Krabbeschmidt6798

The overall duration for this protocol is approximately 8 hours spanning 4 days (an average of 2 h/day per two workers) excluding microscopy and LC-MS/MS analysis.During sexual reproduction in flowering plants, pollen grains germinate on the stigma surface and grow through the stigma-style tissue to reach the ovary and deliver sperm cells for fertilization. Here, we outline a method to test whether a pollen fertility mutation specifically disrupts pollen penetration through the stigma-style barrier. Solutol HS-15 concentration This method surgically removes the stigma-style (stigma decapitation) to test whether transferring pollen directly onto an exposed ovary surface significantly improves the transmission efficiency (TE) of a mutant allele. To illustrate this technique, we applied stigma decapitation to investigate a loss-of-function mutation in Arabidopsis OFT1, a gene encoding a putative o-fucosyl transferase functioning in the secretory pathway. oft1-3 mutant pollen showed a significant decrease in transmission efficiency compared to wild type. Decapitation crosses (described here) indicated that the removal of the stigma-style barrier alleviated the transmission deficiency from 858-fold to a 2.6-fold, providing evidence that most, but not all, oft1 pollen deficiencies can be attributed to a reduced ability to penetrate through the stigma-style barrier. This method outlines a genetic strategy to quantify a mutation's impact on the ability of pollen to navigate through the stigma-style barrier on its journey to the ovule.In hermaphroditic flowering plants, the female pistil serves as the main gatekeeper of mate acceptance as several mechanisms are present to prevent fertilization by unsuitable pollen. The characteristic Brassicaceae dry stigma at the top of pistil represents the first layer that requires pollen recognition to elicit appropriate physiological responses from the pistil. Successful pollen-stigma interactions then lead to pollen hydration, pollen germination, and pollen tube entry into the stigmatic surface. To assess these early stages in detail, our lab has used three experimental procedures to quantitatively and qualitatively characterize the outcome of compatible pollen-stigma interactions that would ultimately lead to the successful fertilization. These assays are also useful for assessing self-incompatible pollinations and mutations that affect these pathways. The model organism, Arabidopsis thaliana, offers an excellent platform for these investigations as loss-of-function or gain-of-function mutants can be easily generated using CRISPR/Cas9 technology, existing T-DNA insertion mutant collections, and heterologous expression constructs, respectively. Here, we provide a detailed description of the methods for these inexpensive assays that can be reliably used to assess pollen-stigma interactions and used to identify new players regulating these processes.The number of pollen grains is a critical part of the reproductive strategies in plants and varies greatly between and within species. In agriculture, pollen viability is important for crop breeding. It is a laborious work to count pollen tubes using a counting chamber under a microscope. Here, we present a method of counting the number of pollen grains using a cell counter. In this method, the counting step is shortened to 3 min per flower, which, in our setting, is more than five times faster than the counting chamber method. This technique is applicable to species with a lower and higher number of pollen grains, as it can count particles in a wide range, from 0 to 20,000 particles, in one measurement. The cell counter also estimates the size of the particles together with the number. Because aborted pollen shows abnormal membrane characteristics and/or a distorted or smaller shape, a cell counter can quantify the number of normal and aborted pollen separately. We explain how to count the number of pollen grains and measure pollen size in Arabidopsis thaliana, Arabidopsis kamchatica, and wheat (Triticum aestivum).Background Healthcare work is a risk factor for coronavirus disease 2019 (COVID-19). Objectives To review risk mitigation strategies in ophthalmology during the COVID-19 pandemic. Material and methods Risk mitigation strategies to maintain ophthalmology care, to prevent collateral damage from care disruption, and to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are summarized based on the example of a tertiary referral center in Germany. Results Clinical management strategies included restricted access to buildings, triage systems, telemedicine approaches, strategies for rapid treat and release, and transparent communication strategies. Strategies to protect patients and staff relied on both standard hygiene precautions and the use of personal protective equipment for prevention of droplet infections. Physical barriers supported risk mitigation strategies. Conclusion To maintain ophthalmological care during the COVID-19 pandemic, a coordinated, multifaceted approach using risk mitigation strategies to protect staff, patients, and the public was initiated.Objective We examined barriers to accessing medical care for migrant US-residing Marshallese Islanders. Methods Cross-sectional analyses were conducted to identify potential inequities. Surveys from largely migrant diabetic Marshallese Islanders (n = 255) were compared with nationally representative data. Two major outcomes were assessed including 1-whether or not one reported having forgone medical care in the past year because of cost-and 2-whether or not one reported not having a usual source of care. Results Overall, 74% and 77% of Marshallese Islanders reported forgone care and no usual source of care, respectively, versus 15% and 7% of the US diabetic population. In multivariable analyses, being younger; uninsured; unemployed; male; of lower education; Native American or Hispanic (versus White); and residing in the South were associated with forgone care nationwide, whereas only lacking insurance was associated with forgone care among Marshallese Islanders. Nationwide being younger; uninsured; unmarried; female; of lower education; Native American or Hispanic (versus White); and residing in the South were associated with not having a usual source of care, whereas only being younger and uninsured were associated with not having a usual source of care among Marshallese Islanders.