Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity.

A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool.

DELFMUT: duplex sequencing-oriented depth estimation model for stable detection of low-frequency mutations.

Duplex sequencing technology has been widely used in the detection of low-frequency mutations in circulating tumor deoxyribonucleic acid (DNA), but how to determine the sequencing depth and other experimental parameters to ensure the stable detection of low-frequency mutations is still an urgent problem to be solved. The mutation detection rules of duplex sequencing constrain not only the number of mutated templates but also the number of mutation-supportive reads corresponding to each forward and reverse strand of the mutated templates. To tackle this problem, we proposed a Depth Estimation model for stable detection of Low-Frequency MUTations in duplex sequencing (DELFMUT), which models the identity correspondence and quantitative relationships between templates and reads using the zero-truncated negative binomial distribution without considering the sequences composed of bases. The results of DELFMUT were verified by real duplex sequencing data. In the case of known mutation frequency and mutation detection rule, DELFMUT can recommend the combinations of DNA input and sequencing depth to guarantee the stable detection of mutations, and it has a great application value in guiding the experimental parameter setting of duplex sequencing technology.

The loss of heterochromatin is associated with multiscale three-dimensional genome reorganization and aberrant transcription during cellular senescence.

Heterochromatin remodeling is critical for various cell processes. In particular, the "loss of heterochromatin" phenotype in cellular senescence is associated with the process of aging and age-related disorders. Although biological processes of senescent cells, including senescence-associated heterochromatin foci (SAHF) formation, chromosome compaction, and redistribution of key proteins, have been closely associated with high-order chromatin structure, the relationship between the high-order chromatin reorganization and the loss of heterochromatin phenotype during senescence has not been fully understood. By using senescent and deep senescent fibroblasts induced by DNA damage harboring the "loss of heterochromatin" phenotype, we observed progressive 3D reorganization of heterochromatin during senescence. Facultative and constitutive heterochromatin marked by H3K27me3 and H3K9me3, respectively, show different alterations. Facultative heterochromatin tends to switch from the repressive B-compartment to the active A-compartment, whereas constitutive heterochromatin shows no significant changes at the compartment level but enhanced interactions between themselves. Both types of heterochromatin show increased chromatin accessibility and gene expression leakage during senescence. Furthermore, increased chromatin accessibility in potential CTCF binding sites accompanies the establishment of novel loops in constitutive heterochromatin. Finally, we also observed aberrant expression of repetitive elements, including LTR (long terminal repeat) and satellite classes. Overall, facultative and constitutive heterochromatin show both similar and distinct multiscale alterations in the 3D map, chromatin accessibility, and gene expression leakage. This study provides an epigenomic map of heterochromatin reorganization during senescence.

Evaluating methylation of human ribosomal DNA at each CpG site reveals its utility for cancer detection using cell-free DNA.

Ribosomal deoxyribonucleic acid (DNA) (rDNA) repeats are tandemly located on five acrocentric chromosomes with up to hundreds of copies in the human genome. DNA methylation, the most well-studied epigenetic mechanism, has been characterized for most genomic regions across various biological contexts. However, rDNA methylation patterns remain largely unexplored due to the repetitive structure. In this study, we designed a specific mapping strategy to investigate rDNA methylation patterns at each CpG site across various physiological and pathological processes. We found that CpG sites on rDNA could be categorized into two types. One is within or adjacent to transcribed regions; the other is distal to transcribed regions. The former shows highly variable methylation levels across samples, while the latter shows stable high methylation levels in normal tissues but severe hypomethylation in tumors. We further showed that rDNA methylation profiles in plasma cell-free DNA could be used as a biomarker for cancer detection. It shows good performances on public datasets, including colorectal cancer [area under the curve (AUC) = 0.85], lung cancer (AUC = 0.84), hepatocellular carcinoma (AUC = 0.91) and in-house generated hepatocellular carcinoma dataset (AUC = 0.96) even at low genome coverage (<1×). Taken together, these findings broaden our understanding of rDNA regulation and suggest the potential utility of rDNA methylation features as disease biomarkers.

Role of sestrins in metabolic and aging-related diseases.

Sestrins are a type of highly conserved stress-inducing protein that has antioxidant and mTORC1 inhibitory functions. Metabolic dysfunction and aging are the main risk factors for development of human diseases, such as diabetes, neurodegenerative diseases, and cancer. Sestrins have important roles in regulating glucose and lipid metabolism, anti-tumor functions, and aging by inhibiting the reactive oxygen species and mechanistic target of rapamycin complex 1 pathways. In this review, the structure and biological functions of sestrins are summarized, and how sestrins are activated and contribute to regulation of the downstream signal pathways of metabolic and aging-related diseases are discussed in detail with the goal of providing new ideas and therapeutic targets for the treatment of related diseases.

Advances and prospects in microbial production of biotin.

Biotin, serving as a coenzyme in carboxylation reactions, is a vital nutrient crucial for the natural growth, development, and overall well-being of both humans and animals. Consequently, biotin is widely utilized in various industries, including feed, food, and pharmaceuticals. Despite its potential advantages, the chemical synthesis of biotin for commercial production encounters environmental and safety challenges. The burgeoning field of synthetic biology now allows for the creation of microbial cell factories producing bio-based products, offering a cost-effective alternative to chemical synthesis for biotin production. This review outlines the pathway and regulatory mechanism involved in biotin biosynthesis. Then, the strategies to enhance biotin production through both traditional chemical mutagenesis and advanced metabolic engineering are discussed. Finally, the article explores the limitations and future prospects of microbial biotin production. This comprehensive review not only discusses strategies for biotin enhancement but also provides in-depth insights into systematic metabolic engineering approaches aimed at boosting biotin production.

De novo engineering riboflavin production Bacillus subtilis by overexpressing the downstream genes in the purine biosynthesis pathway.

BACKGROUND: Bacillus subtilis is widely used in industrial-scale riboflavin production. Previous studies have shown that targeted mutagenesis of the ribulose 5-phosphate 3-epimerase in B. subtilis can significantly enhance riboflavin production. This modification also leads to an increase in purine intermediate concentrations in the medium. Interestingly, B. subtilis exhibits remarkable efficiency in purine nucleoside synthesis, often exceeding riboflavin yields. These observations highlight the importance of the conversion steps from inosine-5'-monophosphate (IMP) to 2,5-diamino-6-ribosylamino-4(3 H)-pyrimidinone-5'-phosphate (DARPP) in riboflavin production by B. subtilis. However, research elucidating the specific impact of these reactions on riboflavin production remains limited. RESULT: We expressed the genes encoding enzymes involved in these reactions (guaB, guaA, gmk, ndk, ribA) using a synthetic operon. Introduction of the plasmid carrying this synthetic operon led to a 3.09-fold increase in riboflavin production compared to the control strain. Exclusion of gmk from the synthetic operon resulted in a 36% decrease in riboflavin production, which was further reduced when guaB and guaA were not co-expressed. By integrating the synthetic operon into the genome and employing additional engineering strategies, we achieved riboflavin production levels of 2702 mg/L. Medium optimization further increased production to 3477 mg/L, with a yield of 0.0869 g riboflavin per g of sucrose. CONCLUSION: The conversion steps from IMP to DARPP play a critical role in riboflavin production by B. subtilis. Our overexpression strategies have demonstrated their effectiveness in overcoming these limiting factors and enhancing riboflavin production.

Reversed-Phase Medium-Pressure Liquid Chromatography Purification of Omega-3 Fatty Acid Ethyl Esters Using AQ-C18.

Omega-3 fatty acids are in high demand due to their efficacy in treating hypertriglyceridemia and preventing cardiovascular diseases. However, the growth of the industry is hampered by low purity and insufficient productivity. This study aims to develop an efficient RP-MPLC purification method for omega-3 fatty acid ethyl esters with high purity and capacity. The results indicate that the AQ-C18 featuring polar end-capped silanol groups outperformed C18 and others in retention time and impurity separation. By injecting pure fish oil esters with a volume equivalent to a 1.25% bed volume on an AQ-C18 MPLC column using a binary isocratic methanol-water (90:10, v:v) mobile phase at 30 mL/min, optimal omega-3 fatty acid ethyl esters were obtained, with the notable purity of 90.34% and a recovery rate of 74.30%. The total content of EPA and DHA produced increased from 67.91% to 85.27%, meeting the acceptance criteria of no less than 84% set by the 2020 edition of the Pharmacopoeia of the People's Republic of China. In contrast, RP-MPLC significantly enhanced the production efficiency per unit output compared to RP-HPLC. This study demonstrates a pioneering approach to producing omega-3 fatty acid ethyl esters with high purity and of greater quantity using AQ-C18 RP-MPLC, showing this method's significant potential for use in industrial-scale manufacturing.

Withdrawn: Ticagrelor reduces myocardial cell injury induced by myocardial ischemia/reperfusion in diabetic rats by activating JAK2/STAT3.

The above article from Cell Biology International, published online on 5 December 2022, on Wiley Online Library (https://doi.org/10.1002/cbin.11940), has been withdrawn by agreement between the journal Editor in Chief, Sergio Schenkman, and John Wiley and Sons Ltd. The withdrawal has been agreed due to a technical error at the publisher that caused the article to be mistakenly published online although it had been rejected due to evidence that the peer review process for this paper was manipulated.

Angiopoietin-1 promotes triple-negative breast cancer cell proliferation by upregulating carboxypeptidase A4.

Angiopoietin-1 (ANG1) is a pro-angiogenic regulator that contributes to the progression of solid tumors by stimulating the proliferation, migration and tube formation of vascular endothelial cells, as well as the renewal and stability of blood vessels. However, the functions and mechanisms of ANG1 in triple-negative breast cancer (TNBC) are unclear. The clinical sample database shows that a higher level of ANG1 in TNBC is associated with poor prognosis compared to non-TNBC. In addition, knockdown of ANG1 inhibits TNBC cell proliferation and induces cell cycle G1 phase arrest and apoptosis. Overexpression of ANG1 promotes tumor growth in nude mice. Mechanistically, ANG1 promotes TNBC by upregulating carboxypeptidase A4 (CPA4) expression. Overall, the ANG1-CPA4 axis can be a therapeutic target for TNBC.

Evaluation of the reliability of the criteria for assessing prescription quality in Chinese hospitals among pharmacists in China.

BACKGROUND: The reliability of Criteria for Assessing Prescription Quality in Chinese Hospitals (CAPQCH) has never been rigorously verified. This study was designed to verify the reliability of the CAPQCH among pharmacists in China. METHODS: Fourteen pharmacists, 5 from hospitals and 9 from the communities were recruited. We randomly selected 200 prescriptions, and made the testing prescriptions including appropriate and inappropriate testing prescriptions. Pharmacists assessed these testing prescriptions according to criteria in CAPQCH. Three test sets (Set 1, Set 2, and Set 3) were evaluated at 6-month intervals. Before administration of Set 3, pharmacists were informed that achievement on Set 3 would be reflected in their performance appraisal. We also evaluated the performance based on prescription comments before and after combining several confusing criteria. Cohen's Kappa statistic, Fleiss' Kappa statistic, and accuracy were employed to evaluate reliability among pharmacists. RESULTS: Median values of Cohen's Kappa were 0.61 in Set 1, 0.66 in Set 2, and 0.80 in Set 3; reliability is thus substantial. Our data indicate no significant differences between Set 1 and Set 2, whereas Set 3 indicates significantly improved performance. Moreover, combinations of confusing criteria contributed little to improvement of performance in prescription comments. CONCLUSION: Our results verified the reliability of CAPQCH application by working pharmacists. Adding performance based on prescription comments to personal appraisals was effective in improving the quality of prescription comments. These findings may be useful when future modification of the CAPQCH is considered. Moreover, this study contributes to improving the understanding of the prescription assessment situation in China.

Identification of a xylose-inducible promoter and its application for improving vitamin B12 production in Sinorhizobium meliloti.

Sinorhizobium meliloti 320 is a vitamin B12 (VB12 ) high-producing strain that has been isolated and identified in our previous study. Because the regulatory toolbox for S. meliloti is limited, we searched for new genetic components and identified the two xylose-inducible promoters PA and PB based on a promoter-probe vector with a green fluorescent protein (GFP) as reporter. Compared with the ParaA promoter from S. meliloti, both promoters exhibited higher induced expression and lower basal expression. Subsequently, the influence of glucose or sucrose on the expression of GFP driven by these three promoters was assayed. Glucose repressed all three promoters, and the expression of ParaA was the lowest in the presence of glucose. Although sucrose repressed the expression of PA by 35% and improved the expression of ParaA by 16%, the expression level of PA was the highest and was 13% higher than that of ParaA . Lastly, we overexpressed the hemA gene in the C4 pathway using the PA promoter in S. meliloti 320, and the VB12 production of the engineered strain increased by 11%. The VB12 production was further increased by 11% by adding 0.1% sodium succinate to the culture medium.

Histone demethylase KDM7A is required for stem cell maintenance and apoptosis inhibition in breast cancer.

Histone demethylase KDM7A regulates neuronal differentiation and development in mammals. In this study, we found that KDM7A was also required for breast cancer stem cells (BCSCs) maintenance. Silencing KDM7A significantly reduced the BCSCs population and mamosphere formation in vitro, and inhibited breast tumor growth in vivo. Restoring KDM7A expression rescued the defect in stem cell maintenance. Our mechanism analysis suggested that KDM7A upregulated the stemness-associated factors KLF4 and c-MYC for BCSCs maintenance. In addition, KDM7A knockdown promoted apoptosis through decreasing BCL2 expression and BAD phosphorylation in breast cancer (BrCa). Furthermore, restoring KDM7A and BCL2 expression rescued apoptosis inhibition in breast cancer, suggesting that KDM7A inhibited apoptosis by upregulating the BCL2 level in breast cancer. In conclusion, KDM7A promotes cancer stem cell maintenance and apoptosis inhibition in breast cancer.

Optimization of hydrogenobyrinic acid biosynthesis in Escherichia coli using multi-level metabolic engineering strategies.

BACKGROUND: Hydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin B12. The introduction of a heterologous de novo vitamin B12 biosynthesis pathway in Escherichia coli offers an alternative approach for its production. Although E. coli avoids major limitations that currently faced by industrial producers of vitamin B12, such as long growth cycles, the insufficient supply of hydrogenobyrinic acid restricts industrial vitamin B12 production. RESULTS: By designing combinatorial ribosomal binding site libraries of the hemABCD genes in vivo, we found that their optimal relative translational initiation rates are 10:1:1:5. The transcriptional coordination of the uroporphyrinogen III biosynthetic module was realized by promoter engineering of the hemABCD operon. Knockdown of competitive heme and siroheme biosynthesis pathways by RBS engineering enhanced the hydrogenobyrinic acid titer to 20.54 and 15.85 mg L-1, respectively. Combined fine-tuning of the heme and siroheme biosynthetic pathways enhanced the hydrogenobyrinic acid titer to 22.57 mg L-1, representing a remarkable increase of 1356.13% compared with the original strain FH215-HBA. CONCLUSIONS: Through multi-level metabolic engineering strategies, we achieved the metabolic balance of the uroporphyrinogen III biosynthesis pathway, eliminated toxicity due to by-product accumulation, and finally achieved a high HBA titer of 22.57 mg L-1 in E. coli. This lays the foundation for high-yield production of vitamin B12 in E. coli and will hopefully accelerate its industrial production.

Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine.

Due to its clear inherited backgrounds as well as simple and diverse genetic manipulation systems, Bacillus subtilis is the key Gram-positive model bacterium for studies on physiology and metabolism. Furthermore, due to its highly efficient protein secretion system and adaptable metabolism, it has been widely used as a cell factory for microbial production of chemicals, enzymes, and antimicrobial materials for industry, agriculture, and medicine. In this mini-review, we first summarize the basic genetic manipulation tools and expression systems for this bacterium, including traditional methods and novel engineering systems. Secondly, we briefly introduce its applications in the production of chemicals and enzymes, and summarize its advantages, mainly focusing on some noteworthy products and recent progress in the engineering of B. subtilis. Finally, this review also covers applications such as microbial additives and antimicrobials, as well as biofilm systems and spore formation. We hope to provide an overview for novice researchers in this area, offering them a better understanding of B. subtilis and its applications.

A multistrategy approach for improving the expression of E. coli phytase in Pichia pastoris.

Phytase is an additive in animal feed that degrades phytic acid in plant material, reducing feeding costs, and pollution from fecal phosphorus excretion. A multistrategy approach was adopted to improve the expression of E. coli phytase in Pichia pastoris. We determined that the most suitable signal peptide for phytase secretion was an α-factor secretion signal with an initial enzyme activity of 153.51 U/mL. Increasing the copy number of this gene to four increased phytase enzyme activity by 234.35%. PDI overexpression and Pep4 gene knockout increased extracellular phytase production by 35.33% and 26.64%, respectively. By combining favorable factors affecting phytase expression and secretion, the enzyme activity of the phytase-engineered strain was amplified 384.60% compared with that of the original strain. We also evaluated the potential for the industrial production of the engineered strain using a 50-L fed-batch fermenter and achieved a total activity of 30,246 U/mL after 180 h of fermentation.

Metabolic engineering of Escherichia coli for production of chemicals derived from the shikimate pathway.

The shikimate pathway is indispensable for the biosynthesis of natural products with aromatic moieties. These products have wide current and potential applications in food, cosmetics and medicine, and consequently have great commercial value. However, compounds extracted from various plants or synthesized from petrochemicals no longer satisfy the requirements of contemporary industries. As a result, an increasing number of studies has focused on this pathway to enable the biotechnological manufacture of natural products, especially in E. coli. Furthermore, the development of synthetic biology, systems metabolic engineering and high flux screening techniques has also contributed to improving the biosynthesis of high-value compounds based on the shikimate pathway. Here, we review approaches based on a combination of traditional and new metabolic engineering strategies to increase the metabolic flux of the shikimate pathway. In addition, applications of this optimized pathway to produce aromatic amino acids and a range of natural products is also elaborated. Finally, this review sums up the opportunities and challenges facing this field.

Metabolic engineering and optimization of the fermentation medium for vitamin B12 production in Escherichia coli.

Vitamin B12 is a crucial fine chemical that is widely used in the pharmaceutical, food and chemical industries, and its production solely dependents on microbial fermentation. We previously constructed an artificial vitamin B12 biosynthesis pathway in Escherichia coli, but the yield of the engineered strains was low. Here, we removed metabolic bottlenecks of the vitamin B12 biosynthesis pathway in engineered E. coli strains. After screening cobB genes from different sources, optimizing the expression of cobN and customizing the ribosome binding sites of cobS and cobT, the vitamin B12 yield increased to 152.29 µg/g dry cell weight (DCW). Optimization of the downstream module, which converts co(II)byrinic acid a,c-diamide into adenosylcobinamide phosphate, elevated the vitamin B12 yield to 249.04 µg/g DCW. A comparison of a variety of equivalent components indicated that glucose and corn steep liquor are optimal carbon and nitrogen sources, respectively. Finally, an orthogonal array design was applied to determine the optimal concentrations of glucose and nitrogen sources including corn steep liquor and yeast extract, through which a vitamin B12 yield of 530.29 µg/g DCW was obtained. The metabolic modifications and optimization of fermentation conditions achieved in this study offer a basis for further improving vitamin B12 production in E. coli and will hopefully accelerate its industrial application.

Investigation of the characteristics of medication errors and adverse drug reactions using pharmacovigilance data in China.

The aim of this study was to investigate the characteristics of medication errors (MEs) and adverse drug reactions (ADRs) using data from the spontaneous reporting system, which is helpful to understand the actual situation of MEs in China. Data from 2015 in a south distinct in Shanghai were gathered from the spontaneous reporting system and analyzed. The general information, cause of errors, severity, primary diseases, involved system and organs, symptoms, and suspected drugs were investigated. A total of 1290 adverse drug events (ADEs), including 1079 ADRs and 211 MEcs (MEs causing ADE), were reported. Older patients suffered from both ADRs and MEcs (age distribution and dosage form were different between ADRs and MEcs). The main causes of errors were inappropriate usage and dosage of drugs and inappropriate indication selection. Most ADR and MEc cases were mild; the possibility of developing a severe adverse event was quite low. The distribution of the top 10 system and organs, and symptoms involved was significantly different between ADRs and MEcs, with J01 drugs (antibacterials for systemic use) being the leading cause in both. Our results suggested that a direct analysis of data from the spontaneous reporting system is a reliable, and convenient method to investigate MEs and ADRs, despite the existing limitations, and contributes to further understanding the current situation of MEs and ADRs in China.

esATAC: an easy-to-use systematic pipeline for ATAC-seq data analysis.

Summary: ATAC-seq is rapidly emerging as one of the major experimental approaches to probe chromatin accessibility genome-wide. Here, we present 'esATAC', a highly integrated easy-to-use R/Bioconductor package, for systematic ATAC-seq data analysis. It covers essential steps for full analyzing procedure, including raw data processing, quality control and downstream statistical analysis such as peak calling, enrichment analysis and transcription factor footprinting. esATAC supports one command line execution for preset pipelines and provides flexible interfaces for building customized pipelines. Availability and implementation: esATAC package is open source under the GPL-3.0 license. It is implemented in R and C++. Source code and binaries for Linux, MAC OS X and Windows are available through Bioconductor (https://www.bioconductor.org/packages/release/bioc/html/esATAC.html). Supplementary information: Supplementary data are available at Bioinformatics online.