Enzymatic Steps Research Articles

Abstract 388: Identifying synthetic lethalities in GCN2 KO tumors using an in vivo targeted CRISPRi screen

Abstract The integrated stress response (ISR) is an intracellular signaling pathway that fosters cell adaptation to diverse stresses. The ISR is mediated by a family of four eIF2 kinases that each sense distinct cellular stresses. Activation of the eIF2 kinases results in reprogrammed gene expression through translational and transcriptional mechanisms that help prevent stress damage. Previously, we reported that prostate cancer (PCa) cells rely on GCN2 eIF2 kinase for maintenance of amino acid (AA) homeostasis to sustain growth (Cordova et al., 2022 eLife 11:e81083). GCN2 inhibition through pharmacological or genetic means results in decreased expression of AA transporters, depletion of intracellular AAs, and reduced growth of PCa cells in culture and mouse xenograft models. Although loss of GCN2 results in a significant reduction in PCa cell growth, minimal cell death is observed. We hypothesize that loss of GCN2 in PCa cells results in vulnerabilities that can be exploited and targeted to induce cell death. To optimize GCN2-targeted therapies for PCa, we utilized a targeted CRISPR-interference (CRISPRi) screen in castration-resistant 22Rv1 cells with functional wild type (WT) GCN2 or those with it deleted (GCN2 KO). The CRISPRi screen was performed in cells grown in culture or grown as tumor xenografts in mice and featured a guide library targeting genes that encode druggable targets, including kinases, phosphatases, and metabolic enzymes. While reduced expression of 152 genes was synthetically lethal with loss of GCN2 in the in vitro screen, more genes (202) were synthetically lethal with loss of GCN2 in vivo. Of note, reduced expression of many metabolic enzymes was synthetically lethal with GCN2 ablation only in the in vivo screen, potentially due to the abundance of nutrients in standard culture media. A group of six metabolism-related genes, identified as hits in the in vivo screen and targets of FDA-approved drugs, were selected for follow-up studies. One of these metabolic enzymes was GART, a trifunctional enzyme that carries out 3 of the 10 enzymatic steps in de novo purine biosynthesis. Based on our findings showing that GCN2 maintains AA levels to support nucleotide pools in PCa cells, we initially focused on this critical enzyme. Inhibition of GART using antifolates, including the FDA-approved Pemetrexed, resulted in minimal cell death of parental and GCN2 KO PCa cells cultured in standard culture media. However, when cultured in reduced nutrient media that better mimicked reduced nutrient availability in vivo, GART inhibition resulted in selective death of GCN2 KO cells. Our CRISPRi screening strategy uncovers new vulnerabilities in GCN2-inhibited PCa cells and reveals a general strategy to identify actionable targets with the potential to combine with GCN2 inhibitors for the treatment of PCa. Citation Format: Noah R. Sommers, Ricardo A. Cordova, Angela J. Klunk, Ronald C. Wek, Kirk A. Staschke. Identifying synthetic lethalities in GCN2 KO tumors using an in vivo targeted CRISPRi screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 388.

Abstract 7008: NEBNext® E5hmC-seqTM: Direct detection of 5-hydroxymethylcytosine at single base resolution

Abstract DNA methylation is a critical epigenetic regulator of gene expression during development and in diseases such as cancer. The modified cytosines, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), can be detected by sequencing libraries generated using existing methods: NEBNext® EM-seq™ or bisulfite sequencing. However, these methods cannot differentiate between 5mC and 5hmC. Interest in the role of 5hmC in regulating gene expression and as a cancer biomarker has increased, resulting in the need for robust methods for the identification of specific 5hmC sites. Methods currently exist to enable discrimination of 5mC and 5hmC (for example, oxBS-seq and TAB-seq), however these are based on modifications of bisulfite sequencing, and suffer from reduced data quality due to fragmentation and loss of DNA. Here we describe an enzymatic method that enables specific detection of 5hmC, termed NEBNext® Enzymatic 5hmC-seq (E5hmC-seqTM). E5hmC-seq libraries were generated for 0.1 ng to 200 ng DNA isolated from human brain and E14 mouse embryonic stem cells. Libraries were prepared using NEBNext® Ultra IITM reagents followed by two enzymatic steps to detect 5hmC. In the first step, 5hmCs are glucosylated, which protects them from subsequent deamination by APOBEC. In contrast, cytosines and 5mCs are deaminated resulting in their conversion to uracil and thymine, respectively. This conversion allows discrimination of 5hmC from cytosine and 5mC. Finally, libraries were PCR amplified using NEBNext® Q5U® and sequenced on the Illumina platform. The E5hmC-seq libraries have similar characteristics to EM-seq libraries, including expected insert sizes due to intact DNA molecules, low duplication rates and minimal GC bias. Moreover, E5hmC-seq libraries were well-correlated between inputs and replicates at higher sequencing depths. Fully hydroxymethylated T4147 phage DNA was used as an internal control, with 98-99% of cytosines identified as 5hmC. 5mC and 5hmC levels were also profiled during E14 cell differentiation for a period of 10 days. Interestingly, 5hmC levels decreased whereas 5mC levels increased particularly during the first five days of differentiation. LC-MS/MS quantification of this same DNA mirrored the changes observed by sequencing. E5hmC-seq libraries provide accurate measurements of 5hmC across a wide input range with expected insert sizes and minimal GC bias. Additionally, subtracting E5hmC-seq data from EM-seq data, which detects both 5mC and 5hmC, enables the precise location of individual 5mC and 5hmC sites. The ability to discriminate between 5mC and 5hmC will allow key insights to be made into the role of cytosine modifications in development and cancer. Citation Format: Garrett M. Cammarata, Daniel J. Evanich, V. K. Chaithanya Ponnaluri, Vaishnavi Panchapakesa, Ariel Erijman, Matt A. Campbell, Nan Dai, Bradley W. Langhorst, Louise Williams. NEBNext® E5hmC-seqTM: Direct detection of 5-hydroxymethylcytosine at single base resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7008.

Exploring Simple Particle-Based Signal Amplification Strategies in a Heterogeneous Sandwich Immunoassay with Optical Detection.

Heterogeneous sandwich immunoassays are widely used for biomarker detection in bioanalysis and medical diagnostics. The high analyte sensitivity of the current "gold standard" enzyme-linked immunosorbent assay (ELISA) originates from the signal-generating enzymatic amplification step, yielding a high number of optically detectable reporter molecules. For future point-of-care testing (POCT) and point-of-need applications, there is an increasing interest in more simple detection strategies that circumvent time-consuming and temperature-dependent enzymatic reactions. A common concept to aim for detection limits comparable to those of enzymatic amplification reactions is the usage of polymer nanoparticles (NP) stained with a large number of chromophores. We explored different simple NP-based signal amplification strategies for heterogeneous sandwich immunoassays that rely on an extraction-triggered release step of different types of optically detectable reporters. Therefore, streptavidin-functionalized polystyrene particles (PSP) are utilized as carriers for (i) the fluorescent dye coumarin 153 (C153) and (ii) hemin (hem) molecules catalyzing the luminol reaction enabling chemiluminescence (CL) detection. Additionally, (iii) NP labeling with hemin-based microperoxidase MP11 was assessed. For each amplification approach, the PSP was first systematically optimized regarding size, loading concentration, and surface chemistry. Then, for an immunoassay for the inflammation marker C-reactive protein (CRP), the analyte sensitivity achievable with optimized PSP systems was compared with the established ELISA concept for photometric and CL detection. Careful optimization led to a limit of detection (LOD) of 0.1 ng/mL for MP11-labeled PSP and CL detection, performing similarly well to a photometric ELISA (0.13 ng/mL), which demonstrates the huge potential of our novel assay concept.

Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing

BackgroundThe abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers.ResultsThe aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L− 1 after 48 h under oxygen limited condition in bioreactor fermentations.ConclusionThis work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast’s expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.

A myo-inositol dehydrogenase involved in aminocyclitol biosynthesis of hygromycin A.

Hygromycin A is a broad-spectrum antibiotic that contains a furanose, cinnamic acid, and aminocyclitol moieties. The biosynthesis of the aminocyclitol has been proposed to proceed through six enzymatic steps from glucose 6-phosphate through myo-inositol to the final methylenedioxy-containing aminocyclitol. Although there is some in vivo evidence for this proposed pathway, biochemical support for the individual enzyme activities is lacking. In this study, we verify the activity for one enzyme in this pathway. We show that Hyg17 is a myo-inositol dehydrogenase that has a unique substrate scope when compared to other myo-inositol dehydrogenases. Furthermore, we analyze sequences from the protein family containing Hyg17 and discuss genome mining strategies that target this protein family to identify biosynthetic clusters for natural product discovery.

Integrated preservation of water activity as key to intensified chemoenzymatic synthesis of bio-based styrene derivatives

The valorization of lignin-derived feedstocks by catalytic means enables their defunctionalization and upgrading to valuable products. However, the development of productive, safe, and low-waste processes remains challenging. This paper explores the industrial potential of a chemoenzymatic reaction performing the decarboxylation of bio-based phenolic acids in wet cyclopentyl methyl ether (CPME) by immobilized phenolic acid decarboxylase from Bacillus subtilis, followed by a base-catalyzed acylation. Key-to-success is the continuous control of water activity, which fluctuates along the reaction progress, particularly at high substrate loadings (triggered by different hydrophilicities of substrate and product). A combination of experimentation, thermodynamic equilibrium calculations, and MD simulations revealed the change in water activity which guided the integration of water reservoirs and allowed process intensification of the previously limiting enzymatic step. With this, the highly concentrated sequential two-step cascade (400 g·L–1) achieves full conversions and affords products in less than 3 h. The chemical step is versatile, accepting different acyl donors, leading to a range of industrially sound products. Importantly, the finding that water activity changes in intensified processes is an academic insight that might explain other deactivations of enzymes when used in non-conventional media.

A passage-free, simplified, and scalable novel method for iPSC generation in three-dimensional culture

Induced pluripotent stem cells (iPSCs) have immense potential for use in disease modeling, etiological studies, and drug discovery. However, the current workflow for iPSC generation and maintenance poses challenges particularly during the establishment phase when specialized skills are required. Although three-dimensional culture systems offer scalability for maintaining established iPSCs, the enzymatic dissociation step is complex and time-consuming. In this study, a novel approach was developed to address these challenges by enabling iPSC generation, maintenance, and differentiation without the need for two-dimensional culture or enzymatic dissociation. This streamlined method offers a more convenient workflow, reduces variability and labor for technicians, and opens up avenues for advancements in iPSC research and broader applications.

Impact of Pickering Emulsions Composition on Lipase Catalyzed Peroxyacetic Acid Synthesis

AbstractDue to the need for environmental protection and sustainability, there has been a rising need to replace the traditionally harsh oxidative functionalization process, which is essential for syntheses of various organic compounds, by biocatalysis. However, the investigated alternative processes are still challenged with enzyme instability and/or low specific reaction rate. Due to these still existing challenges and the recognition of Pickering emulsions as an efficient platform for continuous biphasic biocatalysis, the first, enzymatic step of the lipase catalyzed oxidative functionalization was investigated in bioactive Pickering emulsions. The study focused on the impact of Pickering emulsion composition such as the volume phase fraction, type and concentration of the nanoparticles and enzymes on continuous peroxyacetic acids synthesis. The type of nanoparticles used and Candida antarctica lipase B concentration significantly impacted the process while the other named parameters only had a marginal influence.

Cellular Metabolic Labeling of Nucleic Acids and Its Applications

AbstractNucleic acids are considered as fundamental molecules of living systems, which serve as universal genetic information messengers and repositories. To uncover the multifaceted aspects of nucleic acid function and metabolism within cells, labeling has become indispensable. This labeling technique enables the visualization, isolation, characterization, and even quantification of specific nucleic acid species. This review delves into cellular metabolic approaches for nucleic acid labeling, wherein enzymatic steps are employed to introduce nucleic acid modifications before conjugation with a label for detection or isolation. The discussion begins with metabolic labeling for DNA, RNA with various reactive groups and post‐transcriptional RNA labeling for RNA methylation and acetylation sites, emphasizing recent advancements in the field and then, we spotlighted pertinent applications for cellular imaging and sequencing. of labeling.

Ternary complexes of isopentenyl phosphate kinase from Thermococcus paralvinellae reveal molecular determinants of non-natural substrate specificity.

Isopentenyl phosphate kinases (IPKs) have recently garnered attention for their central role in biocatalytic "isoprenol pathways," which seek to reduce the synthesis of the isoprenoid precursors to two enzymatic steps. Furthermore, the natural promiscuity of IPKs toward non-natural alkyl-monophosphates (alkyl-Ps) as substrates has hinted at the isoprenol pathways' potential to access novel isoprenoids with potentially useful activities. However, only a handful of IPK crystal structures have been solved to date, and even fewer of these contain non-natural substrates bound in the active site. The current study sought to elucidate additional ternary complexes bound to non-natural substrates using the IPK homolog from Thermococcus paralvinellae (TcpIPK). Four such structures were solved, each bound to a different non-natural alkyl-P and the phosphoryl donor substrate/product adenosine triphosphate (ATP)/adenosine diphosphate (ADP). As expected, the quaternary, tertiary, and secondary structures of TcpIPK closely resembled those of IPKs published previously, and kinetic analysis of a novel alkyl-P substrate highlighted the potentially dramatic effects of altering the core scaffold of the natural substrate. Even more interesting, though, was the discovery of a trend correlating the position of two α helices in the active site with the magnitude of an IPK homolog's reaction rate for the natural reaction. Overall, the current structures of TcpIPK highlight the importance of continued structural analysis of the IPKs to better understand and optimize their activity with both natural and non-natural substrates.

Inhibitors of NAD+ Production in Cancer Treatment: State of the Art and Perspectives.

The addiction of tumors to elevated nicotinamide adenine dinucleotide (NAD+) levels is a hallmark of cancer metabolism. Obstructing NAD+ biosynthesis in tumors is a new and promising antineoplastic strategy. Inhibitors developed against nicotinamide phosphoribosyltransferase (NAMPT), the main enzyme in NAD+ production from nicotinamide, elicited robust anticancer activity in preclinical models but not in patients, implying that other NAD+-biosynthetic pathways are also active in tumors and provide sufficient NAD+ amounts despite NAMPT obstruction. Recent studies show that NAD+ biosynthesis through the so-called "Preiss-Handler (PH) pathway", which utilizes nicotinate as a precursor, actively operates in many tumors and accounts for tumor resistance to NAMPT inhibitors. The PH pathway consists of three sequential enzymatic steps that are catalyzed by nicotinate phosphoribosyltransferase (NAPRT), nicotinamide mononucleotide adenylyltransferases (NMNATs), and NAD+ synthetase (NADSYN1). Here, we focus on these enzymes as emerging targets in cancer drug discovery, summarizing their reported inhibitors and describing their current or potential exploitation as anticancer agents. Finally, we also focus on additional NAD+-producing enzymes acting in alternative NAD+-producing routes that could also be relevant in tumors and thus become viable targets for drug discovery.

Purine metabolism in plant pathogenic fungi.

In eukaryotic cells, purine metabolism is the way to the production of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and plays key roles in various biological processes. Purine metabolism mainly consists of de novo, salvage, and catabolic pathways, and some components of these pathways have been characterized in some plant pathogenic fungi, such as the rice blast fungus Magnaporthe oryzae and wheat head blight fungus Fusarium graminearum. The enzymatic steps of the de novo pathway are well-conserved in plant pathogenic fungi and play crucial roles in fungal growth and development. Blocking this pathway inhibits the formation of penetration structures and invasive growth, making it essential for plant infection by pathogenic fungi. The salvage pathway is likely indispensable but requires exogenous purines, implying that purine transporters are functional in these fungi. The catabolic pathway balances purine nucleotides and may have a conserved stage-specific role in pathogenic fungi. The significant difference of the catabolic pathway in planta and in vitro lead us to further explore and identify the key genes specifically regulating pathogenicity in purine metabolic pathway. In this review, we summarized recent advances in the studies of purine metabolism, focusing on the regulation of pathogenesis and growth in plant pathogenic fungi.

Isopropanol production using engineered Corynebacterium glutamicum from waste rice straw biomass

Isopropanol, a well-known biofuel, is a widely used precursor for chemical products that can replace nonrenewable petroleum energy. Here, engineered Corynebacterium glutamicum that can effectively utilize all xylose and glucose in agricultural waste rice straw to produce isopropanol was described. First, codon mutations were introduced into transporters and glycolytic-related genes to decrease the glucose preference of C. glutamicum. A more energetically favorable xylose oxidative pathway was constructed that replaced traditional xylose isomerization pathways, saving twice the number of enzymatic steps. A succinate auxiliary module was incorporated into the tricarboxylic acid cycle (TCA), connecting the xylose-utilized pathway with the isopropanol pathway to maximize xylose orientation towards the product. The final engineered strain successfully consumed 100 % of the xylose from NaOH-pretreated, enzyme-hydrolyzed rice straw and effectively synthesized 4.91 g/L isopropanol. This study showcases the successful conversion of agricultural waste into renewable energy, unveiling new possibilities for advancing biological fermentation technology.

Automated enzymatic hydrolysis of urine samples for improved systematic toxicological analysis of drug-facilitated sexual assault cases.

Drug-facilitated sexual assault (DFSA) is characterized by victim incapacitation due to intoxicating substances. Detection of single drug exposure from DFSA requires a systematic toxicological analysis strategy including sensitive methods covering a broad spectrum of substances. The aim of this study was to develop and validate an UHPLC-MS/MS screening method for analysis of samples from DFSA cases and incorporate an automated enzymatic pre-treatment of urine samples into a robotic sample preparation for an efficient laboratory workflow. The screening method included 144 drugs of abuse, pharmaceuticals, and metabolites relevant to DFSA. The use of a recombinant enzyme showed an efficient glucuronide hydrolysis with an average parent drug recovery of 97%. Investigation of matrix effect showed no pronounced ion enhancement or suppression for most analytes (96%), and extraction recovery was above 80% for 97% of analytes. Process efficiency ranged from 50% to 138% for most analytes. The LODs ranged from 0.0001 mg/L to 2 mg/L depending on analyte, and most analytes met the SOFT recommended minimum performance limits. The validated method was applied to authentic suspected DFSA cases (n = 38). Results showed that drugs of abuse, benzodiazepines, and antidepressants were most commonly found in suspected DFSA cases. Incorporation of an automated enzymatic hydrolysis step during sample preparation enables a fast and simple workflow for simultaneous analysis of blood and urine samples for an improved systematic toxicological analysis strategy for DFSA cases.

Pretreatment and enzymatic hydrolysis optimization of lignocellulosic biomass for ethanol, xylitol, and phenylacetylcarbinol co-production using Candida magnoliae.

Cellulosic bioethanol production generally has a higher operating cost due to relatively expensive pretreatment strategies and low efficiency of enzymatic hydrolysis. The production of other high-value chemicals such as xylitol and phenylacetylcarbinol (PAC) is, thus, necessary to offset the cost and promote economic viability. The optimal conditions of diluted sulfuric acid pretreatment under boiling water at 95°C and subsequent enzymatic hydrolysis steps for sugarcane bagasse (SCB), rice straw (RS), and corn cob (CC) were optimized using the response surface methodology via a central composite design to simplify the process on the large-scale production. The optimal pretreatment conditions (diluted sulfuric acid concentration (% w/v), treatment time (min)) for SCB (3.36, 113), RS (3.77, 109), and CC (3.89, 112) and the optimal enzymatic hydrolysis conditions (pretreated solid concentration (% w/v), hydrolysis time (h)) for SCB (12.1, 93), RS (10.9, 61), and CC (12.0, 90) were achieved. CC xylose-rich and CC glucose-rich hydrolysates obtained from the respective optimal condition of pretreatment and enzymatic hydrolysis steps were used for xylitol and ethanol production. The statistically significant highest (p ≤ 0.05) xylitol and ethanol yields were 65% ± 1% and 86% ± 2% using Candida magnoliae TISTR 5664. C. magnoliae could statistically significantly degrade (p ≤ 0.05) the inhibitors previously formed during the pretreatment step, including up to 97% w/w hydroxymethylfurfural, 76% w/w furfural, and completely degraded acetic acid during the xylitol production. This study was the first report using the mixed whole cells harvested from xylitol and ethanol production as a biocatalyst in PAC biotransformation under a two-phase emulsion system (vegetable oil/1M phosphate (Pi) buffer). PAC concentration could be improved by 2-fold compared to a single-phase emulsion system using only 1M Pi buffer.

Establishment of tumor microenvironment-preserving organoid model from patients with intracranial meningioma

BackgroundAlthough meningioma is the most common primary brain tumor, treatments rely on surgery and radiotherapy, and recurrent meningiomas have no standard therapeutic options due to a lack of clinically relevant research models. Current meningioma cell lines or organoids cannot reflect biological features of patient tumors since they undergo transformation along culture and consist of only tumor cells without microenvironment. We aim to establish patient-derived meningioma organoids (MNOs) preserving diverse cell types representative of the tumor microenvironment.MethodsThe biological features of MNOs were evaluated using WST, LDH, and collagen-based 3D invasion assays. Cellular identities in MNOs were confirmed by immunohistochemistry (IHC). Genetic alteration profiles of MNOs and their corresponding parental tumors were obtained by whole-exome sequencing.ResultsMNOs were established from four patients with meningioma (two grade 1 and two grade 2) at a 100% succession rate. Exclusion of enzymatic dissociation-reaggregation steps endowed MNOs with original histology and tumor microenvironment. In addition, we used a liquid media culture system instead of embedding samples into Matrigel, resulting in an easy-to-handle, cost-efficient, and time-saving system. MNOs maintained their functionality and morphology after long-term culture (> 9 wk) and repeated cryopreserving-recovery cycles. The similarities between MNOs and their corresponding parental tumors were confirmed by both IHC and whole-exome sequencing. As a representative application, we utilized MNOs in drug screening, and mifepristone, an antagonist of progesterone receptor, showed prominent antitumor efficacy with respect to viability, invasiveness, and protein expression.ConclusionTaken together, our MNO model overcame limitations of previous meningioma models and showed superior resemblance to parental tumors. Thus, our model could facilitate translational research identifying and selecting drugs for meningioma in the era of precision medicine.

Whole-Genome Bisulfite Sequencing Protocol for the Analysis of Genome-Wide DNA Methylation and Hydroxymethylation Patterns at Single-Nucleotide Resolution.

The analysis of genome-wide epigenomic alterations including DNA methylation and hydroxymethylation has become a subject of intensive research for many biological and clinical questions. DNA methylation analysis bears the particular promise to supplement or replace biochemical and imaging-based tests for the next generation of personalized medicine. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies is currently considered the gold standard for a comprehensive and quantitative analysis of DNA methylation throughout the genome. However, bisulfite conversion does not allow distinguishing between cytosine methylation and hydroxymethylation requiring an additional chemical or enzymatic step to identify hydroxymethylated cytosines. Here, we provide a detailed protocol based on a commercial kit for the preparation of sequencing libraries for the comprehensive whole-genome analysis of DNA methylation and/or hydroxymethylation. The protocol is based on the construction of sequencing libraries from limited amounts of input DNA by ligation of methylated adaptors to the fragmented DNA prior to bisulfite conversion. For analyses requiring a quantitative distinction between 5-methylcytosine and 5-hydroxymethylcytosines levels, an oxidation step is included in the same workflow to perform oxidative bisulfite sequencing (OxBs-Seq). In this case, two sequencing libraries will be generated and sequenced: a classic methylome following bisulfite conversion and analyzing modified cytosines (not distinguishing between methylated and hydroxymethylated cytosines) and a methylome analyzing only methylated cytosines, respectively. Hydroxymethylation levels are deduced from the differences between the two reactions. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocol has been successfully applied to different human and plant samples and yields robust and reproducible results.

Microfluidic isolation of extrachromosomal circular DNA through selective digestion of plasmids and linear DNA using immobilized nucleases.

Extrachromosomal circular DNA (eccDNA) refers to small circular DNA molecules that are distinct from chromosomal DNA and play diverse roles in various biological processes. They are also explored as potential biomarkers for disease diagnosis and precision medicine. However, isolating eccDNA from tissues and plasma is challenging due to low abundance and the presence of interfering linear DNA, requiring time-consuming processes and expert handling. Our study addresses this by utilizing a microfluidic chip tailored for eccDNA isolation, leveraging microfluidic principles for enzymatic removal of non-circular DNA. Our approach involves integrating restriction enzymes into the microfluidic chip, enabling selective digestion of mitochondrial and linear DNA fragments while preserving eccDNA integrity. This integration is facilitated by an in situ photo-polymerized emulsion inside microchannels, creating a porous monolithic structure suitable for immobilizing restriction and exonuclease enzymes (restriction enzyme MssI and exonuclease ExoV). Evaluation using control DNA mixtures and plasma samples with artificially introduced eccDNA demonstrated that our microfluidic chips reduce linear DNA by over 99%, performing comparable to conventional off-chip methods but with substantially faster digestion times, allowing for a remarkable 76-fold acceleration in overall sample preparation time. This technological advancement holds great promise for enhancing the isolation and analysis of eccDNA from tissue and plasma and the potential for increasing the speed of other molecular methods with multiple enzymatic steps.

Association of HMGCR rs17671591 and rs3761740 with lipidemia and statin response in Uyghurs and Han Chinese.

Dyslipidemia plays a very important role in the occurrence and development of cardiovascular disease (CVD). Genetic factors, including single nucleotide polymorphisms (SNPs), are one of the main risks of dyslipidemia. 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) is not only the rate-limiting enzyme step of endogenous cholesterol production, but also the therapeutic target of statins. We investigated 405 Han Chinese and 373 Uyghur people who took statins for a period of time, recorded their blood lipid levels and baseline data before and after oral statin administration, and extracted DNA from each subject for SNP typing of HMGCR rs17671591 and rs3761740. The effects of HMGCR rs17671591 and rs3761740 on lipid levels and the effect of statins on lipid lowering in Han Chinese and Uyghur ethnic groups were studied. In this study, for rs17671591, the CC vs. TT+CT model was significantly correlated with the level of LDL-C before oral statin in the Uyghur population, but there were no correlations between rs17671591 and the level of blood lipid before oral statin in the Han population. The CC vs. TT+CT and CT vs. CC+TT models were significantly correlated with the level of LDL-C after oral statin in the Uyghur population. There was no significant correlation between rs3761740 with blood lipids before and after oral statin in the Han population. For rs3761740, before oral statin, the CC vs. AA+CA model was significantly correlated with the level of LDL-C, and the CA vs. CC+AA model was significantly correlated with the level of total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), and non-high density lipoprotein cholesterol (HDL-C) in the Uyghur population. After oral statin, the CC vs. AA+CA and CA vs. CC+AA models were significantly correlated with the level of TC, LDL-C, and apolipoprotein (APOB), and the C vs. A model was significantly correlated with the level of TC, triglyceride (TG), LDL-C, and APOB in the Uyghur population. Particularly, the CT vs. CC+TT model of rs17671591 was significantly correlated with the changes of LDL-C after oral statin in the Uyghur population. In this study, we also explored the association of rs17671591 and rs3761740 with the rate of dyslipidemia as a reference. We found that HMGCR rs3761740 was correlated with the levels of TC, LDL-C, and non-HDL-C before and after oral statin in Uyghurs, but not with blood lipid levels in the Han population. In the Uyghur population, HMGCR rs17671591 was associated with the level of LDL-C before and after oral statin, and also affected the changes of LDL-C after oral statin.

Identification of intracellular activation mechanism of rhamnogalacturonan-I type polysaccharide purified from Panax ginseng leaves in macrophages and roles of component sugar chains on activity.

This study aimed to investigate the mechanisms underlying intracellular signaling pathways in macrophages in relation to the structural features of rhamnogalacturonan (RG) I-type polysaccharide (PGEP-I) purified from Panax ginseng leaves. For this investigation, we used several specific inhibitors and antibodies against mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and pattern recognition receptors (PRRs). Furthermore, we investigated the roles of component sugar chains on immunostimulating activity through a sequential enzymatic and chemical degradation steps. We found that PGEP-I effectively induced the phosphorylation of several MAPK- and NF-κB-related proteins, such as p38, cJun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p65. Particularly, immunocytochemistry analysis confirmed the PGEP-I-induced translocation of p65 into the nucleus. Furthermore, the breakdown of PGEP-I side chains and main chain during sequential enzymatic and chemical degradation reduced the PGEP-I-induced macrophage cytokine secretion activity. IL-6, TNF-α, and NO secreted by macrophages are associated with several signaling pathway proteins such as ERK, JNK, and NF-κB and several PRRs such as dectin-1, CD11b, CD14, TLR2, TLR4, and SR. Thus, these findings suggest that PGEP-I exerts potent macrophage-activating effects, which can be attributed to its typical RG-I structure comprising arabinan, type II arabinogalactan, and rhamnose-galacturonic acid repeating units in the main chain.