular, and sarcoma tumor types). Pharmacokinetics showed MORAb-004 accumulation beginning at 4 mg/kg and saturable elimination beginning at 0.25 mg/kg. Exposure increased in a greater-than-dose-proportional manner with terminal half-life increasing proportionally with dose. The MTD was identified as 12 mg/kg. CONCLUSIONS: Preliminary antitumor activity was observed. Safety profile, pharmacokinetics, and early antitumor activity suggest that MORAb-004 is safe at doses up to 12 mg/kg and should be studied further for efficacy.

Herbivory responsive C2H2 zinc finger transcription factor protein StZFP2 from potato.

While C2H2 zinc finger transcription factors (TF) are often regulated by abiotic stress, their role during insect infestation has been overlooked. This study demonstrates that the transcripts of the zinc finger transcription factors StZFP1 and StZFP2 are induced in potato (Solanum tuberosum L.) upon infestation by either the generalist tobacco hornworm (THW, Manduca sexta L.) or the specialist Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). StZFP1 has been previously characterized as conferring salt tolerance to transgenic tobacco and its transcript is induced by Phytophthora infestans and several abiotic stresses. StZFP2 has not been characterized previously, but contains the hallmarks of a C2H2 zinc finger TF, with two conserved zinc finger domains and DLN motif, which encodes a transcriptional repressor domain. Expression studies demonstrate that StZFP2 transcript is also induced by tobacco hornworm and Colorado potato beetle. These observations expand the role of the C2H2 transcription factor in potato to include the response to chewing insect pests.

A soluble form of P74 can act as a per os infectivity factor to the Autographa californica multiple nucleopolyhedrovirus.

The baculovirus occlusion-derived virion (ODV) is required to spread virus infection among insect hosts via the per os route. The Autographa californica multicapsid nucleopolyhedrovirus P74 protein is an ODV envelope protein that is essential for ODVs to be infectious. P74 is anchored in the ODV envelope by a C-terminal transmembrane anchor domain and is N-terminally exposed on the ODV surface. In the present study, a series of N-terminal and C-terminal truncation mutants of P74 were evaluated for their ability to rescue per os infectivity of the P74-null virus, AcLP4. It was discovered that a P74 truncation mutant lacking the C-terminal transmembrane anchor domain of P74 was able to rescue per os infection. This result shows that a soluble form of P74 retains per os infectivity factor function and suggests that P74 may be complexed with other proteins in the ODV envelope.

Trypsin cleavage of the baculovirus occlusion-derived virus attachment protein P74 is prerequisite in per os infection.

Baculovirus occlusion-derived virions (ODVs) contain a number of infectivity factors essential for the initiation of infection in larval midgut cells. Deletion of any of these factors neutralizes infectivity by the per os route. We have observed that P74 of the group I alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is N-terminally cleaved when a soluble form of the protein was incubated with insect midgut tissues under alkaline conditions and that cleavage was prevented by soybean trypsin inhibitor (SBTI). Presently, biological assays were carried out that suggest SBTI inhibits and trypsin enhances baculovirus per os infectivity. We developed a method to rescue per os infectivity of a P74 null virus involving co-transfection of viral DNA with a plasmid that transiently expresses p74. We used this plasmid rescue method to functionally characterize P74. A series of site-directed mutants were generated at the N terminus to evaluate if trypsin cleavage sites were necessary for function. Mutagenesis of R195, R196 and R199 compromised per os infectivity and rendered P74 resistant to midgut trypsin.

Adolescent vs. adult-onset nicotine self-administration in male rats: duration of effect and differential nicotinic receptor correlates.

Adolescence is the life stage when tobacco addiction typically begins. Adolescent neurobehavioral development may be altered by nicotine self-administration in a way that persistently potentiates addiction. Previously, we showed that female adolescent rats self-administer more nicotine than do adults and that the increased nicotine intake then persists through the transition to adulthood [E.D. Levin, A. Rezvani, D. Montoya, J. Rose, H. Swartzwelder, Adolescent-onset nicotine self-administration modeled in female rats, Psychopharmacology 169 (2003) 141-149.]. In the current study, male Sprague-Dawley rats were given access to nicotine via the standard operant IV self-administration procedure (nicotine bitartrate dose of 0.03 mg/kg/infusion). One group of male rats started during adolescence the other group started in young adulthood. After the end of the four-week period of self-administration brain regions of the rats were assessed for alpha4beta2 nicotinic receptor binding. We found that male rats, like females, show higher nicotine self-administration when starting during adolescence as compared to starting in adulthood (p<0.001). Indeed, the effect in adolescent males was even greater than that in females, with more than triple the rate of nicotine self-administration vs. the adult-onset group during the first 2 weeks. The adolescent onset nicotine-self-administering rats also had significantly greater high affinity nicotinic receptor binding in the midbrain and the striatum, whereas hippocampal binding did not differ between the age groups. Striatal values significantly correlated with nicotine self-administration during the first 2 weeks in the adult-onset group but not the adolescent-onset rats, suggesting that the differences in self-administration may depend in part on underlying disparities in synaptic responses to nicotine. After the initial 2 weeks, nicotine self-administration in male rats declined toward adult-like levels, as the adolescent rats approached adulthood. This study showed that adolescent male rats self-administer significantly more nicotine than do male adult rats, but that adolescent-onset nicotine self-administration in male rats declines over weeks of continued use to approach adult-onset levels. In a previous study, we found that female rats also show greater nicotine self-administration with adolescent onset vs. adult onset, but that the females continued higher rates of self-administration into adulthood. Our results thus reinforce the concept that the adolescent brain is unusually receptive to the effects of nicotine in a manner that reinforces the potential for addiction.

Genetic modification of cassava for enhanced starch production.

To date, transgenic approaches to biofortify subsistence crops have been rather limited. This is particularly true for the starchy root crop cassava (Manihot esculenta Crantz). Cassava has one of the highest rates of CO(2) fixation and sucrose synthesis for any C3 plant, but rarely reaches its yield potentials in the field. It was our hypothesis that starch production in cassava tuberous roots could be increased substantially by increasing the sink strength for carbohydrate. To test this hypothesis, we generated transgenic plants with enhanced tuberous root ADP-glucose pyrophosphorylase (AGPase) activity. This was achieved by expressing a modified form of the bacterial glgC gene under the control of a Class I patatin promoter. AGPase catalyses the rate-limiting step in starch biosynthesis, and therefore the expression of a more active bacterial form of the enzyme was expected to lead to increased starch production. To facilitate maximal AGPase activity, we modified the Escherichia coli glgC gene (encoding AGPase) by site-directed mutagenesis (G336D) to reduce allosteric feedback regulation by fructose-1,6-bisphosphate. Transgenic plants (three) expressing the glgC gene had up to 70% higher AGPase activity than control plants when assayed under conditions optimal for plant and not bacterial AGPase activity. Plants having the highest AGPase activities had up to a 2.6-fold increase in total tuberous root biomass when grown under glasshouse conditions. In addition, plants with the highest tuberous root AGPase activity had significant increases in above-ground biomass, consistent with a possible reduction in feedback inhibition on photosynthetic carbon fixation. These results demonstrate that targeted modification of enzymes regulating source-sink relationships in crop plants having high carbohydrate source strengths is an effective strategy for increasing carbohydrate yields in sink tissues.

Increased nicotine self-administration following prenatal exposure in female rats.

There is a significant association between maternal cigarette smoking during pregnancy and greater subsequent risk of smoking in female offspring. In animal models, prenatal nicotine exposure causes persistent alterations in cholinergic and monoaminergic systems, both of which are important for nicotine actions underlying tobacco addiction. Accordingly, the current study was conducted to determine if there is a cause-and-effect relationship between prenatal nicotine exposure and nicotine self-administration starting in adolescence. Pregnant rats were administered nicotine (6 mg/kg/day) by osmotic minipump infusion throughout gestation and then, beginning in adolescence and continuing into adulthood, female offspring were given access to nicotine via a standard operant IV self-administration procedure (0.03 mg/kg/infusion). Gestational nicotine exposure did not alter the initial rate of nicotine self-administration. However, when animals underwent one week of forced abstinence and then had a second opportunity to self-administer nicotine, the prenatally-exposed animals showed a significantly greater rate of self-administration than did the controls. Prenatal nicotine exposure causes increased nicotine self-administration, which is revealed only when the animals are allowed to experience a period of nicotine abstinence. This supports a cause-and-effect relationship between the higher rates of smoking in the daughters of women who smoke cigarettes during pregnancy and implicates a role for nicotine in this effect. Our results further characterize the long-term liabilities of maternal smoking but also point to the potential liabilities of nicotine-based treatments for smoking cessation during pregnancy.

Influence of social environment in smoking among adolescents in Turkey.

BACKGROUND: The aim of this study was to examine the social determinants of smoking among adolescents attending school and/or work. METHODS: A survey was carried out on 6012 adolescents aged between 13 and 17 years in 15 cities, recruited from schools, vocational training centres and work places. A self-completed questionnaire was used for data collection. Single- and multi-level regression analyses were run to estimate models. RESULTS: Ever smoking and current smoking rates were 41.1% and 10.5% among girls, and 57.5% and 25.2% among boys. These rates were 47.0% and 13.3% among those who only attended school, 62.2% and 31.7% among those who attended school and worked simultaneously, and 67.5% and 43.0% among those who worked and did not attend school. In multi-level analysis, the major predictors of current smoking were close friends smoking [odds ratio (OR) 3.48; 95% confidence interval (CI) 1.93-6.27], no knowledge of harmful effects of short-term smoking (OR 2.15; 95% CI 1.74-2.67), vulnerability to peer pressure (OR 1.90; 95% CI 1.48-2.46), negative self-perception (OR 1.69; 95% CI 1.31-2.18) and male sex (OR 1.68; 95% CI 1.30-2.16). Mothers higher education was a predictor for girls' smoking, while mother's lower education was a predictor for boys' smoking. At the school level, smoking prevalence was a predictor of current smoking (OR 1.07; 95% CI 1.05-1.08). CONCLUSIONS: Smoking patterns were similar to Western countries in several aspects, while male prevalence rates were higher and the impact of gender-related predictors was significant. Our findings suggest that youth smoking prevention policies should address personal, familial and educational environmental level requirements, taking into consideration the gender differences in addition to international guidelines.

Evidence for proteolytic cleavage of the baculovirus occlusion-derived virion envelope protein P74.

Baculovirus occlusion-derived virions (ODVs) are released from occlusion bodies by the alkaline environment of the insect midgut. The ODV envelope protein P74 is required for oral infectivity. A soluble form of the Autographa californica multiple nucleopolyhedrovirus P74 protein, P74sol, was engineered as part of a chimeric protein with jellyfish green fluorescent protein (GFP). P74sol-GFP was overproduced by the baculovirus expression system and purified away from the wild-type P74. Brush border membrane vesicles (BBMVs) were prepared from the midguts of third-instar Helicoverpa zea larvae. When P74sol-GFP was incubated under alkaline conditions with BBMVs, a P74sol-GFP product with a smaller molecular mass was produced. Immunoblots indicated that the smaller product was generated by N-terminal cleavage of P74. This cleavage was prevented by soybean trypsin inhibitor. Analysis of the peptide sequences of P74 homologues identified a conserved trypsin cleavage site that could generate the observed P74sol-GFP BBMV-specific cleavage product.

John M. Eisenberg Patient Safety Awards. The LVHHN patient safety video: patients as partners in safe care delivery.

BACKGROUND: In fall 2002, Lehigh Valley Hospital and Health Network (LVHHN), an 800-bed, three-site academic community hospital, embarked on an initiative to produce an educational patient safety video. IMPLEMENTING THE INITIATIVE: The video addresses six topics relevant to optimum patient safety: treatment plan, medication safety, falls, surgical site identification, hand washing, and discharge planning. Each segment outlines strategies that patients may employ or observations they should make to improve patient safety. RESULTS: Analysis of the patient survey data, which were based on 217 surveys, indicated that patients felt more comfortable talking with their health care workers about questions or concerns after viewing the video and that they rated their knowledge of patient safety higher. Patients generally rated the six sections as helpful. DISCUSSION: The video was intended to become an important step in the preadmission process. Releasing the video to patients and staff helped to normalize some practices that initially were not comfortable for staff (repeatedly asking an inpatient for his or her name and date of birth before administering all medications) or patients (inquiring whether a staff member has washed his or her hands). Additional methods were in development to share the video with current and prospective patients and assess its impact. The LVHHN patient safety council plans to share the video with the community at large.

First do no harm: integrating patient safety and quality improvement.

BACKGROUND: Lehigh Valley Hospital's (LVH's; Allentown, Penn) interdisciplinary quality improvement program Primum Non Nocere (PNN), or First Do No Harm, is composed of 12 quality improvement (QI) projects that are a combination of ongoing operations improvement projects and new projects in patient safety. The projects stress delivery of cost-effective medical care while reducing preventable adverse events through improved communication, process redesign, and evidence-based protocol use. EXAMPLE: WRONG-SITE SURGERY: In response to an initial alert warning in 1998, LVH developed a policy of marking "yes" on the surgical site and "no" on the other side. However, several near misses occurred, and a root cause analysis indicated that the policy was not always followed for some very specific reasons. For example, the operative record included no prompt to address laterality, and the procedures in which laterality should be addressed were never specified. Interventions to address these issues were quickly developed that were in keeping with the recommendations outlined in a second alert warning on the issue in December 2001. A year after these stepwise changes, compliance with the policy is almost 100%, and there have been no further near misses. DISCUSSION: Specific project barriers included the initial challenge of changing the mindset in the institution from gradual change on a grand scale to smaller, more rapid changes, analyses, and actions. Another issue identified early in the initiative was the tendency of project groups to outline elaborate process improvements without determining how to measure and monitor success. A project sustainability is inherently linked to its initial strengths and the successful solutions to barriers that are encountered.

A study of the Autographa californica multiple nucleopolyhedrovirus ODV envelope protein p74 using a GFP tag.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) protein p74 is associated with the occlusion-derived virus (ODV) envelope. p74 is essential for oral infectivity of ODV and has been proposed to play a role in midgut attachment and/or fusion. In this study, p74 protein was expressed in-frame with green fluorescent protein (GFP) to create a p74-GFP chimera. The C-terminal GFP portion of the chimera facilitated visualization of the trafficking of p74 in baculovirus-infected Spodoptera frugiperda (Sf-9) cells. p74-GFP chimeric proteins localized in the intranuclear ring zone of the nucleus and were found to co-precipitate with the microvesicle fraction of cell lysates. A series of truncations of p74 was expressed as p74-GFP chimeras in recombinant baculoviruses. When C-terminal region S580-F645 was deleted from p74, p74-GFP chimera localization became non-specific and chimeras became soluble. p74 region S580-F645 directed GFP to the intranuclear ring zone in a similar pattern to full-length p74. The hydrophobic C terminus of p74 plays a role in protein localization and possibly in transmembrane anchoring and insertion.