Benchmarking and optimization of a high-throughput sequencing based method for transgene sequence variant analysis in biotherapeutic cell line development

In recent years, High-Throughput Sequencing (HTS) based methods to detect mutations in biotherapeutic transgene products have become a key quality step deployed during the development of manufacturing cell line clones. Previously we reported on a higher throughput, rapid mutation detection method based on amplicon sequencing (targeting transgene RNA) and detailed its implementation to facilitate cell line clone selection. By gaining experience with our assay in a diverse set of cell line development programs, we improved the computational analysis as well as experimental protocols. Here we report on these improvements as well as on a comprehensive benchmarking of our assay. We evaluated assay performance by mixing amplicon samples of a verified mutated antibody clone with a non-mutated antibody clone to generate spike-in mutations from ∼60% down to ∼0.3% frequencies. We subsequently tested the effect of 16 different sample and HTS library preparation protocols on the assay's ability to quantify mutations and on the occurrence of false-positive background error mutations (artifacts). Our evaluation confirmed assay robustness, established a high confidence limit of detection of ∼0.6%, and identified protocols that reduce error levels thereby significantly reducing a source of false positives that bottlenecked the identification of low-level true mutations.     

First record of organic-walled microfossils from the Tonian Shiwangzhuang Formation of the Tumen Group in western Shandong,North China

Organic-walled microfossils offer important information on the biospheric evolution in pre-Cryogenian and provide biostratigraphic implications for many Proterozoic fossiliferous sequences that are poorly age constrained for the lack of reliable radiometric date. Recently, macroscopic carbonaceous compression fossils have been reported for the first time from the Tonian Shiwangzhuang Formation of the Tumen Group in western Shandong, North China. However, organic-walled microfossils have never been discovered from this formation up till now. To improve our knowledge about Proterozoic biodiversity in North China, we conducted a micropaleontological survey on the argillaceous limestone samples of the Shiwangzhuang Formation, which also contain macroscopic carbonaceous compression fossils, from the Baishicun section in Anqiu, western Shandong, North China. Our investigation shows that the Shiwangzhuang microfossil assemblage is dominated by smooth-walled sphaeromorphic acritarchs and cyanobacterium-like filaments and relatively low abundance of other acritarchs, including 16 taxa, such as Polysphaeroides filliformis, Ostiana microcystis, Simia annulare, ?Jacutianema sp., Arctacellularia tetragonala, Pellicularia tenera, Polythrichoides lineatus, and Navifusa actinomorpha. The Shiwangzhuang organic-walled microfossil assemblage, although consisting of long-ranging and not age diagnostic taxa, is consistent with a Tonian age suggested by macroscopic carbonaceous compression fossils, including the Chuaria-Tawuia and Sinosabellidites-Protoarenicola-Pararenicola assemblages, revealed from the same fossiliferous horizon of the Shiwangzhuang Formation and by organic-walled microfossil assemblage, including the late Mesoproterozoic to Tonian index fossil Trachyhystrichosphaera aimika, from the underlying Tongjiazhuang Formation. However, it is also worth noting that a Cryogenian or Ediacaran age cannot be completely excluded based just on the Shiwangzhuang microfossils because of their limited biostratigraphic utility.     

Five-in-One: Simultaneous isolation of multiple major liver cell types from livers of normal and NASH mice

NASH is a chronic liver disease that affects 3%–6% of individuals and requires urgent therapeutic developments. Isolating the key cell types in the liver is a necessary step towards understanding their function and roles in disease pathogenesis. However, traditional isolation methods through gradient centrifugation can only collect one or a few cell types simultaneously and pose technical difficulties when applied to NASH livers. Taking advantage of identified cell surface markers from liver single-cell RNAseq, here we established the combination of gradient centrifugation and antibody-based cell sorting techniques to isolate five key liver cell types (hepatocytes, endothelial cells, stellate cells, macrophages and other immune cells) from a single mouse liver. This method yielded high purity of each cell type from healthy and NASH livers. Our five-in-one protocol simultaneously isolates key liver cell types with high purity under normal and NASH conditions, enabling for systematic and accurate exploratory experiments such as RNA sequencing.     

Comprehensive landscape and interference of clonal haematopoiesis mutations for liquid biopsy: A Chinese pan-cancer cohort

Tumour-derived DNA found in the plasma of cancer patients provides the probability to detect somatic mutations from circulating cell-free DNA (cfDNA) in plasma samples. However, clonal hematopoiesis (CH) mutations affect the accuracy of liquid biopsy for cancer diagnosis and treatment. Here, we integrated landscape of CH mutations in 11,725 pan-cancer patients of Chinese and explored effects of CH on liquid biopsies in real-world. We first identified 5933 CHs based on panel sequencing of matched DNA of white blood cell and cfDNA on 301 genes for 5100 patients, in which CH number of patients had positive correlation with their diagnosis age. We observed that canonical genes related to CH, including DNMT3A, TET2, ASXL1, TP53, ATM, CHEK2 and SF3B1, were dominant in the Chinese cohort and 13.29% of CH mutations only appeared in the Chinese cohort compared with the Western cohort. Analysis of CH gene distribution bias indicated that CH tended to appear in genes with functions of tyrosine kinase regulation, PI3K-Akt signalling and TP53 activity, suggesting unfavourable effects of CH mutations in cancer patients. We further confirmed effect of driver genes carried by CH on somatic mutations in liquid biopsy of cancer patients. Forty-eight actionable somatic mutations in 17 driver genes were considered CH genes in 92 patients (1.80%) of the Chinese cohort, implying potential impacts of CH on clinical decision-making. Taken together, this study exhibits strong evidence that gene mutations from CH interfere accuracy of liquid biopsies using cfDNA in cancer diagnosis and treatment in real-world.     

RNA-dependent RNA polymerase 1 delays the accumulation of viroids in infected plants

RNA-dependent RNA polymerase 1 (RDR1) is essential for plant antiviral defence, but its role in plant defence against viroid infection remains unknown. The present study aimed to identify the function and mechanism of RDR1 in plant resistance to viroid infection. Overexpression of Nicotiana tabacum RDR1 (NtRDR1) delayed the accumulation of potato spindle tuber viroid (PSTVd) genomic RNA and PSTVd-derived small RNA (sRNA) in Nicotiana benthamiana plants at the early invasion stage, but not in the late stage of infection. Conversely, virus-induced gene silencing of tomato RDR1 (SlRDR1a) increased the susceptibility to PSTVd infection (increased viroid accumulation). Salicylic acid (SA) pretreatment induced SlRDR1a expression and enhanced the defence against PSTVd infection in tomato plants. Our study demonstrated that RDR1 is involved in SA-mediated defence and restricts the early systemic invasion by PSTVd in plants. The decreased PSTVd accumulation in N. benthamiana was not caused by efficient accumulation of PSTVd sRNAs. These results deepen our understanding of the mechanism of RDR1 in plant defence responses to viroid attack.     

Base editing with high efficiency in allotetraploid oilseed rape by A3A‐PBE system

CRISPR/Cas‐base editing is an emerging technology that could convert a nucleotide to another type at the target site. In this study, A3A‐PBE system consisting of human A3A cytidine deaminase fused with a Cas9 nickase and uracil glycosylase inhibitor was established and developed in allotetraploid Brassica napus. We designed three sgRNAs to target ALS, RGA and IAA7 genes, respectively. Base‐editing efficiency was demonstrated to be more than 20% for all the three target genes. Target sequencing results revealed that the editing window ranged from C1 to C10 of the PAM sequence. Base‐edited plants of ALS conferred high herbicide resistance, while base‐edited plants of RGA or IAA7 exhibited decreased plant height. All the base editing could be genetically inherited from T₀ to T₁ generation. Several Indel mutations were confirmed at the target sites for all the three sgRNAs. Furthermore, though no C to T substitution was detected at the most potential off‐target sites, large‐scale SNP variations were determined through whole‐genome sequencing between some base‐edited and wild‐type plants. These results revealed that A3A‐PBE base‐editing system could effectively convert C to T substitution with high‐editing efficiency and broadened editing window in oilseed rape. Mutants for ALS, IAA7 and RGA genes could be potentially applied to confer herbicide resistance for weed control or with better plant architecture suitable for mechanic harvesting.     

Therapeutic efficacy of novel memantine nitrate MN‐08 in animal models of Alzheimer’s disease

Alzheimer''s disease (AD) is a leading cause of dementia in elderly individuals and therapeutic options for AD are very limited. Over‐activation of N‐methyl‐D‐aspartate (NMDA) receptors, amyloid β (Aβ) aggregation, a decrease in cerebral blood flow (CBF), and downstream pathological events play important roles in the disease progression of AD. In the present study, MN‐08, a novel memantine nitrate, was found to inhibit Aβ accumulation, prevent neuronal and dendritic spine loss, and consequently attenuate cognitive deficits in 2‐month‐old APP/PS1 transgenic mice (for a 6‐month preventative course) and in the 8‐month‐old triple‐transgenic (3×Tg‐AD) mice (for a 4‐month therapeutic course). In vitro, MN‐08 could bind to and antagonize NMDA receptors, inhibit the calcium influx, and reverse the dysregulations of ERK and PI3K/Akt/GSK3β pathway, subsequently preventing glutamate‐induced neuronal loss. In addition, MN‐08 had favorable pharmacokinetics, blood‐brain barrier penetration, and safety profiles in rats and beagle dogs. These findings suggest that the novel memantine nitrate MN‐08 may be a useful therapeutic agent for AD.     

Protective Effects of Pidotimod Against Salmonella Infections

International Journal of Peptide Research and Therapeutics - Pidotimod has been shown to exhibit immunomodulatory activities and exert protective effects against bacterial infections. This study...