Inflorescence stem grafting made easy in Arabidopsis

UNLABELLED BACKGROUND Plant grafting techniques have deepened our understanding of the signals facilitating communication between the root and shoot, as well as between shoot and reproductive organs. Transmissible signalling molecules can include hormones, peptides, proteins and metabolites: some of which travel long distances to communicate stress, nutrient status, disease and developmental events. While hypocotyl micrografting techniques have been successfully established for Arabidopsis to explore root to shoot communications, inflorescence grafting in Arabidopsis has not been exploited to the same extent. Two different strategies (horizontal and wedge-style inflorescence grafting) have been developed to explore long distance signalling between the shoot and reproductive organs. We developed a robust wedge-cleft grafting method, with success rates greater than 87%, by developing better tissue contact between the stems from the inflorescence scion and rootstock. We describe how to perform a successful inflorescence stem graft that allows for reproducible translocation experiments into the physiological, developmental and molecular aspects of long distance signalling events that promote reproduction. RESULTS Wedge grafts of the Arabidopsis inflorescence stem were supported with silicone tubing and further sealed with parafilm to maintain the vascular flow of nutrients to the shoot and reproductive tissues. Nearly all (87%) grafted plants formed a strong union between the scion and rootstock. The success of grafting was scored using an inflorescence growth assay based upon the growth of primary stem. Repeated pruning produced new cauline tissues, healthy flowers and reproductive siliques, which indicates a healthy flow of nutrients from the rootstock. Removal of the silicone tubing showed a tightly fused wedge graft junction with callus proliferation. Histological staining of sections through the graft junction demonstrated the differentiation of newly formed vascular connections, parenchyma tissue and lignin accumulation, supporting the presumed success of the graft union between two sections of the primary inflorescence stem. CONCLUSIONS We describe a simple and reliable method for grafting sections of an Arabidopsis inflorescence stem. This step-by-step protocol facilitates laboratories without grafting experience to further explore the molecular and chemical signalling which coordinates communications between the shoot and reproductive tissues

Numerical simulation of droplet formation in a microchannel device

The formation of droplets is a phenomenon with particular importance in the development of industrial emulsions. The quality of these compounds is associated with droplet size and stability over time. Anna et al. (2003) developed a methodology named ¨flow focusing¨ to improve droplet formation processes for engineering applications. In this work, Computational Fluid Dynamics (CFD) based techniques are used to assess the capacity of a pseudo-2D numerical model to reproduce water droplets formation within silicon oil, as obtained in Anna et al.’s experiments. Average time of droplet onset obtained via numerical analysis was 1.5 times larger than observed experimentally, whereas droplets convection velocity and diameter predictions differed by 40-45% and 60%, respectively. Nevertheless, calculated velocity profiles downstream the discharge slot reproduced the expected free-jet shear layer according to outer/inner flow ratio

Numerical simulation of droplet formation in a microchannel device

The formation of droplets is a phenomenon with particular importance in the development of industrial emulsions. The quality of these compounds is associated with droplet size and stability over time. Anna et al. (2003) developed a methodology named ¨flow focusing¨ to improve droplet formation processes for engineering applications. In this work, Computational Fluid Dynamics (CFD) based techniques are used to assess the capacity of a pseudo-2D numerical model to reproduce water droplets formation within silicon oil, as obtained in Anna et al.’s experiments. Average time of droplet onset obtained via numerical analysis was 1.5 times larger than observed experimentally, whereas droplets convection velocity and diameter predictions differed by 40-45% and 60%, respectively. Nevertheless, calculated velocity profiles downstream the discharge slot reproduced the expected free-jet shear layer according to outer/inner flow ratio

miR159 Represses a Constitutive Pathogen Defense Response in Tobacco

MicroR159 (miR159) regulation of GAMYB expression is highly conserved in terrestrial plants; however, its functional role remains poorly understood. In Arabidopsis (Arabidopsis thaliana), although GAMYB-like genes are constitutively transcribed during vegetative growth, their effects are suppressed by strong and constitutive silencing by miR159. GAMYB expression occurs only if miR159 function is inhibited, which results in detrimental pleiotropic defects, questioning the purpose of the miR159-GAMYB pathway. Here, miR159 function was inhibited in tobacco (Nicotiana tabacum) and rice (Oryza sativa) using miRNA MIM159 technology. Similar to observations in Arabidopsis, inhibition of miR159 in tobacco and rice resulted in pleiotropic defects including stunted growth, implying functional conservation of the miR159-GAMYB pathway among angiosperms. In MIM159 tobacco, transcriptome profiling revealed that genes associated with defense and programmed cell death were strongly activated, including a suite of 22 PATHOGENESIS-RELATED PROTEIN (PR) genes that were 100- to 1,000-fold upregulated. Constitutive expression of a miR159-resistant GAMYB transgene in tobacco resulted in phenotypes similar to that of MIM159 tobacco and activated PR gene expression, verifying the dependence of the above-mentioned changes on GAMYB expression. Consistent with the broad defense response, MIM159 tobacco appeared immune to Phytophthora infection. These findings suggest that the tobacco miR159-GAMYB pathway functions in the biotic defense response, which becomes activated upon miR159 inhibition. However, PR gene expression was not upregulated in Arabidopsis or rice when miR159 was inhibited, suggesting that miR159-GAMYB pathway functional differences exist between species, or factors in addition to miR159 inhibition are required in Arabidopsis and rice to activate this broad defense response

Evaluation of low acuity patients discharged from a virtual emergency department at a major urban academic health sciences centre in Toronto, Canada.

Objective In response to the COVID-19 pandemic, Sunnybrook Health Sciences Centre launched the first virtual emergency department (VED) in Toronto, Ontario. The objective of this pilot project was to leverage linked administrative data to describe the healthcare utilization of VED patients compared to matched patients who attended an ED in person. Approach Evaluation of the VED program was supported by the ICES Applied Health Research Question Program, which is funded by the Ontario Ministry of Health to answer questions directly related to Ontario healthcare policy, planning, or practice. VED visit records from December 2020 to May 2021 were linked with Ontario administrative data. VED patients with low acuity complaints were matched 1:1 with in-person ED comparators according to visit date, presenting complaint, and a propensity score that incorporated age, sex, comorbidities, and other important potential confounders. The primary outcomes were healthcare utilization within 7 days and all-cause mortality within 30 days. Results Of the 609 eligible patients discharged from the VED, 600 (98.5%) were successfully matched to a comparator. Mean (SD) age was 43.0 (21.1) and 64.1% were female. In-person ED revisits and hospitalizations were similar for VED and comparator patients at 72 hours (ED: 12.1% vs. 11.3%; Δ 0.8%, 95%: -2.8, 4.5%; hospitalization:  1.2% vs. 1.5%; Δ 0.3%, 95%: -0.7, 1.4%,) and 7 days (ED: 16.1% vs. 14.4%; Δ 1.7, 95%: -2.4, 5.7%; hospitalization: 1.7% vs. 1.8%; Δ 0.2%, 95%: -0.1, 1.4%) following the index visit. The number of patients visiting a primary care provider within 7 days was also similar between groups (36.7% vs. 32.4%; Δ 4.3, 95%: -1.1, 9.8%). No patients died within 30 days. Conclusion/Implications VED patients and their matched comparators had similar healthcare utilization in the 7 days following their index ED visit. Methodology from this study will inform a province-wide evaluation of VED programs across Ontario

On the structure-properties relationship in montmorillonite-filled polyamide 6 nanocomposites

Polyamide 6/montmorillonite (MMT) nanocomposites were prepared by melt compounding method comprising 1–7.5 wt % of Nanomer I.24 TL or 5 and 10 wt % of Cloisite 15A organically modified nanoclays. The composite samples were characterized by synchrotron X-ray, thermal and FT-IR spectroscopy methods looking for changes in the micro- and nanostructure of both PA6 matrix and MMT reinforcement as a function of the clay content and type. These data were discussed in conjunction with the mechanical properties of the respective nanocomposites. Generally, the Young’s modulus was found to increase proportionally to the clay content being the highest in samples with strong aggregation of MMT at micron length scale. The tensile strength passed through a maximum at 2.5 wt % clay load presenting a homogeneous microstructure with almost no agglomeration. Increasing the amount of MMT produced less crystalline PA6 matrices, richer in gamma-PA6 polymorph and resulted in larger long spacings of PA6 due to expansion of both crystalline and amorphous domains.Fundação para a Ciência e a Tecnologia (FCT), Portugal; HASYALb at DESY, Hamburg, German

Global Retinoblastoma Presentation and Analysis by National Income Level.

Importance: Early diagnosis of retinoblastoma, the most common intraocular cancer, can save both a child's life and vision. However, anecdotal evidence suggests that many children across the world are diagnosed late. To our knowledge, the clinical presentation of retinoblastoma has never been assessed on a global scale. Objectives: To report the retinoblastoma stage at diagnosis in patients across the world during a single year, to investigate associations between clinical variables and national income level, and to investigate risk factors for advanced disease at diagnosis. Design, Setting, and Participants: A total of 278 retinoblastoma treatment centers were recruited from June 2017 through December 2018 to participate in a cross-sectional analysis of treatment-naive patients with retinoblastoma who were diagnosed in 2017. Main Outcomes and Measures: Age at presentation, proportion of familial history of retinoblastoma, and tumor stage and metastasis. Results: The cohort included 4351 new patients from 153 countries; the median age at diagnosis was 30.5 (interquartile range, 18.3-45.9) months, and 1976 patients (45.4%) were female. Most patients (n = 3685 [84.7%]) were from low- and middle-income countries (LMICs). Globally, the most common indication for referral was leukocoria (n = 2638 [62.8%]), followed by strabismus (n = 429 [10.2%]) and proptosis (n = 309 [7.4%]). Patients from high-income countries (HICs) were diagnosed at a median age of 14.1 months, with 656 of 666 (98.5%) patients having intraocular retinoblastoma and 2 (0.3%) having metastasis. Patients from low-income countries were diagnosed at a median age of 30.5 months, with 256 of 521 (49.1%) having extraocular retinoblastoma and 94 of 498 (18.9%) having metastasis. Lower national income level was associated with older presentation age, higher proportion of locally advanced disease and distant metastasis, and smaller proportion of familial history of retinoblastoma. Advanced disease at diagnosis was more common in LMICs even after adjusting for age (odds ratio for low-income countries vs upper-middle-income countries and HICs, 17.92 [95% CI, 12.94-24.80], and for lower-middle-income countries vs upper-middle-income countries and HICs, 5.74 [95% CI, 4.30-7.68]). Conclusions and Relevance: This study is estimated to have included more than half of all new retinoblastoma cases worldwide in 2017. Children from LMICs, where the main global retinoblastoma burden lies, presented at an older age with more advanced disease and demonstrated a smaller proportion of familial history of retinoblastoma, likely because many do not reach a childbearing age. Given that retinoblastoma is curable, these data are concerning and mandate intervention at national and international levels. Further studies are needed to investigate factors, other than age at presentation, that may be associated with advanced disease in LMICs

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2,3,4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease