Molecular perspectives on age-related resistance of plants to (viral) pathogens.

Age-related resistance to microbe invasion is a commonly accepted concept in plant pathology. However, the impact of such age-dependent interactive phenomena is perhaps not yet sufficiently recognized by the broader plant science community. Toward cataloging an understanding of underlying mechanisms, this review explores recent molecular studies and their relevance to the concept. Examples describe differences in genetic background, transcriptomics, hormonal balances, protein-mediated events, and the contribution by short RNA-controlled gene silencing events. Throughout, recent findings with viral systems are highlighted as an illustration of the complexity of the interactions. It will become apparent that instead of uncovering a unifying explanation, we unveiled only trends. Nevertheless, with a degree of confidence, we propose that the process of plant age-related defenses is actively regulated at multiple levels. The overarching goal of this control for plants is to avoid a constitutive waste of resources, especially at crucial metabolically draining early developmental stages.

Native Processing of Single Guide RNA Transcripts to Create Catalytic Cas9/Single Guide RNA Complexes in Planta.

The present CRISPR/Cas9 gene editing dogma for single guide RNA (sgRNA) delivery is based on the premise that 5'-and 3'-nucleotide overhangs negate Cas9/sgRNA catalytic activity in vivo. This has led to engineering strategies designed to either avoid or remove extraneous nucleotides at the 5' and 3' termini of sgRNAs. Previously, we used a Tobacco mosaic virus viral vector to express both GFP and a sgRNA from a single virus-derived mRNA in Nicotiana benthamiana This vector yielded high levels of GFP and catalytically active sgRNAs. Here, in an effort to understand the biochemical interactions of this result, we used in vitro assays to demonstrate that nucleotide overhangs 5', but not 3', proximal to the sgRNA do in fact inactivate Cas9 catalytic activity at the specified target site. Next we showed that in planta sgRNAs bound to Cas9 are devoid of the expected 5' overhangs transcribed by the virus. Furthermore, when a plant nuclear promoter was used for expression of the GFP-sgRNA fusion transcript, it also produced indels when delivered with Cas9. These results reveal that 5' auto-processing of progenitor sgRNAs occurs natively in plants. Toward a possible mechanism for the perceived auto-processing, we found, using in vitro-generated RNAs and those isolated from plants, that the 5' to 3' exoribonuclease XRN1 can degrade elongated progenitor sgRNAs, whereas the mature sgRNA end products are resistant. Comparisons with other studies suggest that sgRNA auto-processing may be a phenomenon not unique to plants, but present in other eukaryotes as well.

Fabrication Method for a Mounting Jig and Cast Support for Mounting Complete Dentures.

In complete denture fabrication, the definitive maxillary cast is mounted on an articulator using a facebow transfer or mounting jig, and the mandibular cast is mounted using an interocclusal record. The technique presented describes an easy and inexpensive method for fabrication of a mounting jig and rigid cast supports for mounting complete dentures.

Bias: A 30-Year Review of Randomized Controlled Trials Published in Three Prosthodontic Journals.

PURPOSE: Bias can occur in various phases of an investigation, and its control is an important measure of the validity of results for randomized controlled trials (RCTs). The purpose of this study is to determine if bias control in prosthodontic RCTs published from 2008 to 2017 improved over those published from 1988 to 1997. MATERIALS AND METHODS: MEDLINE was searched for RCTs in The International Journal of Prosthodontics, The Journal of Prosthetic Dentistry, and The Journal of Prosthodontics published from 2008 to 2017. Citations retrieved were included if the trial involved human subjects, included at least 2 treatment groups, and group assignment was by random allocation. Pilot and follow-up studies were excluded. Included RCTs were evaluated on the basis of how control of potential sources of bias in trial methodology were reported. Three areas-control of bias at entry, control of bias in assessment of outcome, and control of bias after entry-were scored 1 or 0, based on whether method of randomization was explicitly reported, blinding was done, and all subjects were accounted for at the end of the study. Thus, the maximum quality score was 3 (good bias control) and the minimum 0 (poor bias control). Frequencies were calculated for each dimension of trial methodology, and overall scores were reported. Results were compared to those of RCTs published from 1988 to 1997 reported in a previous study. RESULTS: Ninety-six RCTs published from 2008 to 2017 met the inclusion criteria and were reviewed. Method of randomization was explicit in 68% of RCTs, 50% reported blinding, and 85% accounted for all subjects; 32% scored the maximum 3 points for good bias control. In comparison, 62 RCTs published from 1988 to 1997 had frequencies of 47%, 40%, and 76% in the 3 areas examined, respectively; only 16% had maximum scores for good bias control. CONCLUSION: Control of bias and overall quality scores have improved for RCTs published in the 3 studied prosthodontic journals.

Reporting of Sample Size Parameters in Randomized Controlled Trials Published in Prosthodontic Journals.

PURPOSE: An important consideration in the proper planning of randomized controlled trials (RCTs) is the determination of sample size. A study may fail to answer its research question if the sample size is inadequate, while a large enough sample size may be impractical to implement. The purpose of this study is to describe the reporting of sample size methodology and parameters used in RCTs published in prosthodontic journals. MATERIALS AND METHODS: A MEDLINE search for publications categorized as RCTs was conducted for articles in The Journal of Prosthodontics and The Journal of Prosthetic Dentistry published from 2008 to 2017. The Abstract and Methodology sections of RCTs identified were reviewed, and the following data recorded: reporting of method used to calculate sample size and reporting of parameters used for sample size calculation - type I error (α), type II error (ß) or power, minimal clinically relevant difference (MCRD), and variability. RESULTS: The search strategy retrieved 96 articles; 42 met inclusion criteria for RCTs and were reviewed. Fifty percent (21) of RCTs reported how sample size was determined, but only 17% (7) of RCTs reported all 4 parameters. Type I error (α) was reported in 90% (38) of RCTs, 38% (16) reported power, while only 26% (11) and 12% (5) reported MCRD and variability, respectively. CONCLUSION: Methodology and parameters used for sample size determination are inadequately reported in RCTs published in prosthodontic journals.

Multiplexed Gene Editing and Protein Overexpression Using a Tobacco mosaic virus Viral Vector.

Development of CRISPR/Cas9 transient gene editing screening tools in plant biology has been hindered by difficulty of delivering high quantities of biologically active single guide RNAs (sgRNAs). Furthermore, it has been largely accepted that in vivo generated sgRNAs need to be devoid of extraneous nucleotides, which has limited sgRNA expression by delivery vectors. Here, we increased cellular concentrations of sgRNA by transiently delivering sgRNAs using a Tobacco mosaic virus-derived vector (TRBO) designed with 5' and 3' sgRNA proximal nucleotide-processing capabilities. To demonstrate proof-of-principle, we used the TRBO-sgRNA delivery platform to target GFP in Nicotiana benthamiana (16c) plants, and gene editing was accompanied by loss of GFP expression. Surprisingly, indel (insertions and deletions) percentages averaged nearly 70% within 7 d postinoculation using the TRBO-sgRNA constructs, which retained 5' nucleotide overhangs. In contrast, and in accordance with current models, in vitro Cas9 cleavage assays only edited DNA when 5' sgRNA nucleotide overhangs were removed, suggesting a novel processing mechanism is occurring in planta. Since the Cas9/TRBO-sgRNA platform demonstrated sgRNA flexibility, we targeted the N. benthamiana NbAGO1 paralogs with one sgRNA and also multiplexed two sgRNAs using a single TRBO construct, resulting in indels in three genes. TRBO-mediated expression of an RNA transcript consisting of an sgRNA adjoining a GFP protein coding region produced indels and viral-based GFP overexpression. In conclusion, multiplexed delivery of sgRNAs using the TRBO system offers flexibility for gene expression and editing and uncovered novel aspects of CRISPR/Cas9 biology.

Tobacco rattle virus (TRV)-Mediated Silencing of Nicotiana benthamiana ARGONAUTES (NbAGOs) Reveals New Antiviral Candidates and Dominant Effects of TRV-NbAGO1.

The objective of this study was to determine the contribution of different ARGONAUTE proteins in Nicotiana benthamiana (NbAGOs) to the defense against silencing sensitive GFP-expressing viral constructs based on Tomato bushy stunt virus (TBSV) (Tombusvirus), Sunn-hemp mosaic virus (Tobamovirus), and Foxtail mosaic virus (Potexvirus). Upon Tobacco rattle virus (TRV)-mediated down-regulation of NbAGO1, 4, 5, or 6, no effects were noted on susceptibility to any virus construct, whereas knockdown of NbAGO2 specifically prevented silencing of P19-defective TBSV (TGdP19). Down-regulation of a new gene referred to as NbAGO5L showed some reduced silencing for TGdP19 but not for the other two virus constructs, whereas silencing of NbAGO7 gave rise to a subtle increase in susceptibility to all three viruses. Co-infiltrating different TRV-NbAGO constructs simultaneously did not enhance virus susceptibility. However, an unexpected finding was that whenever the TRV-NbAGO1 construct was present, this compromised silencing of genes targeted by co-infiltrated constructs, as shown upon co-infiltration of TRV-NbAGO1 with either TRV-NbAGO2 or TRV-Sul (targeting Magnesium chelatase I). Only after a prolonged period (approximately 2 months) did TRV-Sul-mediated systemic bleaching occur in these co-infected plants, suggesting that TRV-NbAGO1 hinders the silencing ability of other TRV-NbAGO constructs. In conclusion, this study revealed new antiviral NbAGOs and dominant effects of silencing NbAGO1.

Epac2 Mediates cAMP-Dependent Potentiation of Neurotransmission in the Hippocampus.

Presynaptic terminal cAMP elevation plays a central role in plasticity at the mossy fiber-CA3 synapse of the hippocampus. Prior studies have identified protein kinase A as a downstream effector of cAMP that contributes to mossy fiber LTP (MF-LTP), but the potential contribution of Epac2, another cAMP effector expressed in the MF synapse, has not been considered. We investigated the role of Epac2 in MF-CA3 neurotransmission using Epac2(-/-) mice. The deletion of Epac2 did not cause gross alterations in hippocampal neuroanatomy or basal synaptic transmission. Synaptic facilitation during short trains was not affected by loss of Epac2 activity; however, both long-term plasticity and forskolin-mediated potentiation of MFs were impaired, demonstrating that Epac2 contributes to cAMP-dependent potentiation of transmitter release. Examination of synaptic transmission during long sustained trains of activity suggested that the readily releasable pool of vesicles is reduced in Epac2(-/-) mice. These data suggest that cAMP elevation uses an Epac2-dependent pathway to promote transmitter release, and that Epac2 is required to maintain the readily releasable pool at MF synapses in the hippocampus.

Impact of an Electronic Clinical Decision Support Tool for Emergency Department Patients With Pneumonia.

STUDY OBJECTIVE: Despite evidence that guideline adherence improves clinical outcomes, management of pneumonia patients varies in emergency departments (EDs). We study the effect of a real-time, ED, electronic clinical decision support tool that provides clinicians with guideline-recommended decision support for diagnosis, severity assessment, disposition, and antibiotic selection. METHODS: This was a prospective, controlled, quasi-experimental trial in 7 Intermountain Healthcare hospital EDs in Utah's urban corridor. We studied adults with International Classification of Diseases, Ninth Revision codes and radiographic evidence for pneumonia during 2 periods: baseline (December 2009 through November 2010) and post-tool deployment (December 2011 through November 2012). The tool was deployed at 4 intervention EDs in May 2011, leaving 3 as usual care controls. We compared 30-day, all-cause mortality adjusted for illness severity, using a mixed-effect, logistic regression model. RESULTS: The study population comprised 4,758 ED pneumonia patients; 14% had health care-associated pneumonia. Median age was 58 years, 53% were female patients, and 59% were admitted to the hospital. Physicians applied the tool for 62.6% of intervention ED study patients. There was no difference overall in severity-adjusted mortality between intervention and usual care EDs post-tool deployment (odds ratio [OR]=0.69; 95% confidence interval [CI] 0.41 to 1.16). Post hoc analysis showed that patients with community-acquired pneumonia experienced significantly lower mortality (OR=0.53; 95% CI 0.28 to 0.99), whereas mortality was unchanged among patients with health care-associated pneumonia (OR=1.12; 95% CI 0.45 to 2.8). Patient disposition from the ED postdeployment adhered more to tool recommendations. CONCLUSION: This study demonstrates the feasibility and potential benefit of real-time electronic clinical decision support for ED pneumonia patients.

Regulatory acceptance and use of 3R models for pharmaceuticals and chemicals: expert opinions on the state of affairs and the way forward.

Pharmaceuticals and chemicals are subjected to regulatory safety testing accounting for approximately 25% of laboratory animal use in Europe. This testing meets various objections and has led to the development of a range of 3R models to Replace, Reduce or Refine the animal models. However, these models must overcome many barriers before being accepted for regulatory risk management purposes. This paper describes the barriers and drivers and options to optimize this acceptance process as identified by two expert panels, one on pharmaceuticals and one on chemicals. To untangle the complex acceptance process, the multilevel perspective on technology transitions is applied. This perspective defines influences at the micro-, meso- and macro level which need alignment to induce regulatory acceptance of a 3R model. This paper displays that there are many similar mechanisms within both sectors that prevent 3R models from becoming accepted for regulatory risk assessment and management. Shared barriers include the uncertainty about the value of the new 3R models (micro level), the lack of harmonization of regulatory requirements and acceptance criteria (meso level) and the high levels of risk aversion (macro level). In optimizing the process commitment, communication, cooperation and coordination are identified as critical drivers.

A tomato bushy stunt virus-based vector for simultaneous editing and sensing to survey the host antiviral RNA silencing machinery.

A tomato bushy stunt virus (TBSV)-derived vector system was applied for the delivery of CRISPR/Cas9 gene editing materials, to facilitate rapid, transient assays of host-virus interactions involved in the RNA silencing pathway. Toward this, single guide RNAs designed to target key components of the virus-induced host RNA silencing pathway (AGO2, DCL2, HEN1) were inserted into TBSV-based GFP-expressing viral vectors TBSV-GFP (TG) and its P19 defective mutant TGΔP19. This produced rapid, efficient, and specific gene editing in planta. Targeting AGO2, DCL2, or HEN1 partially rescued the lack of GFP accumulation otherwise associated with TGΔP19. Since the rescue phenotypes are normally only observed in the presence of the P19 silencing suppressor, the results support that the DCL2, HEN1, and AGO2 proteins are involved in anti-TBSV RNA silencing. Additionally, we show that knockdown of the RNA silencing machinery increases cargo expression from a nonviral binary Cas9 vector. The TBSV-based gene editing technology described in this study can be adapted for transient heterologous expression, rapid gene function screens, and molecular interaction studies in many plant species considering the wide host range of TBSV. In summary, we demonstrate that a plant virus can be used to establish gene editing while simultaneously serving as an accumulation sensor for successful targeting of its homologous antiviral silencing machinery components.

Novel protease-based diagnostic devices for detection of wound infection.

A gelatinase-based device for fast detection of wound infection was developed. Collective gelatinolytic activity in infected wounds was 23 times higher (p ≤ 0.001) than in noninfected wounds and blisters according to the clinical and microbiological description of the wounds. Enzyme activities of critical wounds showed 12-fold elevated enzyme activities compared with noninfected wounds and blisters. Upon incubation of gelatin-based devices with infected wound fluids, an incubation time of 30 minutes led to a clearly visible dye release. A 32-fold color increase was measured after 60 minutes. Both matrix metalloproteinases and elastases contributed to collective gelatinolytic enzyme activity as shown by zymography and inhibition experiments. The metalloproteinase inhibitor 1,10-phenanthroline (targeting matrix metalloproteinases) and the serine protease inhibitor phenylmethlysulfonyl fluoride (targeting human neutrophil elastase) inhibited gelatinolytic activity in infected wound fluid samples by 11-37% and 60-95%, respectively. Staphylococcus aureus and Pseudomonas aeruginosa, both known for gelatinase production, were isolated in infected wound samples.

Leadership lessons from the Chilean mine rescue.

Three years ago, when a cave-in at the San José mine in Chile trapped 33 men under 700,000 metric tons of rock, experts estimated the probability of getting them out alive at less than 1%. Yet, after spending a record 69 days underground, all 33 were hoisted up to safety. The inspiring story of their rescue is a case study in how to lead in situations where the stakes, risk, and uncertainty are incredibly high and time pressure is intense. Today executives often find themselves in similar straits. When they do, many feel torn. Should they be directive, taking charge and commanding action? Or should they be empowering, enabling innovation and experimentation? As the successful example of André Sougarret, the chief of the mine rescue operation, shows, the answer is yes--to both. The choice is a false dichotomy. Implementing this dual approach involves three key tasks. Each has directive and enabling components. The first task is envisioning, which requires instilling both realism and hope. The second task is enrolling, which means setting clear boundaries for who is on and off the team, but inviting in helpful collaborators. The third task is engaging--leading disciplined execution while encouraging innovation and experimentation. The authors of this article describe how Sougarret ably juggled all of these tasks, orchestrating the efforts of hundreds of people from different organizations, areas of expertise, and countries in an extraordinary mission that overcame impossible odds.

Plant virology: an RNA treasure trove.

Key principles pertaining to RNA biology not infrequently have their origins in plant virology. Examples have arisen from studies on viral RNA-intrinsic properties and the infection process from gene expression, replication, movement, and defense evasion to biotechnological applications. Since RNA is at the core of the central dogma in molecular biology, how plant virology assisted in the reinforcement or adaptations of this concept, while at other instances shook up elements of the doctrine, is discussed. Moreover, despite the negative effects of viral diseases in agriculture worldwide, plant viruses can be considered a scientific treasure trove. Today they remain tools of discovery for biotechnology, studying evolution, cell biology, and host-microbe interactions.

Identification of an ARGONAUTE for antiviral RNA silencing in Nicotiana benthamiana.

ARGONAUTE proteins (AGOs) are known to be key components of the RNA silencing mechanism in eukaryotes that, among other functions, serves to protect against viral invaders. Higher plants encode at least 10 individual AGOs yet the role played by many in RNA silencing-related antiviral defense is largely unknown, except for reports that AGO1, AGO2, and AGO7 play an antiviral role in Arabidopsis (Arabidopsis thaliana). In the plant virus model host Nicotiana benthamiana, Tomato bushy stunt virus (TBSV) P19 suppressor mutants are very susceptible to RNA silencing. Here, we report that a N. benthamiana AGO (NbAGO) with similarity to Arabidopsis AGO2, is involved in antiviral defense against TBSV. The activity of this NbAGO2 is shown to be directly associated with anti-TBSV RNA silencing, while its inactivation does not influence silencing of transiently expressed transgenes. Thus, the role of NbAGO2 might be primarily for antiviral defense.

Plant responses against invasive nucleic acids: RNA silencing and its suppression by plant viral pathogens.

RNA silencing is a common strategy shared by eukaryotic organisms to regulate gene expression, and also operates as a defense mechanism against invasive nucleic acids such as viral transcripts. The silencing pathway is quite sophisticated in higher eukaryotes but the distinct steps and nature of effector complexes vary between and even within species. To counteract this defense mechanism viruses have evolved the ability to encode proteins that suppress silencing to protect their genomes from degradation. This review focuses on our current understanding of how individual components of the plant RNA silencing mechanism are directed against viruses, and how in turn virus-encoded suppressors target one or more key events in the silencing cascade.

Induction and suppression of RNA silencing: insights from plant viral infections--a BARD workshop report.

An international workshop on ''Induction and Suppression of RNA Silencing: Insights from Plant Viral Infections'' was sponsored by the United States-Israel Binational Agricultural Research and Development Fund (BARD) and organized in Eilat, Israel in March 2010. The focus of this workshop was on molecular mechanisms employed by viruses or their hosts, and their interactions, for the regulation of virus-induced silencing and suppression. Several of the talks also served as potent reminders of scientific hubris and the need to be attentive to earlier results, both for analyses and perspective regarding new findings.

Improved foreign gene expression in plants using a virus-encoded suppressor of RNA silencing modified to be developmentally harmless.

Endeavours to obtain elevated and prolonged levels of foreign gene expression in plants are often hampered by the onset of RNA silencing that negatively affects target gene expression. Plant virus-encoded suppressors of RNA silencing are useful tools for counteracting silencing but their wide applicability in transgenic plants is limited because their expression often causes harmful developmental effects. We hypothesized that a previously characterized tombusvirus P19 mutant (P19/R43W), typified by reduced symptomatic effects while maintaining the ability to sequester short-interfering RNAs, could be used to suppress virus-induced RNA silencing without the concomitant developmental effects. To investigate this, transient expression in Nicotiana benthamiana was used to evaluate the ability of P19/R43W to enhance heterologous gene expression. Although less potent than wt-P19, P19/R43W was an effective suppressor when used to enhance protein expression from either a traditional T-DNA expression cassette or using the CPMV-HT expression system. Stable transformation of N. benthamiana yielded plants that expressed detectable levels of P19/R43W that was functional as a suppressor. Transgenic co-expression of green fluorescent protein (GFP) and P19/R43W also showed elevated accumulation of GFP compared with the levels found in the absence of a suppressor. In all cases, transgenic expression of P19/R43W caused no or minimal morphological defects and plants produced normal-looking flowers and fertile seed. We conclude that the expression of P19/R43W is developmentally harmless to plants while providing a suitable platform for transient or transgenic overexpression of value-added genes in plants with reduced hindrance by RNA silencing.

RNA silencing suppressor-influenced performance of a virus vector delivering both guide RNA and Cas9 for CRISPR gene editing.

We report on further development of the agroinfiltratable Tobacco mosaic virus (TMV)-based overexpression (TRBO) vector to deliver CRISPR/Cas9 components into plants. First, production of a Cas9 (HcoCas9) protein from a binary plasmid increased when co-expressed in presence of suppressors of gene silencing, such as the TMV 126-kDa replicase or the Tomato bushy stunt virus P19 protein. Such suppressor-generated elevated levels of Cas9 expression translated to efficient gene editing mediated by TRBO-G-3'gGFP expressing GFP and also a single guide RNA targeting the mgfp5 gene in the Nicotiana benthamiana GFP-expressing line 16c. Furthermore, HcoCas9 encoding RNA, a large cargo insert of 4.2 kb, was expressed from TRBO-HcoCas9 to yield Cas9 protein again at higher levels upon co-expression with P19. Likewise, co-delivery of TRBO-HcoCas9 and TRBO-G-3'gGFP in the presence of P19 also resulted in elevated levels percentages of indels (insertions and deletions). These data also revealed an age-related phenomenon in plants whereby the RNA suppressor P19 had more of an effect in older plants. Lastly, we used a single TRBO vector to express both Cas9 and a sgRNA. Taken together, we suggest that viral RNA suppressors could be used for further optimization of single viral vector delivery of CRISPR gene editing parts.

Diverse and newly recognized effects associated with short interfering RNA binding site modifications on the Tomato bushy stunt virus p19 silencing suppressor.

The Tomato bushy stunt virus-encoded P19 forms dimers that bind duplex short interfering RNAs (siRNAs) to suppress RNA silencing. P19 is also involved in multiple host-specific activities, including the elicitation of symptoms, and in local and/or systemic spread. To study the correlation between those various roles and the siRNA binding by P19, predicted siRNA-interacting sites were modified. Twenty-two mutants were generated and inoculated onto Nicotiana benthamiana plants, to reveal that (i) they were all infectious, (ii) symptom differences did not correlate strictly with mutation-associated variation in P19 accumulation, and (iii) substitutions affecting a central domain of P19 generally exhibited symptoms more severe than for mutations affecting peripheral regions. Three mutants selected to represent separate phenotypic categories all displayed a substantially reduced ability to sequester siRNA. Consequently, these three mutants were compromised for systemic virus spread in P19-dependent hosts but had differential plant species-dependent effects on the symptom severity. One mutant in particular caused relatively exacerbated symptoms, exemplified by extensive morphological leaf deformations in N. benthamiana; this was especially remarkable because P19 was undetectable. Another striking feature of this mutant was that only within a few days after infection, viral RNA was cleared by silencing. One more original property was that host RNAs and proteins (notably, the P19-interactive Hin19 protein) were also susceptible to degradation in these infected N. benthamiana plants but not in spinach. In conclusion, even though siRNA binding by P19 is a key functional property, compromised siRNA sequestration can result in novel and diverse host-dependent properties.