A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize.

Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots.

Foxtail mosaic virus: A tool for gene function analysis in maize and other monocots.

Many plant viruses have been engineered into vectors for use in functional genomics studies, expression of heterologous proteins, and, most recently, gene editing applications. The use of viral vectors overcomes bottlenecks associated with mutagenesis and transgenesis approaches often implemented for analysis of gene function. There are several engineered viruses that are demonstrated or suggested to be useful in maize through proof-of-concept studies. However, foxtail mosaic virus (FoMV), which has a relatively broad host range, is emerging as a particularly useful virus for gene function studies in maize and other monocot crop or weed species. A few clones of FoMV have been independently engineered, and they have different features and capabilities for virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX) of proteins. In addition, FoMV can be used to deliver functional guide RNAs in maize and other plants expressing the Cas9 protein, demonstrating its potential utility in virus-induced gene editing applications. There is a growing number of studies in which FoMV vectors are being applied for VIGS or VOX in maize and the vast majority of these are related to maize-microbe interactions. In this review, we highlight the biology and engineering of FoMV as well as its applications in maize-microbe interactions and more broadly in the context of the monocot functional genomics toolbox.

Characterization of a foxtail mosaic virus vector for gene silencing and analysis of innate immune responses in Sorghum bicolor.

Sorghum is vulnerable to many biotic and abiotic stresses, which cause considerable yield losses globally. Efforts to genetically characterize beneficial sorghum traits, including disease resistance, plant architecture, and tolerance to abiotic stresses, are ongoing. One challenge faced by sorghum researchers is its recalcitrance to transformation, which has slowed gene validation efforts and utilization for cultivar development. Here, we characterize the use of a foxtail mosaic virus (FoMV) vector for virus-induced gene silencing (VIGS) by targeting two previously tested marker genes: phytoene desaturase (PDS) and ubiquitin (Ub). We additionally demonstrate VIGS of a subgroup of receptor-like cytoplasmic kinases (RLCKs) and report the role of these genes as positive regulators of early defence signalling. Silencing of subgroup 8 RLCKs also resulted in higher susceptibility to the bacterial pathogens Pseudomonas syringae pv. syringae (B728a) and Xanthomonas vasicola pv. holcicola, demonstrating the role of these genes in host defence against bacterial pathogens. Together, this work highlights the utility of FoMV-induced gene silencing in the characterization of genes mediating defence responses in sorghum. Moreover, FoMV was able to systemically infect six diverse sorghum genotypes with high efficiency at optimal temperatures for sorghum growth and therefore could be extrapolated to study additional traits of economic importance.

Assessing surgeon and novice force skill on a haptic stiffness simulator for laparoscopic surgery.

Though several simulators and training methods are available for basic laparoscopic skills, few have addressed force-based skills. In this work, we discuss a haptic simulator that renders virtual materials of different stiffness profiles to be used for haptic skills differentiation. A force-based task was designed on the simulator and the performance of surgeons and novices was analyzed. Results indicate that surgeons and novices differ in their ability to use the haptic device to reproduce target stiffness levels. This work provides an important step towards quantifying haptic skill metrics for the design of surgical skills training simulators.

Haptic tasks for physical laparoscopic ("Box") trainers to differentiate surgeon skill.

In this work, we present four tasks, primarily testing haptic laparoscopic skill that can be simulated in a conventional box trainer. Results from examining expert surgeon and novice performance is presented as evidence that these tasks can be used for training haptic skills for laparoscopy in a box trainer.

Impacts of RNA Mobility Signals on Virus Induced Somatic and Germline Gene Editing.

Viral vectors are being engineered to deliver CRISPR/Cas9 components systemically in plants to induce somatic or heritable site-specific mutations. It is hypothesized that RNA mobility signals facilitate entry of viruses or single guide RNAs (sgRNAs) into the shoot apical meristem where germline mutations can occur. Our objective was to understand the impact of RNA mobility signals on virus-induced somatic and germline gene editing in Nicotiana benthamiana and Zea mays. Previously, we showed that foxtail mosaic virus (FoMV) expressing sgRNA induced somatic mutations in N. benthamiana and Z. mays expressing Cas9. Here, we fused RNA mobility signals to sgRNAs targeting the genes encoding either N. benthamiana phytoene desaturase (PDS) or Z. mays high affinity potassium transporter 1 (HKT1). Addition of Arabidopsis thaliana Flowering Locus T (AtFT) and A. thaliana tRNA-Isoleucine (AttRNAIle) did not improve FoMV-induced somatic editing, and neither were sufficient to facilitate germline mutations in N. benthamiana. Maize FT homologs, Centroradialus 16 (ZCN16) and ZCN19, as well as AttRNAIle were found to aid somatic editing in maize but did not enable sgRNAs delivered by FoMV to induce germline mutations. Additional viral guide RNA delivery systems were assessed for somatic and germline mutations in N. benthamiana with the intention of gaining a better understanding of the specificity of mobile signal-facilitated germline editing. Potato virus X (PVX), barley stripe mosaic virus (BSMV), and tobacco rattle virus (TRV) were included in this comparative study, and all three of these viruses delivering sgRNA were able to induce somatic and germline mutations. Unexpectedly, PVX, a potexvirus closely related to FoMV, expressing sgRNA alone induced biallelic edited progeny, indicating that mobility signals are dispensable in virus-induced germline editing. These results show that PVX, BSMV, and TRV expressing sgRNA all have an innate ability to induce mutations in the germline. Our results indicate that mobility signals alone may not be sufficient to enable virus-based delivery of sgRNAs using the viruses, FoMV, PVX, BSMV, and TRV into cell types that result in germline mutations.

The Performance and Trajectory of Medical Students With Disabilities: Results From a Multisite, Multicohort Study.

PURPOSE: To conduct a post-Americans with Disabilities Act Amendments Act of 2008 multisite, multicohort study called the Pathways Project to assess the performance and trajectory of medical students with disabilities (SWDs). METHOD: From June to December 2020, the authors conducted a matched cohort study of SWDs and nondisabled controls from 2 graduating cohorts (2018 and 2019) across 11 U.S. MD-granting medical schools. Each SWD was matched with 2 controls, one from their institution and, whenever possible, one from their cohort for Medical College Admission Test score and self-reported gender. Outcome measures included final attempt Step 1 and Step 2 Clinical Knowledge scores, time to graduation, leave of absence, matching on first attempt, and matching to primary care. RESULTS: A total of 171 SWDs and 341 controls were included; the majority of SWDs had cognitive/learning disabilities (118/171, 69.0%). Compared with controls, SWDs with physical/sensory disabilities had similar times to graduation (88.6%, 95% confidence interval [CI]: 77.0, 100.0 vs 95.1%, 95% CI: 90.3, 99.8; P = .20), Step 1 scores (229.6 vs 233.4; P = .118), and match on first attempt (93.9%, 95% CI: 86.9, 100.0 vs 94.6%, 95% CI: 91.8, 97.4; P = .842), while SWDs with cognitive/learning disabilities had lower Step 1 scores (219.4; P < .001) and were less likely to graduate on time (81.2%, 95% CI: 69.2, 93.2; P = .003) and match on first attempt (85.3%, 95% CI: 78.0, 92.7; P = .009). Accommodated SWDs had Step 1 scores that were 5.9 points higher than nonaccommodated SWDs (95% CI: -0.7, 12.5; P = .08). CONCLUSIONS: Structural barriers remain for SWDs with cognitive/learning disabilities, which could be partially mitigated by accommodations on high-stakes exams.

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones.

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22) or phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.

Protein expression and gene editing in monocots using foxtail mosaic virus vectors.

Plant viruses can be engineered to carry sequences that direct silencing of target host genes, expression of heterologous proteins, or editing of host genes. A set of foxtail mosaic virus (FoMV) vectors was developed that can be used for transient gene expression and single guide RNA delivery for Cas9-mediated gene editing in maize, Setaria viridis, and Nicotiana benthamiana. This was accomplished by duplicating the FoMV capsid protein subgenomic promoter, abolishing the unnecessary open reading frame 5A, and inserting a cloning site containing unique restriction endonuclease cleavage sites immediately after the duplicated promoter. The modified FoMV vectors transiently expressed green fluorescent protein (GFP) and bialaphos resistance (BAR) protein in leaves of systemically infected maize seedlings. GFP was detected in epidermal and mesophyll cells by epifluorescence microscopy, and expression was confirmed by Western blot analyses. Plants infected with FoMV carrying the bar gene were temporarily protected from a glufosinate herbicide, and expression was confirmed using a rapid antibody-based BAR strip test. Expression of these proteins was stabilized by nucleotide substitutions in the sequence of the duplicated promoter region. Single guide RNAs expressed from the duplicated promoter mediated edits in the N. benthamiana Phytoene desaturase gene, the S. viridis Carbonic anhydrase 2 gene, and the maize HKT1 gene encoding a potassium transporter. The efficiency of editing was enhanced in the presence of synergistic viruses and a viral silencing suppressor. This work expands the utility of FoMV for virus-induced gene silencing (VIGS), virus-mediated overexpression (VOX), and virus-enabled gene editing (VEdGE) in monocots.

Learning to perceive haptic distance-to-break in the presence of friction.

Two experiments employed attunement and calibration training to investigate whether observers are able to identify material break points in compliant materials through haptic force application. The task required participants to attune to a recently identified haptic invariant, distance-to-break (DTB), rather than haptic stimulation not related to the invariant, including friction. In the first experiment participants probed simulated force-displacement relationships (materials) under 3 levels of friction with the aim of pushing as far as possible into the materials without breaking them. In a second experiment a different set of participants pulled on the materials. Results revealed that participants are sensitive to DTB for both pushing and pulling, even in the presence of varying levels of friction, and this sensitivity can be improved through training. The results suggest that the simultaneous presence of friction may assist participants in perceiving DTB. Potential applications include the development of haptic training programs for minimally invasive (laparoscopic) surgery to reduce accidental tissue damage. (PsycINFO Database Record

Control of Western Corn Rootworm (Diabrotica virgifera virgifera) Reproduction through Plant-Mediated RNA Interference.

RNA interference (RNAi) in transgenic maize has recently emerged as an alternative mode of action for western corn rootworm (Diabrotica virgifera virgifera) control which can be combined with protein-based rootworm control options for improved root protection and resistance management. Currently, transgenic RNAi-based control has focused on suppression of genes that when silenced lead to larval mortality. We investigated control of western corn rootworm reproduction through RNAi by targeting two reproductive genes, dvvgr and dvbol, with the goal of reducing insect fecundity as a new tool for pest management. The results demonstrated that exposure of adult beetles, as well as larvae to dvvgr or dvbol dsRNA in artificial diet, caused reduction of fecundity. Furthermore, western corn rootworm beetles that emerged from larval feeding on transgenic maize roots expressing dvbol dsRNA also showed significant fecundity reduction. This is the first report of reduction of insect reproductive fitness through plant-mediated RNAi, demonstrating the feasibility of reproductive RNAi as a management tool for western corn rootworm.

Discovery of midgut genes for the RNA interference control of corn rootworm.

RNA interference (RNAi) is a promising new technology for corn rootworm control. This paper presents the discovery of new gene targets - dvssj1 and dvssj2, in western corn rootworm (WCR). Dvssj1 and dvssj2 are orthologs of the Drosophila genes snakeskin (ssk) and mesh, respectively. These genes encode membrane proteins associated with smooth septate junctions (SSJ) which are required for intestinal barrier function. Based on bioinformatics analysis, dvssj1 appears to be an arthropod-specific gene. Diet based insect feeding assays using double-stranded RNA (dsRNA) targeting dvssj1 and dvssj2 demonstrate targeted mRNA suppression, larval growth inhibition, and mortality. In RNAi treated WCR, injury to the midgut was manifested by "blebbing" of the midgut epithelium into the gut lumen. Ultrastructural examination of midgut epithelial cells revealed apoptosis and regenerative activities. Transgenic plants expressing dsRNA targeting dvssj1 show insecticidal activity and significant plant protection from WCR damage. The data indicate that dvssj1 and dvssj2 are effective gene targets for the control of WCR using RNAi technology, by apparent suppression of production of their respective smooth septate junction membrane proteins located within the intestinal lining, leading to growth inhibition and mortality.

Pharmacokinetic interaction between intravenous phenytoin and amiodarone in healthy volunteers.

To determine the mechanism of the amiodarone-phenytoin interaction, seven healthy male subjects were given intravenous phenytoin, 5 mg/kg, before (phase I) and after (phase II) 3 weeks of oral amiodarone, 200 mg/day. Serum AUC increased from 245 +/- 37.6 to 342 +/- 87.3 mg.hr/L (p = 0.007); area under the first moment curve increased from 5666 +/- 1003 to 11,632 +/- 4198 mg.hr2/L (p = 0.008); the time-averaged total body clearance decreased from 1.57 +/- 0.3 to 1.17 +/- 0.33 L/hr (p = 0.0004); and the apparent elimination half-life increased from 16.1 +/- 1.32 to 22.6 +/- 3.8 hours (p = 0.001) for phenytoin during phase II. The volume of distribution at steady state and the unbound fraction for phenytoin remained unchanged. However, the formation of p-hydroxyphenytoin as a function of serum phenytoin concentration decreased during phase II. These findings suggest that amiodarone inhibits phenytoin metabolism. These observations also suggest that phenytoin doses will need to be reduced when coadministered with amiodarone. The magnitude of this reduction is difficult to predict because of the saturable pharmacokinetics of phenytoin, and therapeutic monitoring is recommended if amiodarone is added to the phenytoin regimen.

Steady-state interaction between amiodarone and phenytoin in normal subjects.

Amiodarone has been reported to increase phenytoin levels. This study was designed to evaluate the pharmacokinetic basis of this interaction at steady-state. Pharmacokinetic parameters for phenytoin were determined after 14 days of oral phenytoin, 2 to 4 mg/kg/day, before and after oral amiodarone, 200 mg daily for 6 weeks in 7 healthy male subjects. During amiodarone therapy, area under the serum concentration time curve for phenytoin was increased from 208 +/- 82.8 (mean +/- standard deviation) to 292 +/- 108 mg.hr/liter (p = 0.015). Both the maximum and 24-hour phenytoin concentrations were increased from 10.75 +/- 3.75 and 6.67 +/- 3.51 micrograms/ml to 14.26 +/- 3.97 (p = 0.016) and 10.27 +/- 4.67 micrograms/ml (p = 0.012), respectively, during concomitant amiodarone treatment. Amiodarone caused a decrease in the oral clearance of phenytoin from 1.29 +/- 0.30 to 0.93 +/- 0.25 liters/hr (p = 0.002). These results were due to a reduction in phenytoin metabolism by amiodarone as evidenced by a decrease in the urinary excretion of the principal metabolite of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, 149 +/- 39.7 to 99.3 +/- 40.0 mg (p = 0.041) and no change in the unbound fraction of the total phenytoin concentration expressed as a percentage, 10.3 +/- 2.7 versus 10.7 +/- 2.1% (p = 0.28) during coadministration of amiodarone. The alterations in phenytoin pharmacokinetics suggest that steady-state doses of phenytoin of 2 to 4 mg/kg/day should be reduced at least 25% when amiodarone is concurrently administered. All dosage reductions should be guided by clinical and therapeutic drug monitoring.

Network structure and hospital financial performance in New York State: 1991-1995.

As networks have proliferated, questions have arisen regarding which structure is optimal. To obtain an answer from the hospital perspective, the authors conducted a survey of New York State hospitals to determine how network integration, complexity, and financial risk sharing relate to measures of financial performance during the period of 1991-1995. Of the 64 hospitals indicating a network affiliation by 1995, 67.2 percent listed some network risk-sharing activity. The least integrated networks were associated with the smallest improvements in throughput, and the most complex were associated with the largest negative changes in operating margins. During the first 2 years of network membership, hospitals joining risk-sharing networks experienced operating margin gains averaging 12 percentage points higher than hospitals joining networks without risk sharing; however, this difference dissipated in later years. Networks with higher levels of integration, lower levels of complexity, and which involve some risk-sharing between affiliates are most likely to experience improved hospital financial performance during the network's initial years.

Resources, the family and voluntary euthanasia.

Ethnological studies show that the care which societies are able to provide for their old people depends largely on available resources. However, the concept of resource depends on contemporary requirements and expectations. Modern families still try to look after their old people, but increasing longevity is making this more difficult. There is a finite ability of populations, however wealthy, to support dependent members. Resources provided to look after old people must necessarily be subtracted from those available for the other, still more important dependent group, the children, with potentially disastrous results in underfunding of social support and education. The sociobiological theory of inclusive fitness emphasizes the importance of the ways in which family members interact to help each other and try to ensure their genetic survival, even if this involves sacrificing their own interests and occasionally, their lives. Many old people do not wish for further longevity after they have become too disabled to be of service to their families, and would prefer to see limited resources being used for the young. In the USA, loss of autonomy of patients and their families owing to the practice of defensive medicine has resulted in the development of the 'living will', a legal document in which people can specify in advance what treatment they wish to accept in the event of life threatening illness. It is to be hoped that improved understanding of family relationships will make this generally unnecessary in the future and that, unless specified to the contrary, families will be allowed to decide about treatment for members who are unable to decide for themselves.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary and biliary metabolic patterns of chlorothalonil in germ-free and conventional rats.

The metabolic fate of chlorothalonil, a broad spectrum fungicide that is known to be metabolized via glutathione conjugation, was examined through the analysis of urine and bile metabolites. The role of digestive microflora in the metabolism of chlorothalonil was assessed by comparing the metabolic patterns in germ-free and conventional rats. Low urinary and biliary excretion of radioactivity was observed in both conventional and germ-free rats. However, the urinary excretion of radioactivity was higher in conventional than in germ-free rats. Radio-HPLC analysis of urine and bile showed a complex metabolic profile in both conventional and germ-free rats. Methylthio metabolites of chlorothalonil were determined in ethyl acetate extracts of urine and bile of conventional and germ-free rats. These metabolites were excreted in a higher amount in the urine of conventional rats than in the urine of germ-free rats. This study shows the complexity of chlorothalonil metabolism and the role of the digestive microflora in chlorothalonil metabolism.

Readiness for Medicaid managed care reform initiatives: a profile of rural family physicians.

Medicaid managed care is now an important factor in the financing of rural health care delivery. The participation of rural family physicians in Medicaid managed care is vital for the rural poor to access health services. This study examined 855 family physicians practicing in nonmetropolitan counties across the United States to determine their readiness to participate in Medicaid managed care. Physicians were asked about their experience with prepaid programs and the factors that would influence their participation in such a program. A shortage of health care providers and low reimbursement rates were most frequently cited as barriers to successful implementation. Physicians who had participated in prepaid programs in the past but were no longer participating had the most negative opinions about the potential for Medicaid managed care programs to enhance care for the poor in their communities. Overall, physicians reported potential for the program to improve access and quality of care, but they also expressed reservations about the financial and administrative effects on their practices. These results reveal that negative attitudes were associated with prepaid programs that failed to meet expectations, but physicians also expressed an optimism about the potential to serve the poor within a managed care model.

Managed care in rural markets: availability and enrollment.

The objective of this research was to test the hypothesis that urban-rural differences in managed care availability and enrollment are primarily due to differences in population socioeconomic and health system characteristics rather than geographic location, population size, or density. These two groups of variables were entered into a regression equation to determine which group could best account for the variance in managed care availability and enrollment. In general, the results of these analyses indicated that socioeconomic and health system characteristics did a much better job of explaining differences in managed care availability and enrollment. Therefore, focusing on factors such as adjacency to metropolitan areas or population size or density in making managed care policy decisions may be less productive than focusing on the socioeconomic and health system characteristics of an area.