A PXY-Mediated Transcriptional Network Integrates Signaling Mechanisms to Control Vascular Development in Arabidopsis.

The cambium and procambium generate the majority of biomass in vascular plants. These meristems constitute a bifacial stem cell population from which xylem and phloem are specified on opposing sides by positional signals. The PHLOEM INTERCALATED WITH XYLEM (PXY) receptor kinase promotes vascular cell division and organization. However, how these functions are specified and integrated is unknown. Here, we mapped a putative PXY-mediated transcriptional regulatory network comprising 690 transcription factor-promoter interactions in Arabidopsis (Arabidopsis thaliana). Among these interactions was a feedforward loop containing transcription factors WUSCHEL HOMEOBOX RELATED14 (WOX14) and TARGET OF MONOPTEROS6 (TMO6), each of which regulates the expression of the gene encoding a third transcription factor, LATERAL ORGAN BOUNDARIES DOMAIN4 (LBD4). PXY signaling in turn regulates the WOX14, TMO6, and LBD4 feedforward loop to control vascular proliferation. Genetic interaction between LBD4 and PXY suggests that LBD4 marks the phloem-procambium boundary, thus defining the shape of the vascular bundle. These data collectively support a mechanism that influences the recruitment of cells into the phloem lineage, and they define the role of PXY signaling in this context in determining the arrangement of vascular tissue.

Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo.

Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.

The Lhx9-integrin pathway is essential for positioning of the proepicardial organ.

The development of the vertebrate embryonic heart occurs by hyperplastic growth as well as the incorporation of cells from tissues outside of the initial heart field. Amongst these tissues is the epicardium, a cell structure that develops from the precursor proepicardial organ on the right side of the septum transversum caudal to the developing heart. During embryogenesis, cells of the proepicardial organ migrate, adhere and envelop the maturing heart, forming the epicardium. The cells of the epicardium then delaminate and incorporate into the heart giving rise to cardiac derivatives, including smooth muscle cells and cardiac fibroblasts. Here, we demonstrate that the LIM homeodomain protein Lhx9 is transiently expressed in Xenopus proepicardial cells and is essential for the position of the proepicardial organ on the septum transversum. Utilizing a small-molecule screen, we found that Lhx9 acts upstream of integrin-paxillin signaling and consistently demonstrate that either loss of Lhx9 or disruption of the integrin-paxillin pathway results in mis-positioning of the proepicardial organ and aberrant deposition of extracellular matrix proteins. This leads to a failure of proepicardial cell migration and adhesion to the heart, and eventual death of the embryo. Collectively, these studies establish a requirement for the Lhx9-integrin-paxillin pathway in proepicardial organ positioning and epicardial formation.

Jasmonate and auxin perception: how plants keep F-boxes in check.

Phytohormones regulate the plasticity of plant growth and development, and responses to biotic and abiotic stresses. Many hormone signal transduction cascades involve ubiquitination and subsequent degradation of proteins by the 26S proteasome. The conjugation of ubiquitin to a substrate is facilitated by the E1 activating, E2 conjugating, and the substrate-specifying E3 ligating enzymes. The most prevalent type of E3 ligase in plants is the Cullin-RING ligase (CRL)-type, with F-box proteins (FBPs) as the substrate recognition component. The activity of these SKP-Cullin-F-box (SCF) complexes needs to be tightly regulated in time and place. Here, we review the regulation of SCF function in plants on multiple levels, with a focus on the auxin and jasmonate SCF-type receptor complexes. We discuss in particular the relevance of protein-protein interactions and post-translational modifications as mechanisms to keep SCF functioning under control. Additionally, we highlight the unique property of SCFTIR1/AFB and SCFCOI1 to recognize substrates by forming co-receptor complexes. Finally, we explore how engineered selective agonists can be used to study and uncouple the outcomes of the complex auxin and jasmonate signaling networks that are governed by these FBPs.

A brief history of the TDIF-PXY signalling module: balancing meristem identity and differentiation during vascular development.

474 I. 474 II. 475 III. 475 IV. 477 V. 477 VI. 477 VII. 479 VIII. 481 482 References 482 SUMMARY: A significant proportion of terrestrial biomass is constituted of xylem cells that make up woody plant tissue. Xylem is required for water transport, and is present in the vascular tissue with a second conductive tissue, phloem, required primarily for nutrient transport. Both xylem and phloem are derived from cell divisions in vascular meristems known as the cambium and procambium. One major component that influences several aspects of plant vascular development, including cell division in the vascular meristem, vascular organization and differentiation of vascular cell types, is a signalling module characterized by a peptide ligand called TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) and its cognate receptor, PHLOEM INTERCALATED WITH XYLEM (PXY). In this review, we explore the literature that describes signalling components, phytohormones and transcription factors that interact with these two central factors, to control the varying outputs required in vascular tissues for normal organization and elaboration of plant vascular tissue.

Dynamic DNA methylation turnover in gene bodies is associated with enhanced gene expression plasticity in plants.

BACKGROUND: In several eukaryotes, DNA methylation occurs within the coding regions of many genes, termed gene body methylation (GbM). Whereas the role of DNA methylation on the silencing of transposons and repetitive DNA is well understood, gene body methylation is not associated with transcriptional repression, and its biological importance remains unclear. RESULTS: We report a newly discovered type of GbM in plants, which is under constitutive addition and removal by dynamic methylation modifiers in all cells, including the germline. Methylation at Dynamic GbM genes is removed by the DRDD demethylation pathway and added by an unknown source of de novo methylation, most likely the maintenance methyltransferase MET1. We show that the Dynamic GbM state is present at homologous genes across divergent lineages spanning over 100 million years, indicating evolutionary conservation. We demonstrate that Dynamic GbM is tightly associated with the presence of a promoter or regulatory chromatin state within the gene body, in contrast to other gene body methylated genes. We find Dynamic GbM is associated with enhanced gene expression plasticity across development and diverse physiological conditions, whereas stably methylated GbM genes exhibit reduced plasticity. Dynamic GbM genes exhibit reduced dynamic range in drdd mutants, indicating a causal link between DNA demethylation and enhanced gene expression plasticity. CONCLUSIONS: We propose a new model for GbM in regulating gene expression plasticity, including a novel type of GbM in which increased gene expression plasticity is associated with the activity of DNA methylation writers and erasers and the enrichment of a regulatory chromatin state.

Interference between ER stress-related bZIP-type and jasmonate-inducible bHLH-type transcription factors in the regulation of triterpene saponin biosynthesis in Medicago truncatula.

Triterpene saponins (TS) are a structurally diverse group of metabolites that are widely distributed in plants. They primarily serve as defense compounds and their production is often triggered by biotic stresses through signaling cascades that are modulated by phytohormones such as the jasmonates (JA). Two JA-modulated basic helix-loop-helix (bHLH) transcription factors (TFs), triterpene saponin biosynthesis activating regulator 1 (TSAR1) and TSAR2, have previously been identified as direct activators of TS biosynthesis in the model legume Medicago truncatula. Here, we report on the involvement of the core endoplasmic reticulum (ER) stress-related basic leucine zipper (bZIP) TFs bZIP17 and bZIP60 in the regulation of TS biosynthesis. Expression and processing of M. truncatula bZIP17 and bZIP60 proteins were altered in roots with perturbed TS biosynthesis or treated with JA. Accordingly, such roots displayed an altered ER network structure. M. truncatula bZIP17 and bZIP60 proteins were shown to localize in the nucleus and appeared to be capable of interfering with the TSAR-mediated transactivation of TS biosynthesis genes. Furthermore, interference between ER stress-related bZIP and JA-modulated bHLH TFs in the regulation of JA-dependent terpene biosynthetic pathways may be widespread in the plant kingdom, as we demonstrate that it also occurs in the regulation of monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.

Corrigendum: Interference between ER stress-related bZIP-type and jasmonate-inducible bHLH-type transcription factors in the regulation of triterpene saponin biosynthesis in Medicago truncatula.

[This corrects the article DOI: 10.3389/fpls.2022.903793.].

LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples.

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2-3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

Wnt regulation: exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex.

Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis and is altered in human cancer. Signaling is turned on and off by regulating stability of the effector ß-catenin (ß-cat). The multiprotein destruction complex binds and phosphorylates ß-cat and transfers it to the SCF-TrCP E3-ubiquitin ligase for ubiquitination and destruction. Wnt signals act though Dishevelled to turn down the destruction complex, stabilizing ß-cat. Recent work clarified underlying mechanisms, but important questions remain. We explore ß-cat transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin. We find that Slimb/TrCP is a dynamic component of the destruction complex biomolecular condensate, while other E3 proteins are not. Recruitment requires Axin and not APC, and Axin's RGS domain plays an important role. We find that elevating Dishevelled levels in Drosophila embryos has paradoxical effects, promoting the ability of limiting levels of Axin to turn off Wnt signaling. When we elevate Dishevelled levels, it forms its own cytoplasmic puncta, but these do not recruit Axin. Superresolution imaging in mammalian cells raises the possibility that this may result by promoting Dishevelled:Dishevelled interactions at the expense of Dishevelled: Axin interactions when Dishevelled levels are high.

IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2.

Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.

Propofol versus thiopental: effects on peri-induction intraocular pressures in normal dogs.

OBJECTIVE: To determine the effects of propofol or thiopental induction on intraocular pressures (IOP) in normal dogs. STUDY DESIGN: Prospective randomized experimental study. ANIMALS: Twenty-two random-source dogs weighing 19.5 +/- 5.3 kg. METHODS: Dogs were randomly assigned to receive propofol 8 mg kg(-1) IV (group P) or thiopental 18 mg kg(-1) IV (group T) until loss of jaw tone. Direct arterial blood pressure, arterial blood gasses, and IOP were measured at baseline, after pre-oxygenation but before induction, before endotracheal intubation, and after intubation. RESULTS: There were no significant differences between groups with regard to weight, body condition score, breed group, or baseline or before-induction IOP, arterial blood pressure, or blood gases. The baseline IOP was 12.9 mmHg. Before endotracheal intubation, IOP was significantly higher compared to baseline and before induction in dogs receiving propofol. After intubation with propofol, IOP was significantly higher compared to thiopental and was significantly higher compared to before induction. After intubation, IOP was significantly lower compared to before intubation in dogs receiving thiopental. Propofol increased IOP before intubation by 26% over the before-induction score and thiopental increased IOP by 6% at the same interval. The IOP in group P remained 24% over the before induction score whereas thiopental ultimately decreased IOP 9% below baseline after intubation. There was no significant relationship between any cardiovascular or blood gas parameter and IOP at any time. There was no significant relationship between the changes in any cardiovascular or blood gas parameter and the changes in IOP between time points. CONCLUSIONS AND CLINICAL RELEVANCE Propofol caused a significant increase in IOP compared to baseline and thiopental. Thiopental caused an insignificant increase in IOP which decreased after intubation. Propofol should be avoided when possible in induction of anesthesia in animals where a moderate increase in IOP could be harmful.

A Dual sgRNA Approach for Functional Genomics in Arabidopsis thaliana.

Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows the generation of knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain for several different genes Cas9 null-segregants with bi-allelic mutations in the T2 generation. While somatic mutations were predominantly generated by the canonical non-homologous end joining (cNHEJ) pathway, we observed inherited mutations that were the result of synthesis-dependent microhomology-mediated end joining (SD-MMEJ), a repair pathway linked to polymerase θ (PolQ). We also demonstrate that our workflow is compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method results in more reliable loss-of-function alleles that lack a large essential part of the gene. The ease of the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.

Endosurgical treatment of a retrobulbar abscess in a rabbit.

CASE DESCRIPTION: A 1-year-old sexually intact female Netherland dwarf rabbit was examined because of a 3-week history of signs of lethargy, decreased appetite, left unilateral exophthalmia, a previous draining sinus from a left maxillary facial abscess, and bilateral nasal discharge. CLINICAL FINDINGS: The rabbit weighed 1.0 kg (2.2 lb) and had a body condition score of 1.5/5. Physical examination revealed generalized muscle atrophy, bilateral mucopurulent nasal discharge, and severe left-sided exophthalmia. Diagnostic investigation revealed anemia, neutrophilia, severe dental disease, a superficial corneal ulcer of the left eye, and a retrobulbar abscess. TREATMENT AND OUTCOME: Stomatoscopy-aided dental trimming, tooth removal, and abscess debridement were performed. Antimicrobials were flushed into the tooth abscess cavity, and antimicrobial treatment was initiated on the basis of cytologic findings and results of bacterial culture and susceptibility testing. Two months after the initial surgery, minimal exophthalmia was evident and no further physical, radiographic, or ultrasonographic changes were evident. CLINICAL RELEVANCE: Stomatoscopy is a valuable technique that can facilitate diagnosis, treatment, and serial reevaluation of rabbits with dental disease.

Influence of lidocaine and diazepam on peri-induction intraocular pressures in dogs anesthetized with propofol-atracurium.

The purpose of this study was to evaluate the effects on the intraocular pressure (IOP) of lidocaine or diazepam administered intravenously (IV) before induction of anesthesia with propofol-atracurium and orotracheal intubation in normal dogs, as well as the effects on the IOP of lidocaine applied topically to the larynx after induction with propofol-atracurium. We randomly assigned 32 random-source dogs, obtained from municipal pounds, to receive the following: lidocaine, 2 mg/kg IV, with saline, 0.1 mL/kg topically applied to the larynx (LIDOsal); saline, 0.1 mL/kg IV, with lidocaine, 2 mg/kg topically applied to the larynx (SALlido); diazepam (Valium), 0.25 mg/kg IV, with saline, 0.1 mL/kg topically applied to the larynx (VALsal); or saline, 0.1 mL/kg IV, with saline, 0.1 mL/kg topically applied to the larynx (SALsal). We measured arterial pressure directly, by means of an indwelling catheter placed in a peripheral artery. Anesthesia was induced with propofol, 8 mg/kg IV, until loss of jaw tone, followed by atracurium, 0.3 mg/kg IV. We measured the IOP in triplicate in each eye before premedication, before induction, before intubation, and after intubation. After induction, the IOP was significantly increased except in the VALsal group, in which the IOP was significantly lower than in the negative-control group before intubation. After intubation, the IOP was significantly elevated in all the groups compared with the values before induction. Cardiovascular parameters were essentially similar in all the groups, except for a significant increase in blood pressure after intubation in the SALlido group. Thus, propofol-atracurium anesthesia causes an increase in IOP that is blunted by diazepam. However, diazepam does not blunt the increase in IOP observed with intubation.

Altitude training experiences and perspectives: survey of 67 professional pilots.

INTRODUCTION: Pilots and crewmembers of flights exceeding 7620 m/mean sea level (msl) are required to complete ground training in high-altitude physiology, including hypoxia training. However, regulations do not require altitude chamber training (ACT). METHOD: An anonymous questionnaire concerning their experiences and perceptions of hypoxia training was filled out by 67 pilots attending an aviation safety conference. All pilots had logged professional business flight hours in the previous 6 mo. RESULTS: There were 62 pilots who reported receiving hypoxia training, and of these, 71% reported having initial ACT. Most of the pilots surveyed agreed that all pilots should receive introductory hypoxia training (92%), recurrent hypoxia training (86%), initial ACT (85%), and recurrent ACT (70%). Initial ACT received lower endorsements for private (32%) or recreational (10%) pilots than for commercial (74%) and air transport (90%) pilots. When asked if ACT should be based on the altitude capability of an aircraft, 59% responded affirmatively. Apparently, the perceived need for ACT was based on the likelihood of flying at higher altitudes and not simply the level of certification. When asked if the current regulations (i.e., not requiring ACT) addressing high-altitude flying (above 7620 m/msl) are sufficient, 52% of the current sample disagreed or strongly disagreed. DISCUSSION: Generally, these professional pilots perceived that pilot training should include introductory hypoxia training, recurrent hypoxia training, and ACT. Exceptions were initial ACT for recreational pilots and private pilots. Generalizability of these results may be affected by the specificity and size of the sample. Distributing the survey to a wider audience of pilots would provide additional information regarding perceptions of hypoxia training.

The impact of an AIDS vaccine in developing countries: a new model and initial results.

A new model was developed to examine the potential impacts of an AIDS vaccine in developing countries. The findings suggest that even a modestly efficacious first-generation vaccine could have a profound effect on the AIDS pandemic. A vaccine with 50 percent efficacy provided to 30 percent of the population would reduce new annual infections by 34 percent (seventeen million infections avoided) over fifteen years and result in substantial financial savings. A more efficacious vaccine, combined with expanded delivery, would do even more to control the pandemic. It therefore makes sense to continue investing in AIDS vaccine research and development and the eventual manufacture and widespread distribution of a vaccine.