Search

Publication bias threatens meta-analysis validity. It is often assessed via the funnel plot; an asymmetric plot implies small-study effects, and publication bias is one cause of the asymmetry. Egger's regression test is a widely used tool to quantitatively assess such asymmetry. It examines the association between the observed effect sizes and their sample SEs; a strong association indicates small-study effects. However, its false positive rates may be inflated if such an association intrinsically exists even if no small-study effects appear, particularly in meta-analyses of odds ratios (ORs). Various alternatives are available to address this problem. They usually replace Egger's regression predictor or response with different measures; consequently, they are powerful only in specific cases. We propose a Bayesian approach to assessing small-study effects in meta-analyses of ORs. It controls false positive rates by using latent "true" SEs, rather than sample SEs, in the Egger-type regression to avoid the intrinsic association between ORs and their SEs. Although "true" SEs are unknown in practice, they can be modeled under the Bayesian framework. We use simulated and real data to compare various methods. When ORs are away from 1, the proposed method may have high powers with controlled false positive rates, while Egger's test has seriously inflated false positive rates; nevertheless, in other situations, some other methods may be superior. In general, the proposed method may serve as an alternative to rule out potential confounding effects caused by the intrinsic association between ORs and their SEs in the assessment of small-study effects.

Approximately 10% of monogenic diseases are caused by nonsense point mutations that generate premature termination codons (PTCs), resulting in a truncated protein and nonsense-mediated decay of the mutant mRNAs. Here, we demonstrate a mini-dCas13X-mediated RNA adenine base editing (mxABE) strategy to treat nonsense mutation-related monogenic diseases via A-to-G editing in a genetically humanized mouse model of Duchenne muscular dystrophy (DMD). Initially, we identified a nonsense point mutation (c.4174C>T, p.Gln1392*) in the DMD gene of a patient and validated its pathogenicity in humanized mice. In this model, mxABE packaged in a single adeno-associated virus (AAV) reached A-to-G editing rates up to 84% in vivo, at least 20-fold greater than rates reported in previous studies using other RNA editing modalities. Furthermore, mxABE restored robust expression of dystrophin protein to over 50% of WT levels by enabling PTC read-through in multiple muscle tissues. Importantly, systemic delivery of mxABE by AAV also rescued dystrophin expression to averages of 37%, 6%, and 54% of WT levels in the diaphragm, tibialis anterior, and heart muscle, respectively, as well as rescued muscle function. Our data strongly suggest that mxABE-based strategies may be a viable new treatment modality for DMD and other monogenic diseases., Introduction The majority of monogenic diseases are caused by point mutations (1), among which approximately 50% can be reversed by A-to-G conversion with adenine base editors (ABEs). Specifically, A-to-G conversion [...]

Background: In forensic investigations, accurate estimation of the postmortem interval (PMI) is an important task, but also an ongoing challenge. Especially in cases where the cadaver has been specially treated, for example, by boiling, the determination of PMI becomes extremely difficult. Previous studies have shown that the succession of the microbial community after decomposition of the cadaver can be used to infer PMI. However, the feasibility of determining the PMI of boiled cadavers has not yet been demonstrated. Aims and Objectives: The main objective of this study was to test whether we can infer PMI of boiled cadavers based on the succession of microbial communities. Materials and Methods: SD rats were killed by cervical dislocation. Subsequently, the rat cadavers were divided into the case (boiled cadavers) and control (unboiled cadavers) groups. Rectal samples were collected from the rats for 45 days and at nine time points. High-throughput sequencing of the 16S rRNA gene was performed to characterize the microbial community in the rectum. Results: The results showed that the composition and relative abundance of bacterial communities at the phylum level were significantly different between the case and control groups. The alpha diversity of the microbial community showed a decreasing trend with the decomposition process. Principal coordinate analysis showed that the case and control groups had obvious patterns along the succession of microbial communities. The rectal microbial communities showed a significant linear trend in the time course of decomposition. A random forest model was used to infer PMI. The goodness-of-fit (R2) of the model was 68.00% and 84.00%, and the mean absolute errors were 2.05 and 1.48 days within 45 days of decomposition for the case and control groups, respectively. Conclusions: Our results suggest that microbial community succession could be a potential method to infer PMI of boiled cadavers. [ABSTRACT FROM AUTHOR]

Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available at present. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1500 fold. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e. G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base editing approach by endowing the engineered DNA glycosylase the capability to selectively excise a new type of substrate. [ABSTRACT FROM AUTHOR]

Catalytically inactive CRISPR‐Cas13 (dCas13)‐based base editors can achieve the conversion of adenine‐to‐inosine (A‐to‐I) or cytidine‐to‐uridine (C‐to‐U) at the RNA level, however, the large size of dCas13 protein limits its in vivo applications. Here, a compact and efficient RNA base editor (ceRBE) is reported with high in vivo editing efficiency. The larger dCas13 protein is replaced with a 199‐amino acid EcCas6e protein, derived from the Class 1 CRISPR family involved in pre‐crRNA processing, and conducted optimization for toxicity and editing efficiency. The ceRBE efficiently achieves both A‐to‐I and C‐to‐U base editing with low transcriptome off‐target in HEK293T cells. The efficient repair of the DMD Q1392X mutation (68.3±10.1%) is also demonstrated in a humanized mouse model of Duchenne muscular dystrophy (DMD) after AAV delivery, achieving restoration of expression for gene products. The study supports that the compact and efficient ceRBE has great potential for treating genetic diseases. [ABSTRACT FROM AUTHOR]

Sperm and eggs carry distinctive epigenetic modifications that are adjusted by reprogramming after fertilization (1). The paternal genome in a zygote undergoes active DNA demethylation before the first mitosis (2,3). The biological significance and mechanisms of this paternal epigenome remodelling have remained unclear (4). Here we report that, within mouse zygotes, oxidation of 5-methylcytosine (5mC) occurs on the paternal genome, changing 5mC into 5-hydroxymethylcytosine (5hmC). Furthermore, we demonstrate that the dioxygenase Tet3 (ref. 5) is enriched specifically in the male pronucleus. In Tet3-deficient zygotes from conditional knockout mice, paternal-genome conversion of 5mC into 5hmC fails to occur and the level of 5mC remains constant. Deficiency of Tet3 also impedes the demethylation process of the paternal Oct4 and Nanog genes and delays the subsequent activation of a paternally derived Oct4 transgene in early embryos. Female mice depleted of Tet3 in the germ line show severely reduced fecundity and their heterozygous mutant offspring lacking maternal Tet3 suffer an increased incidence of developmental failure. Oocytes lacking Tet3 also seem to have a reduced ability to reprogram the injected nuclei from somatic cells. Therefore, Tet3-mediated DNA hydroxylation is involved in epigenetic reprogramming of the zygotic paternal DNA following natural fertilization and may also contribute to somatic cell nuclear reprogramming during animal cloning., To investigate whether loss of DNA methylation in the male pronucleus coincides with oxidation of 5mC to 5hmC, a recently reported type of modification of mammalian DNA (5,6), we performed [...]