Synthetic messenger RNA (mRNA)−based therapeutics are an increasingly popular approach to gene and cell therapies, genome engineering, enzyme replacement therapy, and now, during the global SARS‐CoV‐2 pandemic, vaccine development. mRNA for such purposes can be synthesized through an enzymatic in vitro transcription (IVT) reaction and formulated for in vivo delivery. Mature mRNA requires a 5′‐cap for gene expression and mRNA stability. There are two methods to add a cap in vitro: via a two‐step multi‐enzymatic reaction or co‐transcriptionally. Co‐transcriptional methods minimize reaction steps and enzymes needed to make mRNA when compared to enzymatic capping. CleanCap® AG co‐transcriptional capping results in 5 mg/ml of IVT with 94% 5′‐cap 1 structure. This is highly efficient compared to first‐generation cap analogs, such as mCap and ARCA, that incorporate cap 0 structures at lower efficiency and reaction yield. This article describes co‐transcriptional capping using TriLink Biotechnology's CleanCap® AG in IVT. © 2021 The Authors. Basic Protocol 1: IVT with CleanCap Basic Protocol 2: mRNA purification and analysis

In the above-referenced article, an error was corrected for the concentration of Triton X-100 in the recipe for 10 × in vitro transcription (IVT) buffer in the Reagents and Solutions section, which now reads 0.02% (not 0.2%).The current version online now includes this correction and may be considered the authoritative version of record.

A non-HLA autoantibody combined with a preformed HLA DSA is associated with an increased mortality risk. Isolated de novo anti-angiotensin II type-1 receptor and anti-endothelin-1 type A receptor autoantibodies are associated with an increased risk of rejection and fibrosis progression. The novel location of C4d staining in proximity to liver sinusoidal endothelial cell capillarization and stellate cell activation demonstrates allograft injury in proximity to non-HLA autoantibody binding.

Patients with chronic hepatitis C virus (HCV) infection vary in their rates of fibrosis progression. The renin-angiotensin system (RAS) regulates fibrosis. Polymorphisms in the genes of the RAS may contribute to the outcome of renal and cardiovascular disease. We studied four RAS gene polymorphisms in 195 patients with chronic HCV infection. Patients were grouped by Ishak stage of fibrosis on liver biopsy: group 1 (fibrosis score 0 or 1; n = 97), group 2 (fibrosis score 2 or 3; n = 73) and group 3 (fibrosis score 4-6; n = 25). Polymorphisms of the angiotensinogen (AGT) gene (M235T and AT-6), the angiotensin I converting enzyme gene and the type 1 angiotensin II receptor gene were assayed. There was no difference in the distribution of these polymorphisms of the RAS between the fibrosis groups. There did not appear to be any increased prevalence of fibrosis if two or even three of the polymorphisms associated with increased RAS effect were present. On multivariate analysis factors significantly associated with fibrosis were necroinflammatory activity (P < 0.001) and age (P < 0.001). No association was identified between these four RAS polymorphisms and fibrosis in chronic HCV infection.

The cytochrome P450-dependent monooxygenases play a central role in the metabolism of chemical carcinogens. The action of these enzymes can lead to either carcinogen detoxication or activation. Differences in P450 expression in animal models give rise to large differences in susceptibility to chemical carcinogens, so genetic polymorphisms in P450 expression may be expected to be an important factor in individual human susceptibility to cancer. Of particular interest is the genetic polymorphism at the cytochrome P450-debrisoquine/sparteine hydroxylase locus (CYP2D6). Although this is a minor liver P450, its polymorphic expression is associated with the abnormal metabolism of at least 30 therapeutic drugs, including P-blockers and tricyclic antidepressants. Conflicting reports have been made on the association of this polymorphism with cancer susceptibility. This disagreement may be attributable to limitations of the phenotyping assay used to identify affected individuals (poor metabolizers, PMs). In order to clarify these anomalies, we have developed a simple DNA-based assay with which we can identify the majority of PMs. The assay is centered around the primary gene defect responsible for the polymorphism, a G to A transition at the junction of intron 3/exon 4 which results in a frame-shift in the resultant mRNA. The frequency of this mutation is 70-80% in PMs. We have studied the frequency of mutated alleles in a control population and in a wide range of cancer patients. No association between this polymorphism and lung cancer susceptibility was observed; however, in other populations of cancer patients some very interesting shifts were found in the proportion of PMs and heterozygotes from that in the normal population.