Biology Methods and Protocols最新文献

Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) followed by the 2^-ΔΔCt method is the most common way to measure transcript levels for relative gene expression assays. The quality of an RT-qPCR assay is dependent upon the identification and validation of reference genes to normalize gene expression data. The so-called housekeeping genes are commonly used as internal reference genes because they are assumed to be ubiquitously expressed at stable levels. Commonly, researchers do not validate their reference genes but rely on historical reference genes or previously validated genes from an unrelated experiment. Using previously validated reference genes to assess gene expression changes occurring during malting resulted in extensive variability. Therefore, a new method was tested and validated to circumvent the use of internal reference genes. Total mouse RNA was chosen as the external reference RNA and a suite of primer sets to putatively stable mouse genes was created to identify stably expressed genes for use as an external reference gene. cDNA was created by co-amplifying total mouse RNA, as an RNA spike-in, and barley RNA. When using the external reference genes to normalize malting gene expression data, standard deviations were significantly reduced and significant differences in transcript abundance were observed, whereas when using the internal reference genes, standard deviations were larger with no significant differences seen. Furthermore, external reference genes were more accurate at assessing expression levels in malting and developing grains, whereas the internal reference genes overestimated abundance in developing grains and underestimated abundance in malting grains.

Foldscopes are ultra-low-cost paper microscopes invented by Manu Prakash and Jim Cybulski at Stanford University. They are about as light as a pencil and waterproof, all whilst offering similar optic quality to traditional microscopes. Foldscopes do not require electricity or glass slides to be used, which increases the possibilities of their use in education and outreach activities with children or people with disabilities. In 2019, thanks to a material grant of 100 foldscopes from One World Science and additional purchased foldscopes, I designed and implemented a science workshop called Exploradores del Microcosmos, or Explorers of Microcosmos in English. The aim of the workshop was to help make microscopy more accessible, in particular at underfunded schools, and stimulate active learning about ecosystems and evolution in the participants. Within this article, I describe the workshop and relay my personal insights and reflections on its execution across multiple schools and groups in Mexico.

Human asparagine synthetase (ASNS) catalyzes the conversion of aspartate to asparagine in an ATP-dependent reaction that utilizes glutamine as a nitrogen source while generating glutamate, AMP, and pyrophosphate as additional products. Asparagine Synthetase Deficiency (ASNSD) is an inborn error of metabolism in which children present with homozygous or compound heterozygous mutations in the ASNS gene. These mutations result in ASNS variant protein expression. It is believed that these variant ASNS proteins have reduced enzymatic activity or stability resulting in a lack of sufficient asparagine production for cell function. Reduced asparagine production by ASNS appears to severely hinder fetal brain development. Although a variety of approaches for assaying ASNS activity have been reported, we present here a straightforward method for the in vitro enzymatic analysis by detection of AMP production. Our method overcomes limitations in technical feasibility, signal detection, and reproducibility experienced by prior methods like high-performance liquid chromatography, ninhydrin staining, and radioactive tracing. After purification of FLAG-tagged R49Q, G289A, and T337I ASNS variants from stably expressing HEK 293T cells, this method revealed a reduction in activity of 90, 36, and 96%, respectively. Thus, ASNS protein expression and purification, followed by enzymatic activity analysis, has provided a relatively simple protocol to evaluate structure-function relationships for ASNS variants reported for ASNSD patients.

Pandemics are, by definition, temporary intervals of substantially increased mortality rates experienced across a wide geographic area. One way of assessing the magnitude of the COVID-19 pandemic in the USA has been to compute the differences in life expectancy at birth during a pandemic year and the year before the pandemic. Such comparisons are misleading because they do not account for the duration of the pandemic. The computation of life expectancy in 2019 assumes that people spend their entire lives experiencing prepandemic mortality rates. The computation of life expectancy in 2021 assumes that people live their entire lives in a permanent pandemic. However, people do not live their entire lives experiencing the elevated mortality rates of 2021. This article introduces a method for calculating life expectancy that reflects the experience of people enduring pandemic-level mortality rates for fixed durations. We call the new quantity hybrid life expectancy because it integrates both pandemic and prepandemic mortality rates. The difference in life expectancy at birth in the USA in 2019 with and without a 3-year-long pandemic is 0.01 years. This is because mortality rates at ages 0, 1, and 2 in the pandemic were essentially unchanged from their prepandemic levels. Life expectancy at age 65 incorporating a 3-year pandemic is 0.18 years lower than life expectancy would have been without it. Reductions in life expectancy due to the COVID-19 pandemic using hybrid life expectancy are dramatically lower than differences in life expectancy that do not take the duration of the pandemic into account.

Genetic association signals have been mostly found in noncoding regions through genome-wide association studies (GWAS), suggesting the roles of gene expression regulation in human diseases and traits. However, there has been limited success in colocalizing expression quantitative trait locus (eQTL) with disease-associated variants. Mediated expression score regression (MESC) is a recently proposed method to quantify the proportion of trait heritability mediated by genetically regulated gene expressions (GReX). Applications of MESC to GWAS results have yielded low estimation of mediated heritability for many traits. As MESC relies on stringent independence assumptions between cis-eQTL effects, gene effects, and nonmediated SNP effects, it may fail to characterize the true relationships between those effect sizes, which leads to biased results. Here, we consider the robustness of MESC to investigate whether the low fraction of mediated heritability inferred by MESC reflects biological reality for complex traits or is an underestimation caused by model misspecifications. Our results suggest that MESC may lead to biased estimates of mediated heritability with misspecification of gene annotations leading to underestimation, whereas misspecification of SNP annotations may lead to overestimation. Furthermore, errors in eQTL effect estimates may lead to underestimation of mediated heritability.