This study aims to assess the potential clinical application of targeted next generation sequencing (NGS)-based deep sequencing for the detection of clinically relevant mutations in circulating tumor DNA (ctDNA) obtained from non-small cell lung cancer (NSCLC) patients. Targeted deep sequencing was performed to identify High Confidence Somatic Variants (HCSVs) in matched tumor tissue DNA (tDNA) and ctDNA in 50 NSCLC patients. Our results demonstrated that NSCLC patients with Stage IV (61.5%) exhibited a higher concordance rate at the mutation level between plasma ctDNA and tDNA samples than patients with Stage I-III (14.5%). Moreover, it is noteworthy that the allele frequency of these detected HCSVs in ctDNA increased with the advance in tumor stage. Besides, using tDNA as a reference, the sensitivity of plasma ctDNA analyzed by deep NGS for actionable EGFR was much higher in patients with Stage IV (66.6%) than in patients with Stage I-III (7.7%). In conclusion, it appears that ctDNA NGS-based deep sequencing is a feasible approach to identify mutations in patients with Stage IV NSCLC. However, additional methods with higher sensitivity and specificity are needed to improve the successful application of this platform in the earlier stages of NSCLC.

1. Fusidic acid (FA) is widely used for the treatment of infections of sensitive osteomyelitis or skin and soft tissue caused by bacteria. However, the role of cytochrome P450s (CYPs) in the metabolism of FA is unclear. In the present study, we screened the main CYPs for the metabolism of FA and studied its interactions with isoform-selective substrates in vitro. 2. The main CYP450s were screened according to the inhibitory effect of specific inhibitors on the metabolism of FA in human liver microsomes (HLMs) or recombinant CYP isoforms. Enzyme kinetic parameters including K, K, V, and IC were calculated to determine the potential of FA to affect CYP-mediated metabolism of isoform-selective substrates. 3. FA metabolism rate was inhibited by 49.8% and 83.1% under CYP2D6, CYP3A4 selective inhibitors in HLMs. In recombinant experiment, the inhibitory effects on FA metabolism were 83.3% for CYP2D6 and 58.9% for CYP3A4, respectively. FA showed inhibition on CYP2D6 and CYP3A4 with Ks of 13.9 and 38.6 μM, respectively. Other CYP isoforms including CYP1A2, CYP2A6, CYP2C9, CYP2E1, and CYP2C19 showed minimal or no effect on the metabolism of FA. 4. FA was primarily metabolized by CYP2D6 and CYP3A4 and showed a noncompetitive inhibition on CYP2D6 and a mixed competitive inhibition on CYP3A4. Drug-drug interactions between FA and other chemicals, especially with substrates of CYP2D6 and CYP3A4, are phenomena that clinicians need to be aware of and cautious about.

12Reliable identification of brain cell types is necessary for studying brain cell 13 biology. Many brain cell marker genes have been proposed, but their reliability 14 has not been fully validated. We evaluated 540 commonly-used marker genes 15 of astrocyte, microglia, neuron, and oligodendrocyte with six transcriptome and 16 proteome datasets from purified human and mouse brain cells (n=125). By 17 setting new criteria of cell-specific fold change, we identified 22 gold standard 18 marker genes (GSM) with stable cell-specific expression. Our results call into 19 question the specificity of many proposed marker genes. We used two single-20 cell transcriptome datasets from human and mouse brains to explore the co-21 expression of marker genes (n=3337). The mouse co-expression modules were 22 perfectly preserved in human transcriptome, but the reverse was not. Also, we 23 proposed new criteria for identifying marker genes based on both differential 24 expression and co-expression data. We identified 16 novel candidate marker 25 genes (NCM) for mouse and 18 for human independently, which have the 26 potential for use in cell sorting or other tagging techniques. We validated the 27 specificity of GSM and NCM by in-silico deconvolution analysis. Our systematic 28 evaluation provides a list of credible marker genes to facilitate correct cell 29 identification, cell labeling, and cell function studies. 30 31 development of marker genes, which are sets of genes that express specifically 41 in a cell type. Thousands of genes have been proposed as marker genes 2 . One 42 well-known marker gene, RBFOX3 (gene of NeuN), is only expressed in nuclei 43 of most neuronal cell types 3 . Marker genes can be used in several applications. 44Protein products of marker genes can be used to label different cell types, which 45 may be used in fluorescence activated cell sorting (FACS). Marker genes also 46 can be used to determine cell composition in bulk tissue samples. A 47 computational method known as supervised deconvolution was developed to 48 infer cell proportions in bulk tissue samples based on the expression of marker 49 genes 4-6 . This method has been applied to studying the composition of bulk 50 brain samples 7,8 . High specificity of marker genes is critical for generating 51 reliable results in all of these applications. 52Differential gene expression (DGE) analysis of transcriptome or proteome 53 data is the most straightforward way to define the specificity of marker genes 9-54 15 . One of the drawbacks of DGE is that the outcomes is study-dependent. The 55 outcomes are affected by many factors such as species, cell or tissue source, 56 and the data generation platform. Human and mouse genomes are 80% 57 orthologous 16 , but differences in gene expression between species are often 58 greater than those between tissues within one species 17 . Within a species, cells 59 isolated from primary culture or acutely from tissue showed different gene 60 expression patterns 18 . Also, the expression estimates of the mar...

Study question What’s the etiology of severe teratozoospermia characteristic as bubble-shaped acrosome (BSA)? Summary answer Severe teratozoospermia characterized as BSA caused by mutation (c.1024G>A) in actin-like 7A (ACTL7A). What is known already Teratozoospermia is a common cause of male infertility, defined by having a proportion of morphologically normal sperm at less than 4%. It exhibits aberrant sperm phenotypes in the head, neck, midpiece, and endpiece of sperm. Teratozoospermia with ephalic abnormalities are among the most severe and characteristic sperm defects. Some genetic factors are reported to be associated with ephalic abnormalities such as globozoospermia and macrozoospermia. However, other phenotypes and the causative genes of ephalic abnormalities, especially in acrosomal structure, and were largely unknown. Study design, size, duration Severe teratozoospermia were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya from Jan 2019 to Dec 2021. Participants/materials, setting, methods Whole-exome sequencing analysis was used to analyze the genetic factor of man. An Actl7a-mutated mouse model was generated by CRISPER-Cas9. Transmission electron microscopy was used to detect the abnormality of ultrastructure during acrosome biogenesis. Immunostaining was used to analyze the localization of ACTL7A and PLCζ. Immunoprecipitation followed by liquid chromatography-mass spectrometry (LC-MS) was used to select the differentially expressed proteins. ICSI with calcium ionophore exposure was performed in couple with ACTL7A mutation. Main results and the role of chance We found a man with severe teratozoospermia characterized as BSA carrying a mutation (c.1024G>A) in ACTL7A. Homozygous Actl7a-mutated male mice were sterile, and all of sperm showed acrosomal abnormalities. During acrosomal biogenesis, it detected the acrosome detach from the nuclear in Actl7a-mutated mice. Furthermore, mutant ACTL7A failed to attach to the acroplaxome and was discharged by cytoplasmic droplets, which led to the absence of ACTL7A in mature sperm. The mutant sperm failed to activate the oocyte, and PLCζ discharge accompanied by ACTL7A was observed, leading to total fertilization failure (TFF). Immunoprecipitation followed by LC-MS showed that several differentially expressed proteins participate in acrosome assembly and actin filament organization. Furthermore, assisted oocyte activation by calcium ionophore exposure successfully overcame TFF in a couple with an ACTL7A mutation. Limitations, reasons for caution More cases are needed to demonstrate the relationship between mutation and phenotype. Wider implications of the findings Our study defined a novel phenotype of the acrosomal abnormality characterized as BSA and revealed the underlying mechanism of mutation in ACTL7A and provided a genetic marker and a therapeutic option for male infertility. Trial registration number Not Applicable