Tutorial on how to build non-Markovian dynamic models from molecular dynamics simulations for studying protein conformational changes.

Protein conformational changes play crucial roles in their biological functions. In recent years, the Markov State Model (MSM) constructed from extensive Molecular Dynamics (MD) simulations has emerged as a powerful tool for modeling complex protein conformational changes. In MSMs, dynamics are modeled as a sequence of Markovian transitions among metastable conformational states at discrete time intervals (called lag time). A major challenge for MSMs is that the lag time must be long enough to allow transitions among states to become memoryless (or Markovian). However, this lag time is constrained by the length of individual MD simulations available to track these transitions. To address this challenge, we have recently developed Generalized Master Equation (GME)-based approaches, encoding non-Markovian dynamics using a time-dependent memory kernel. In this Tutorial, we introduce the theory behind two recently developed GME-based non-Markovian dynamic models: the quasi-Markov State Model (qMSM) and the Integrative Generalized Master Equation (IGME). We subsequently outline the procedures for constructing these models and provide a step-by-step tutorial on applying qMSM and IGME to study two peptide systems: alanine dipeptide and villin headpiece. This Tutorial is available at https://github.com/xuhuihuang/GME_tutorials. The protocols detailed in this Tutorial aim to be accessible for non-experts interested in studying the biomolecular dynamics using these non-Markovian dynamic models.

Submillisecond Atomistic Molecular Dynamics Simulations Reveal Hydrogen Bond-Driven Diffusion of a Guest Peptide in Protein-RNA Condensate.

Liquid-liquid phase separation mediated by proteins and/or nucleic acids is believed to underlie the formation of many distinct condensed phases, or membraneless organelles, within living cells. These condensates have been proposed to orchestrate a variety of important processes. Despite recent advances, the interactions that regulate the dynamics of molecules within a condensate remain poorly understood. We performed accumulated 564.7 µs all-atom molecular dynamics (MD) simulations (system size ∼200k atoms) of model condensates formed by a scaffold RNA oligomer and a scaffold peptide rich in arginine (Arg). These model condensates contained one of three possible guest peptides: the scaffold peptide itself or a variant in which six Arg residues were replaced by lysine (Lys) or asymmetric dimethyl arginine (ADMA). We found that the Arg-rich peptide can form the largest number of hydrogen bonds and bind the strongest to the scaffold RNA in the condensate, relative to the Lys- and ADMA-rich peptides. Our MD simulations also showed that the Arg-rich peptide diffused more slowly in the condensate relative to the other two guest peptides, which is consistent with a recent fluorescence microscopy study. There was no significant increase in the number of cation-π interactions between the Arg-rich peptide and the scaffold RNA compared to the Lys-rich and ADMA-rich peptides. Our results indicate that hydrogen bonds between the peptides and the RNA backbone, rather than cation-π interactions, play a major role in regulating peptide diffusion in the condensate.

Goal-Directed, Evidence-Based Care Reduces the Incidence of Perioperative Stress Injury.

Objective: This study aimed to investigate the clinical impact of goal-oriented, evidence-based nursing in preventing perioperative stress injuries. Methods: A total of 380 patients undergoing surgery were allocated into either the control or study group. The study group received goal-oriented, evidence-based nursing, while the control group received routine nursing care. Various perioperative indicators, including operating time, position change time, intraoperative bleeding, and length of hospitalization, were assessed and compared between the two groups. Additionally, the Mini-Nutritional Assessment (MNA) score, Munro score, incidence of stress injuries, and nursing satisfaction rate were compared. Patients with perioperative pressure sores (PS) were further evaluated using the Pressure Ulcer Healing Score (PUSH), Braden Stress Injury Scale (Braden), visual analogue scale of pain (VAS), and wound healing time. Results: The study group exhibited higher MNA levels during and after the operation, while Munro levels were lower compared to the control group (P < .05). The study group demonstrated a shorter length of stay and quicker body position changes than the control group. Incidence of pressure sores (PS) was lower in the study group, accompanied by higher nursing satisfaction. PS patients in the study group had lower VAS and PUSH scores, higher Braden scores, and shorter wound healing times than those in the control group. Conclusion: This study highlights the efficacy of goal-oriented, evidence-based nursing in reducing perioperative stress injuries, advocating its adoption for improved care and patient outcomes. However, the single-center design limits generalizability, necessitating further validation. Ultimately, this approach signifies a step forward in nursing practice, promising better patient recovery and satisfaction.

In Silico-Predicted Dynamic Oxlipidomics MS/MS Library: High-Throughput Discovery and Characterization of Unknown Oxidized Lipids.

Nontargeted lipidomics using liquid chromatography-tandem mass spectrometry can detect thousands of molecules in biological samples. However, the annotation of unknown oxidized lipids is limited to the structures present in libraries, restricting the analysis and interpretation of experimental data. Here, we describe Doxlipid, a computational tool for oxidized lipid annotation that predicts a dynamic MS/MS library for every experiment. Doxlipid integrates three key simulation algorithms to predict libraries and covers 32 subclasses of oxidized lipids from the three main classes. In the evaluation, Doxlipid achieves very high prediction and characterization performance and outperforms the current oxidized lipid annotation methods. Doxlipid, combined with a molecular network, further annotates unknown chemical analogs in the same reaction or pathway. We demonstrate the broad utility of Doxlipid by analyzing oxidized lipids in ferroptosis hepatocellular carcinoma, tissue samples, and other biological samples, substantially advancing the discovery of biological pathways at the trace oxidized lipid level.

All-trans-retinoic acid inhibits hepatocellular carcinoma progression by targeting myeloid-derived suppressor cells and inhibiting angiogenesis.

Hepatocellular carcinoma is characterized by a high infiltration of myeloid-derived suppressor cells (MDSC), which are key drivers of maintaining the immunosuppressive tumor microenvironment. Therefore, targeting MDSCs will improve immunotherapies for cancers. It has been shown that all-trans retinoic acid (ATRA) can differentiate MDSCs into mature myeloid cells. However, whether ATRA suppression of MDSCs function could inhibit the growth of liver cancer remains unknown. Here we found that ATRA significantly inhibited hepatocellular carcinoma promotion, tumor cell proliferation, and angiogenesis markers. Moreover, ATRA decreased the number of mononuclear myeloid-derived suppressor cells (M-MDSCs), granulocytic myeloid-derived suppressor cells (G-MDSCs) and tumor-associated macrophages (TAMs) in spleens. In addition, ATRA significantly reduced the intratumoral infiltrating G-MDSCs and the expression of protumor immunosuppressive molecules (arginase 1, iNOS, IDO and S100A8 + A9), which was accompanied by increased cytotoxic T cell infiltration. Our study demonstrates that ATRA not only has direct intrinsic inhibitory effect on tumor angiogenesis and fibrosis, but also reeducates the tumor microenvironment toward an antitumor phenotype by altering the relative proportion between protumor and antitumor immune cells. This information introduces ATRA as a potential druggable target for treatment of hepatocellular carcinoma.

Disease mutations and phosphorylation alter the allosteric pathways involved in autoinhibition of protein phosphatase 2A.

Mutations in protein phosphatase 2A (PP2A) are connected to intellectual disability and cancer. It has been hypothesized that these mutations might disrupt the autoinhibition and phosphorylation-induced activation of PP2A. Since they are located far from both the active and substrate binding sites, it is unclear how they exert their effect. We performed allosteric pathway analysis based on molecular dynamics simulations and combined it with biochemical experiments to investigate the autoinhibition of PP2A. In the wild type (WT), the C-arm of the regulatory subunit B56δ obstructs the active and substrate binding sites exerting a dual autoinhibition effect. We find that the disease mutant, E198K, severely weakens the allosteric pathways that stabilize the C-arm in the WT. Instead, the strongest allosteric pathways in E198K take a different route that promotes exposure of the substrate binding site. To facilitate the allosteric pathway analysis, we introduce a path clustering algorithm for lumping pathways into channels. We reveal remarkable similarities between the allosteric channels of E198K and those in phosphorylation-activated WT, suggesting that the autoinhibition can be alleviated through a conserved mechanism. In contrast, we find that another disease mutant, E200K, which is in spatial proximity of E198, does not repartition the allosteric pathways leading to the substrate binding site; however, it may still induce exposure of the active site. This finding agrees with our biochemical data, allowing us to predict the activity of PP2A with the phosphorylated B56δ and provide insight into how disease mutations in spatial proximity alter the enzymatic activity in surprisingly different mechanisms.

Alcohol remodels the immunosuppressive tumor microenvironment by targeting myeloid-derived suppressor cells.

The tumor immunosuppressive microenvironment plays an important role in tumor progression. Alcohol is well-known as a regulator of the immune system and several studies have also reported that chronic alcohol intake can activate the immune system. However, it is unclear whether alcohol can affect liver cancer progression by regulating the immunosuppressive microenvironment. In this study, we investigated the effects of different alcohol concentrations on the growth of liver cancer and tumor immune microenvironment. We examined the growth of tumors in mice provided with water, or alcohol (for 2 weeks before tumor injection, and for 3 weeks after tumor injection). We found that alcohol consumption at 5% and 20% inhibited the growth of subcutaneous tumors in hepatocellular carcinoma-bearing mice, whereas 2% alcohol concentration did not significantly inhibit liver cancer growth. The ratio of myeloid-derived suppressor cells (MDSCs) in peripheral blood and spleen of mice treated with 5% or 20% alcohol for 2 weeks before tumor inoculation was downregulated. After tumor inoculation, the proportion of MDSCs in peripheral blood, spleen, and tumor of mice treated with 5% or 20% alcohol for another 3 weeks also decreased and the proportion of CD4+ T cells and CD8+ T cells increased. In addition, Alcohol consumption of 20% reduced levels of the inflammatory factor IL-6 by inhibiting JAK/STAT3 signaling. These results indicate that chronic alcohol consumption may inhibit the growth of liver cancer by regulating MDSCs.

Pouring hot water through drip bags releases thousands of microplastics into coffee.

Microplastics (MPs) released from food packaging have attracted widespread attention. In this study, drip bags made from polyethylene (PE), polypropylene (PP), polyester (PET), and rayon selected from eight brands were employed to investigate MPs releasing. Fourier-transform infrared microspectroscopy (µ-FTIR), optical microscope and scanning electron microscope (SEM) were used to study the effects of brewing time and temperature on the release of MPs. The results showed that a single plastic coffee bag steeped at 95 ℃ for 5 min could release more than 10,000 MPs particles into a cup of coffee. Irregular blocks, long strips, and size range of 10-500 µm MPs were easier to be released, implying that consuming 3-4 cups of coffee will lead to an intake of 50 thousand MPs particles daily. Rayon was the primary type of released MPs, accounting for over 80% of the total amount of the released MPs. Our results are hoped to provide evaluation standards of material selection for processing coffee bags.

Two weeks of high glucose intake is enough to induce intestinal mucosal damage and disturb the balance of the gut microbiota of rats.

High glucose plays a critical role in diabetes. However, the point when high glucose induces diabetes and the organ that triggers the initiation of diabetes remain to be elucidated. The aim of the present study was to clarify the damage induced on different organs of rats, when administered a 2-week infusion of dietary glucose. SD rats (12 weeks old) were randomly divided into normal diet, high glucose infusion (IHG) and oral high glucose (OHG) groups. The levels of fasting blood sugar, tumor necrosis factor (TNF)-α and interleukin (IL)-6 were assessed. Intestine, kidney and liver samples were collected for pathological examination. Feces were collected from the rats for gut microbiota assessment. The results indicated that short-term high glucose induced hyperglycemia that lasted for at least 2 weeks after cessation of high glucose intake. Short-term high glucose also clearly increased the serum levels of IL-6 and TNF-α, led to jejunum mucosa injury and obvious steatosis in hepatocytes, and disturbed the balance of the gut microbiota. OHG led to swelling and necrosis of individual intestinal villi. IHG led to the necrosis and disappearance of cells in the upper layer of the intestinal mucosa. The lesions were confined to the mucosa. A degree of glomerular cell swelling and apoptosis were also observed. Short-term high glucose intake induced lesions in the liver, kidney and intestine, disturbed the balance of the gut microbiota and may consequently induce diabetes complications.

The Ion-Dipole Correction of the 3DRISM Solvation Model to Accurately Compute Water Distributions around Negatively Charged Biomolecules.

The 3D reference interaction site model (3DRISM) provides an efficient grid-based solvation model to compute the structural and thermodynamic properties of biomolecules in aqueous solutions. However, it remains challenging for existing 3DRISM methods to correctly predict water distributions around negatively charged solute molecules. In this paper, we first show that this challenge is mainly due to the orientation of water molecules in the first solvation shell of the negatively charged solute molecules. To properly consider this orientational preference, position-dependent two-body intramolecular correlations of solvent need to be included in the 3DRISM theory, but direct evaluations of these position-dependent two-body intramolecular correlations remain numerically intractable. To address this challenge, we introduce the Ion-Dipole Correction (IDC) to the 3DRISM theory, in which we incorporate the orientation preference of water molecules via an additional solute-solvent interaction term (i.e., the ion-dipole interaction) while keeping the formulism of the 3DRISM equation unchanged. We prove that this newly introduced IDC term is equivalent to an effective direct correlation function which can effectively consider the orientation effect that arises from position dependent two-body correlations. We first quantitatively validate our 3DRISM-IDC theory combined with the PSE3 closure on Cl-, [ClO]- (a two-site anion), and [NO2]- (a three-site anion). For all three anions, we show that our 3DRISM-IDC theory significantly outperforms the 3DRISM theory in accurately predicting the solvation structures in comparison to MD simulations, including RDFs and 3D water distributions. Furthermore, we have also demonstrated that the 3DRISM-IDC can improve the accuracy of hydration free-energy calculation for Cl-. We further demonstrate that our 3DRISM-IDC theory yields significant improvements over the 3DRISM theory when applied to compute the solvation structures for various negatively charged solute molecules, including adenosine triphosphate (ATP), a short peptide containing 19 residues, a DNA hairpin containing 24 nucleotides, and a riboswitch RNA molecule with 77 nucleotides. We expect that our 3DRISM-IDC-PSE3 solvation model holds great promise to be widely applied to study solvation properties for nucleic acids and other biomolecules containing negatively charged functional groups.

Risk Factors for Tigecycline-Associated Hepatotoxicity in Patients in the Intensive Care Units of 2 Tertiary Hospitals: A Retrospective Study.

Tigecycline is a broad-spectrum antibacterial agent. As the incidence of multidrug-resistant bacterial infections has increased in intensive care units (ICUs) over the past decades, tigecycline is often used in ICUs. Information about tigecycline-associated hepatotoxicity in ICU patients is limited. To investigate the potential risk factors for tigecycline-associated hepatotoxicity in ICU patients, 148 patients from 2 centers who had received tigecycline for at least 4 days were retrospectively analyzed. Hepatotoxicity was classified according to the National Cancer Institute Common Terminology Criteria for Adverse Events (5.0) grading system. As a result, 33.8% of patients experienced hepatotoxicity events in the ICU. The multivariate analysis showed that an albumin concentration <25 g/L at baseline (odds ratio, 3.714; 95%CI, 1.082-12.744; P = .037) and treatment duration (odds ratio, 1.094; 95%CI, 1.032-1.160; P = .003) were significantly correlated with tigecycline-associated hepatotoxicity. The median time to onset of hepatotoxicity was 8.0 days. The median duration ICU stay and the in-hospital mortality rate were not different between the hepatotoxicity group and the nonhepatotoxicity group (33.5 days (interquartile range, 21.0-72.0) vs 31.0 days (interquartile range, 21-62.5), P = .850; 38.0% vs 43.8%; P = .504). Therefore, close monitoring of liver function is recommended for patients with baseline albumin concentrations <25 g/L or for patients who receive tigecycline therapy for >8 days.

SynLethDB 2.0: a web-based knowledge graph database on synthetic lethality for novel anticancer drug discovery.

Two genes are synthetic lethal if mutations in both genes result in impaired cell viability, while mutation of either gene does not affect the cell survival. The potential usage of synthetic lethality (SL) in anticancer therapeutics has attracted many researchers to identify synthetic lethal gene pairs. To include newly identified SLs and more related knowledge, we present a new version of the SynLethDB database to facilitate the discovery of clinically relevant SLs. We extended the first version of SynLethDB database significantly by including new SLs identified through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) screening, a knowledge graph about human SLs, a new web interface, etc. Over 16 000 new SLs and 26 types of other relationships have been added, encompassing relationships among 14 100 genes, 53 cancers, 1898 drugs, etc. Moreover, a brand-new web interface has been developed to include modules such as SL query by disease or compound, SL partner gene set enrichment analysis and knowledge graph browsing through a dynamic graph viewer. The data can be downloaded directly from the website or through the RESTful Application Programming Interfaces (APIs). Database URL:  https://synlethdb.sist.shanghaitech.edu.cn/v2.

Circular RNA CircITCH (has-circ-0001141) suppresses hepatocellular carcinoma (HCC) progression by sponging miR-184.

Aberrant expression of circular RNA (circRNA) is involved in the occurrence of various diseases and tumor development, in which plays a vital role, including hepatocellular carcinoma (HCC). Nevertheless, the regulation mechanism and biological function of circITCH in hepatocellular carcinoma (HCC) remain unclear. The expression level of circular RNA itchy E3 ubiquitin protein ligase (circ-ITCH) was identified and validated by real-time polymerase-chain reaction (RT-qPCR) in HCC cell lines. The stability of circITCH was confirmed by Ribonuclease R (RNase R) assay. Subsequently, through silencing and overexpression of circITCH to investigate the functional roles of circITCH in HCC proliferation, invasion, and apoptosis. We also carried out bioinformatics analysis, luciferase reporter assays to define the relationship between microRNA (miR)-184 and circITCH. Moreover, xenograft mouse models and immunohistochemistry were employed to assess the function of circITCH in HCC. CircITCH (hsa_circ_0001141) was a stable circRNA and downregulated in HCC cells. Overexpression of circITCH inhibited cell proliferation, migration, invasion, and promoted apoptosis in vitro and in vivo, whereas knockdown of circITCH had the opposite effects. Mechanistically, miR-184 could be sponged by circITCH, and its overexpression could mitigate the suppressive effects of circITCH overexpression on HCC progression. Through biological website to predict the target genes of miR-184 may be combined. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to investigate mRNAs with significant functional enrichment and pathways, also which its relationship with HCC-related pathway and immune cells. Our findings reveal that circITCH served as a repressor to restrain HCC malignancy via miR-184. Therefore, circITCH may serve as a potential prognostic marker and therapeutic target for HCC.Abbreviations: HCC: hepatocellular carcinoma; CircRNA: Circular RNA; miRNA: MicroRNA; Circ-ITCH: circular RNA itchy E3 ubiquitin protein ligase; RT-qPCR: real-time polymerase-chain reaction; RNase R: Ribonuclease R; CeRNA: competing endogenous RNAs; SiRNA: small interfering RNA.

Myeloid-derived suppressor cells promote tumor growth and sorafenib resistance by inducing FGF1 upregulation and fibrosis.

BACKGROUND: Considerable evidence implicates myeloid-derived suppressor cells (MDSCs) promote tumor progression and drug resistance. Sorafenib is the standard first-line therapy for advanced hepatocellular carcinoma (HCC). Clinical evidence indicates that sorafenib resistance is associated with increased MDSCs, by which MDSCs exerts these effects is obscure. This study aimed to investigate the mechanism of sorafenib resistance mediated by MDSCs. METHODS: A syngeneic mouse-liver cancer cell line BNL was subcutaneously injected to build a tumor-bearing mouse model, and syngeneic MDSCs were adoptive transferred into the tumor-bearing mouse. Tumor tissue was obtained, and transcriptomic analysis of the tumor was carried out on RNAseq data. A coculture system was used to verify the crosstalk between MDSCs and BNL cells. RESULTS: Adoptive MDSCs transfer into tumor-bearing mice induced an increase of tumor-infiltrating MDSCs, which led to tumor growth and impaired antitumor activity of sorafenib in BNL HCC models. MDSCs transfer contributed to tumor fibrosis and tumor-associated fibroblast (CAF) activation, associated with fibroblast growth factor (FGF1) upregulation. In contrast, MDSC depletion by anti-Ly6G+ reduced fibrosis and increased sorafenib antitumor efficacy. Intriguingly, tumor-infiltrating MDSCs barely expressed FGF1. IL-6 derived from MDSCs increased FGF1 expression in BNL liver cancer cells, and anti-IL-6 attenuated this effect in vitro. MAPK pathway, one of the sorafenib targets, is the downstream signaling of FGF1 and is reactivated by MDSCs-mediated FGF1 upregulation. CONCLUSIONS: Our finding demonstrated that MDSCs led to tumor growth and sorafenib resistance via FGF1 upregulation and subsequent indirect CAF activation. We offered a novel mechanism of MDSCs-driven HCC progression and sorafenib resistance.

Free amino acid, 5'-Nucleotide, and lipid distribution in different tissues of blue mussel (Mytilis edulis L.) determined by mass spectrometry based metabolomics.

Blue mussel (Mytilus edulis L.) is a popular, nutritional, and tasty mollusk. To better understand the composition of nutrients and improve further processing of the mussels, metabolomic approaches were used to analyze the free amino acids, 5'-nucleotides, and lipid compositions of different tissues. Our results showed that the viscera and gonad were rich in glutamine and glycine. Adenosine 5'-monophosphate, uridine 5'-monophosphate, guanosine 5'-monophosphate, and inosine 5'-monophosphate were abundant in the mantle, foot, and adductor muscle. Three main types of lipids, phospholipids (PLs), glycerides, and fatty acids (FAs), were semi-quantified. PLs were mainly distributed in the gonad of male mussels and viscera, gonad, and mantles of female mussels. FAs were relatively high in the viscera of males and in the gonad and viscera of females. The viscera of females were rich in phosphatidylcholine, such as 16:0/22:6 and 16:0/20:5. Triglycerides were the key lipids for distinguishing different tissues, especially 16:0/18:1/18:3 and 16:0/18:4/20:5.

NUDT15 genetic testing-guided 6-mercaptopurine dosing in children with ALL likely to be cost-saving in China.

OBJECTIVE: The cost-effectiveness of NUDT15 genetic testing-guided initial 6-mercaptopurine (6-MP) dosing in children with acute lymphoblastic leukemia (ALL) was evaluated. METHODS: A decision tree model was used to evaluate the cost to China's medical system per quality-adjusted life-year (QALY) gained and cost per case of severe leukopenia avoided of NUDT15 genetic testing using public clinical data. RESULTS: Genetic testing-guided initial 6-MP dosing reduced overall costs by $518.61, and prevented 0.221 cases of Grade III-IV leukopenia and increased QALY by 0.00136 per patient. Results were robust in one-way analyses and probabilistic sensitivity analyses. CONCLUSION: NUDT15 genetic testing prior to the initial administration of 6-MP in pediatric ALL patients in China is less expensive than standard dosing without genetic testing.

1'-Ribose cyano substitution allows Remdesivir to effectively inhibit nucleotide addition and proofreading during SARS-CoV-2 viral RNA replication.

COVID-19 has recently caused a global health crisis and an effective interventional therapy is urgently needed. Remdesivir is one effective inhibitor for SARS-CoV-2 viral RNA replication. It supersedes other NTP analogues because it not only terminates the polymerization activity of RNA-dependent RNA polymerase (RdRp), but also inhibits the proofreading activity of intrinsic exoribonuclease (ExoN). Even though the static structure of Remdesivir binding to RdRp has been solved and biochemical experiments have suggested it to be a "delayed chain terminator", the underlying molecular mechanisms is not fully understood. Here, we performed all-atom molecular dynamics (MD) simulations with an accumulated simulation time of 24 microseconds to elucidate the inhibitory mechanism of Remdesivir on nucleotide addition and proofreading. We found that when Remdesivir locates at an upstream site in RdRp, the 1'-cyano group experiences electrostatic interactions with a salt bridge (Asp865-Lys593), which subsequently halts translocation. Our findings can supplement the current understanding of the delayed chain termination exerted by Remdesivir and provide an alternative molecular explanation about Remdesivir's inhibitory mechanism. Such inhibition also reduces the likelihood of Remdesivir to be cleaved by ExoN acting on 3'-terminal nucleotides. Furthermore, our study also suggests that Remdesivir's 1'-cyano group can disrupt the cleavage site of ExoN via steric interactions, leading to a further reduction in the cleavage efficiency. Our work provides plausible and novel mechanisms at the molecular level of how Remdesivir inhibits viral RNA replication, and our findings may guide rational design for new treatments of COVID-19 targeting viral replication.

EmPC-seq: Accurate RNA-sequencing and Bioinformatics Platform to Map RNA Polymerases and Remove Background Error.

Transcription errors can substantially affect metabolic processes in organisms by altering the epigenome and causing misincorporations in mRNA, which is translated into aberrant mutant proteins. Moreover, within eukaryotic genomes there are specific Transcription Error-Enriched genomic Loci (TEELs) which are transcribed by RNA polymerases with significantly higher error rates and hypothesized to have implications in cancer, aging, and diseases such as Down syndrome and Alzheimer's. Therefore, research into transcription errors is of growing importance within the field of genetics. Nevertheless, methodological barriers limit the progress in accurately identifying transcription errors. Pro-Seq and NET-Seq can purify nascent RNA and map RNA polymerases along the genome but cannot be used to identify transcriptional mutations. Here we present background Error Model-coupled Precision nuclear run-on Circular-sequencing (EmPC-seq), a method combining a nuclear run-on assay and circular sequencing with a background error model to precisely detect nascent transcription errors and effectively discern TEELs within the genome.

Targeting Myeloid-Derived Suppressor Cells to Enhance the Antitumor Efficacy of Immune Checkpoint Blockade Therapy.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are activated under pathological conditions, such as cancer, or mature myeloid cells that are converted immune-suppressive cells via tumor-derived exosomes, and potently support the tumor processes at different levels. Currently, multiple studies have demonstrated that MDSCs induce immune checkpoint blockade (ICB) therapy resistance through their contribution to the immunosuppressive network in the tumor microenvironment. In addition, non-immunosuppressive mechanisms of MDSCs such as promotion of angiogenesis and induction of cancer stem cells also exert a powerful role in tumor progression. Thus, MDSCs are potential therapeutic targets to enhance the antitumor efficacy of ICB therapy in cases of multiple cancers. This review focuses on the tumor-promoting mechanism of MDSCs and provides an overview of current strategies that target MDSCs with the objective of enhancing the antitumor efficacy of ICB therapy.

Curcumin attenuates lncRNA H19­induced epithelial­mesenchymal transition in tamoxifen­resistant breast cancer cells.

The H19 long non­coding RNA is involved in the development of tamoxifen resistance in breast cancer. However, the relationship between H19 and the metastatic potential and treatment options for tamoxifen­resistant (TAMR) breast cancer is not completely understood. Curcumin inhibits cellular proliferation, migration and invasiveness in several cancer types, including pancreatic cancer, breast cancer and chronic myeloid leukemia. The present study aimed to investigate the role of H19 in MCF­7/TAMR cell epithelial­mesenchymal transition (EMT), migration and invasiveness, and to assess the ability of curcumin to inhibit H19­mediated effects. Reverse transcription­quantitative PCR and western blot analysis were conducted to detect the gene or protein expression. Cell Counting Kit­8, wound healing and Transwell invasion assays were performed to estimate the capabilities of cell viability, invasion and migration. H19 overexpression enhanced MCF­7/TAMR cell EMT, invasion and migration by upregulating Snail. Furthermore, curcumin notably decreased the expression levels of epithelial marker E­cadherin and markedly increased the expression levels of mesenchymal marker N­cadherin in MCF­7/TAMR cells compared with the control group. In addition, following treatment with curcumin for 48 h, H19 expression was decreased in a dose­dependent manner. Moreover, curcumin treatment for 48 h significantly attenuated H19­induced alterations in N­cadherin and E­cadherin expression levels. Curcumin also prevented H19­induced invasion and migration. The present study indicated that H19 may serve as a promoting factor of EMT, invasion and migration in MCF­7/TAMR cells, suggesting that curcumin may prevent H19­associated metastasis. Therefore, curcumin may serve as a promising therapeutic drug for patients with TAMR breast cancer.