Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry

ABSTRACT: Understanding protein and gene function requires identifying interaction partners usingbiochemical, molecular or genetic tools. In plants, searching for novel protein-proteininteractions is limited to protein purification assays, heterologous in vivo systems such as the yeast-two-hybrid or mutant screens. Ideally one would be able to search for novel proteinpartners in living plant cells. We demonstrate that it is possible to screen for novel proteinproteininteractions from a random library in protoplasted Arabidopsis plant cells and recoversome of the interacting partners. Our screen is based on capturing the bi-molecularcomplementation of mYFP between an YN-bait fusion partner and a completely random preyYC-cDNA library with FACS. The candidate interactions were confirmed using in plantaBiFC assays and in planta FRET-FLIM assays. From this work, we show that the wellcharacterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3,HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This isone of the first random in planta screens to be successfully employed.     

Cardiopulmonary effects of diazepam/ketamine/isoflurane or xylazine/ketamine/isoflurane in foals undergoing abdominal surgery

The purpose of this study was to evaluate the cardiopulmonary effects of anesthetic induction with diazepam/ketamine or xylazine/ketamine with subsequent maintenance of anesthesia using isoflurane in foals undergoing abdominal surgery. Seventeen foals underwent laparotomy at 7–10 days of age and a laparoscopy 7–10 days later. Foals were randomly assigned to receive xylazine (0.8 mg kg^?1)/ketamine (2 mg kg^?1) (X/K)(n = 9) or diazepam (0.2 mg kg^?1)/ketamine (2 mg kg^?1) (D/K)(n = 8) for induction of anesthesia for both procedures. In all foals, anesthesia was maintained with isoflurane in oxygen with the inspired concentration adjusted to achieve adequate depth of anesthesia as assessed by an individual blinded to the treatments. IPPV was employed throughout using a tidal volume of 10 mL kg^?1 adjusting the frequency to maintain eucapnia (PaCO₂ 35–45 mm Hg, 4.7–6.0 kPa). Cardiopulmonary variables were measured after induction of anesthesia prior to, during, and following surgery. To compare the measured cardiopulmonary variables between the two anesthetic regimes for both surgical procedures, results were analyzed using a three‐way factorial anova for repeated measures (p < 0.05). During anesthesia for laparotomy, mean CI and MAP ranged from 110 to 180 mL kg^?1 minute^?1 and 57–81 mm Hg, respectively, in the D/K foals and 98–171 mL kg^?1 minute^?1 and 50–66 mm Hg in the X/K foals. Overall, CI, HR, SAP, DAP, and MAP were significantly higher in foals in the D/K group versus the X/K group during this anesthetic period. During anesthesia for laparoscopy, mean CI and MBP ranged from 85 to 165 mL kg^?1 minute^?1 and 67–83 mm Hg, respectively, in the D/K group, and 98–171 mL kg^?1 minute^?1 and 48–67 mm Hg in the X/K group. Only HR, SAP, DAP, and MAP were significantly higher in the D/K group versus X/K group during this latter anesthetic period. There were no significant differences between groups during either surgical procedure for end‐tidal isoflurane, PaO₂, PaCO₂, or pH. In conclusion, anesthesia of foals for laparotomy and laparoscopy with diazepam/ketamine/isoflurane is associated with less hemodynamic depression than with xylazine/ketamine/isoflurane.     

Climate Change and North American Rangelands: Assessment of Mitigation and Adaptation Strategies

Recent climatic trends and climate model projections indicate that climate change will modify rangeland ecosystem functions and the services and livelihoods that they provision. Recent history has demonstrated that climatic variability has a strong influence on both ecological and social components of rangeland systems and that these systems possess substantial capacity to adapt to climatic variability. Specific objectives of this synthesis are to: 1) evaluate options to mitigate greenhouse gas emissions and future climate change; 2) survey actions that individuals, enterprises, and social organizations can use to adapt to climate change; and 3) assess options for system transformation when adaptation is no longer sufficient to contend with climate change. Mitigation for carbon sequestration does not appear economically viable, given the small and highly variable carbon dioxide fluxes of rangeland ecosystems and the high transaction costs that would be incurred. In contrast, adaptation strategies are numerous and provide a means to manage risks associated with climate change. Adaptation strategies are diverse, including altered risk perception by individuals, greater flexibility of production enterprises, and modifications to social organizations that emphasize climatic variability, rather than consistency. Many adaptations represent “no regrets” actions because their implementation can be justified without emphasis on pending climate change. Adaptations specific to livestock production systems can include flexible herd management, alternative livestock breeds or species, innovative pest management, modified enterprise structures, and geographic relocation. Social-ecological systems in which adaptation is insufficient to counter the adverse consequences of climate change might undergo transformative change to produce alternative ecosystem services, production enterprises, and livelihoods. The rangeland profession is in a pivotal position to provide leadership on this global challenge because it represents the intersection of management and scientific knowledge, includes diverse stakeholders who derive their livelihoods from rangelands, and interacts with organizations responsible for rangeland stewardship.     

Forage and breed effects on behavior and temperament of pregnant beef heifers

Background

Integration of behavioral observations with traditional selection schemes may lead to enhanced animal well-being and more profitable forage-based cattle production systems. Brahman-influenced (BR; n = 64) and Gelbvieh × Angus (GA; n = 64) heifers consumed either toxic endophyte-infected tall fescue (E+) or one of two nontoxic endophyte-infected tall fescue (NT) cultivars during two yr. Heifers were weighed at midpoint and termination of grazing. Grazing behavior (grazing, resting in the shade, lying, or standing without grazing) was recorded (n = 13 visual observations per yr in June and July) for each pasture. During yr 2, exit velocity (EV) and serum prolactin (PRL) were determined.

Results

Grazing behavior was influenced (P < 0.05) by an interaction between fescue cultivar and breed type. Gelbvieh × Angus heifers assigned to E+ pastures had the lowest percentage of animals grazing and the largest percentage of animals resting in the shade. Brahman-influenced heifers had faster EV (P < 0.001) than GA heifers (0.52 vs. 0.74 ± 0.04 s/m, respectively). Body weight (BW) was affected (P < 0.01) by an interaction of tall fescue cultivar and d, and an interaction of tall fescue cultivar and breed type. Heifers grazing NT pastures were heavier (P < 0.01) than heifers grazing E+ pastures at midpoint and termination. Gelbvieh × Angus heifers grazing NT pastures were heavier (P < 0.01) than GA and BR heifers grazing E+ and BR heifers grazing NT pastures. An interaction of forage cultivar and breed type occurred on serum PRL (P < 0.01).

Conclusion

Collectively fescue cultivar, EV, and concentrations of serum PRL were associated with grazing behavior. Heifers grazing NT pastures were observed to be grazing more than heifers assigned to E+ pastures, regardless of breed type, which may have contributed to changes in BW and average daily gain (ADG) in heifers. Integration of behavioral observations along with traditional selection schemes may lead to enhanced animal well-being and more profitable forage-based cattle production systems.     

Caribbean Plant Quarantine Officers’ Self-Perceived Competencies and Training Needs for Regional Food Security

Plant quarantine is a key strategy in international plant protection efforts. This study identified the areas for knowledge and skills improvement among 108 Caribbean plant protection personnel from 20 countries. The Borich (1980) model of needs assessment was used. Results showed that respondents perceived all areas assessed as very important to their job functions. However, they perceived their proficiencies as low to average. Areas with highest training needs, as well as lowest need areas, were identified. The results can be used to revise the curricula for the regional training of plant quarantine personnel.     

Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, Bartonella vinsonii berkhoffii, and Rickettsia spp. in dogs from Grenada

To identify the tick-borne pathogens in dogs from Grenada, we conducted a serologic survey for Ehrlichia canis in 2004 (104 dogs) and a comprehensive serologic and molecular survey for a variety of tick-borne pathogens in 2006 (73 dogs). In 2004 and 2006, 44 and 32 dogs (42.3% and 43.8%) were seropositive for E. canis, respectively. In 2006, several tick-borne pathogens were identified by serology and PCR. DNA of E. canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, and Bartonella sp. were identified in 18 (24.7%), 14 (19.2%), 5 (7%), 5 (7%), and 1 (1.4%) dogs, respectively. Six (8.2%) dogs were seropositive for Bartonella vinsonii subsp. berkhoffii. All dogs were seronegative and PCR-negative for Rickettsia spp. Coinfection with two or three pathogens was observed in eight dogs. Partial 16S rRNA E. canis and A. platys sequences were identical to sequences in GenBank. Partial 18S rRNA gene sequences from the Grenadian H. canis were identical to each other and had one possible mismatch (ambiguous base) from H. canis detected from Spain and Brazil. Grenadian B. c. vogeli sequences were identical to B. c. vogeli from Brazil and Japan. All of the detected pathogens are transmitted, or suspected to be transmitted, by Rhipicephalus sanguineus. Results of this study indicate that dogs from Grenada are infected with multiple tick-borne pathogens; therefore, tick-borne diseases should be included as differentials for dogs exhibiting thrombocytopenia, leukopenia, fever, or lethargy. One pathogen, E. canis, is also of potential public health significance.     

Sedative and cardiopulmonary effects of medetomidine hydrochloride and xylazine hydrochloride and their reversal with atipamezole hydrochloride in calves

OBJECTIVE: To assess the sedative and cardiopulmonary effects of medetomidine and xylazine and their reversal with atipamezole in calves. ANIMALS: 25 calves. PROCEDURES: A 2-phase (7-day interval) study was performed. Sedative characteristics (phase I) and cardiopulmonary effects (phase II) of medetomidine hydrochloride and xylazine hydrochloride administration followed by atipamezole hydrochloride administration were evaluated. In both phases, calves were randomly allocated to receive 1 of 4 treatments IV: medetomidine (0.03 mg/kg) followed by atipamezole (0.1 mg/kg; n = 6), xylazine (0.3 mg/kg) followed by atipamezole (0.04 mg/kg; 7), medetomidine (0.03 mg/kg) followed by saline (0.9% NaCl; 6) solution (10 mL), and xylazine (0.3 mg/kg) followed by saline solution (10 mL; 6). Atipamezole or saline solution was administered 20 minutes after the first injection. Cardiopulmonary variables were recorded at intervals for 35 minutes after medetomidine or xylazine administration. RESULTS: At the doses evaluated, xylazine and medetomidine induced a similar degree of sedation in calves; however, the duration of medetomidine-associated sedation was longer. Compared with pretreatment values, heart rate, cardiac index, and PaO(2) decreased, whereas central venous pressure, PaCO(2), and pulmonary artery pressures increased with medetomidine or xylazine. Systemic arterial blood pressures and vascular resistance increased with medetomidine and decreased with xylazine. Atipamezole reversed the sedative and most of the cardiopulmonary effects of both drugs. CONCLUSIONS AND CLINICAL RELEVANCE: At these doses, xylazine and medetomidine induced similar degrees of sedation and cardiopulmonary depression in calves, although medetomidine administration resulted in increases in systemic arterial blood pressures. Atipamezole effectively reversed medetomidine- and xylazine-associated sedative and cardiopulmonary effects in calves.