IFDlong: an isoform and fusion detector for accurate annotation and quantification of long-read RNA-seq data.

Advancements in long-read transcriptome sequencing (long-RNA-seq) technology have revolutionized the study of isoform diversity. These full-length transcripts enhance the detection of various transcriptome structural variations, including novel isoforms, alternative splicing events, and fusion transcripts. By shifting the open reading frame or altering gene expressions, studies have proved that these transcript alterations can serve as crucial biomarkers for disease diagnosis and therapeutic targets. In this project, we proposed IFDlong, a bioinformatics and biostatistics tool to detect isoform and fusion transcripts using bulk or single-cell long-RNA-seq data. Specifically, the software performed gene and isoform annotation for each long-read, defined novel isoforms, quantified isoform expression by a novel expectation-maximization algorithm, and profiled the fusion transcripts. For evaluation, IFDlong pipeline achieved overall the best performance when compared with several existing tools in large-scale simulation studies. In both isoform and fusion transcript quantification, IFDlong is able to reach more than 0.8 Spearman's correlation with the truth, and more than 0.9 cosine similarity when distinguishing multiple alternative splicing events. In novel isoform simulation, IFDlong can successfully balance the sensitivity (higher than 90%) and specificity (higher than 90%). Furthermore, IFDlong has proved its accuracy and robustness in diverse in-house and public datasets on healthy tissues, cell lines and multiple types of diseases. Besides bulk long-RNA-seq, IFDlong pipeline has proved its compatibility to single-cell long-RNA-seq data. This new software may hold promise for significant impact on long-read transcriptome analysis. The IFDlong software is available at https://github.com/wenjiaking/IFDlong .

TIM22 and TIM29 inhibit HBV replication by up-regulating SRSF1 expression.

Hepatitis B virus (HBV) infection is a serious global health problem. After the viruses infect the human body, the host can respond to the virus infection by coordinating various cellular responses, in which mitochondria play an important role. Evidence has shown that mitochondrial proteins are involved in host antiviral responses. In this study, we found that the overexpression of TIM22 and TIM29, the members of the inner membrane translocase TIM22 complex, significantly reduced the level of intracellular HBV DNA and RNA and secreted HBV surface antigens and E antigen. The effects of TIM22 and TIM29 on HBV replication and transcription is attributed to the reduction of core promoter activity mediated by the increased expression of SRSF1 which acts as a suppressor of HBV replication. This study provides new evidence for the critical role of mitochondria in the resistance of HBV infection and new targets for the development of treatment against HBV infection.

Tick salivary protein Cystatin: structure, anti-inflammation and molecular mechanism.

Ticks are blood-sucking ectoparasites that secrete immunomodulatory substances in saliva to hosts during engorging. Cystatins, a tick salivary protein and natural inhibitor of Cathepsins, are attracting growing interest globally because of the immunosuppressive activities and the feasibility as an antigen for developing anti-tick vaccines. This review outlines the classification and the structure of tick Cystatins, and focuses on the anti-inflammatory effects and molecular mechanisms. Tick Cystatins can be divided into four families based on structures and cystatin 1 and cystatin 2 are the most abundant. They are injected into hosts during blood feeding and effectively mitigate the host inflammatory response. Mechanically, tick Cystatins exert anti-inflammatory properties through the inhibition of TLR-NF-κb, JAK-STAT and p38 MAPK signaling pathways. Further investigations are crucial to confirm the reduction of inflammation in other cell types like neutrophils and mast cells, and fully elucidate the underlying mechanism (like the structural mechanism) to make Cystatin a potential candidate for the development of novel anti-inflammation agents.

[Key Prediction Genes of Nasopharyngeal Carcinoma:Screening Based on Systematic Bioinformatics and Validation by Cell Experiments].

Objective To screen out the potential prediction genes for nasopharyngeal carcinoma(NPC)from the gene microarray data of NPC samples and then verify the genes by cell experiments.Methods The NPC dataset was downloaded from Gene Expression Omnibus,and limma package was employed to screen out the differentially expressed genes.Weighted correlation network analysis package was used for weighted gene co-expression network analysis,and Venn diagram was drawn to find the common genes.The gene ontology annotation and Kyoto encyclopedia of genes and genomes pathway enrichment were then performed for the common genes.The biomarkers for NPC were further explored by protein-protein interaction network,LASSO regression,and non-parametric tests.Real-time quantitative PCR and Western blotting were employed to determine the mRNA and protein levels of key predictors of NPC,so as to verify the screening results.Results There were 622 up-regulated genes and 351 down-regulated genes in the GSE12452 dataset.A total of 116 common genes were obtained by limma analysis and weighted gene co-expression network analysis.The common genes were mainly involved in the biological processes of cell proliferation and regulation and regulation of intercellular adhesion.They were mainly enriched in Rap1,Ras,and tumor necrosis factor signaling pathways.Six key genes were screened out,encoding angiopoietin-2(ANGPT2),dual oxidase 2(DUOX2),coagulation factor Ⅲ(F3),interleukin-15(IL-15),lipocalin-2,and retinoic acid receptor-related orphan receptor B(RORB).Real-time quantitative PCR and Western blotting showed that the NPC cells had up-regulated mRNA and protein levels of ANGPT2 and IL-15 and down-regulated mRNA and protein levels of DUOX2,F3,and RORB,which was consistent with the results predicted by bioinformatics.Conclusion ANGPT2,DUOX2,F3,IL-15 and RORB are potential predictive molecular markers and therapeutic targets for NPC,which may be involved in Rap1,Ras,tumor necrosis factor and other signaling pathways.

Combining virtual screening with cis-/trans-cleavage enzymatic assays effectively reveals broad-spectrum inhibitors that target the main proteases of SARS-CoV-2 and MERS-CoV.

The main protease (Mpro) of SARS-CoV-2 is essential for viral replication, which suggests that the Mpro is a critical target in the development of small molecules to treat COVID-19. This study used an in-silico prediction approach to investigate the complex structure of SARS-CoV-2 Mpro in compounds from the United States National Cancer Institute (NCI) database, then validate potential inhibitory compounds against the SARS-CoV-2 Mpro in cis- and trans-cleavage proteolytic assays. Virtual screening of ∼280,000 compounds from the NCI database identified 10 compounds with highest site-moiety map scores. Compound NSC89640 (coded C1) showed marked inhibitory activity against the SARS-CoV-2 Mpro in cis-/trans-cleavage assays. C1 strongly inhibited SARS-CoV-2 Mpro enzymatic activity, with a half maximal inhibitory concentration (IC50) of 2.69 µM and a selectivity index (SI) of >74.35. The C1 structure served as a template to identify structural analogs based on AtomPair fingerprints to refine and verify structure-function associations. Mpro-mediated cis-/trans-cleavage assays conducted with the structural analogs revealed that compound NSC89641 (coded D2) exhibited the highest inhibitory potency against SARS-CoV-2 Mpro enzymatic activity, with an IC50 of 3.05 µM and a SI of >65.57. Compounds C1 and D2 also displayed inhibitory activity against MERS-CoV-2 with an IC50 of <3.5 µM. Thus, C1 shows potential as an effective Mpro inhibitor of SARS-CoV-2 and MERS-CoV. Our rigorous study framework efficiently identified lead compounds targeting the SARS-CoV-2 Mpro and MERS-CoV Mpro.

The Accurate Imaging of Collective Gold Nanorods with a Polarization-Dependent Dark-Field Light Scattering Microscope.

Anisotropic nanomaterials, such as gold nanorods (AuNRs), could be employed as an orientation platform due to their polarization-dependent surface plasmon resonance. However, a variety of factors would affect the dark-field light scattering imaging of anisotropic nanomaterials, resulting in an unstable signal, which is not advantageous to its further application. In this work, the localized surface plasmon resonance properties of a few AuNRs at different angles were excited by polarization with a conventional dark-field microscope, in which it was found that the ratio of AuNRs' light scattering intensity at different polarization angles (I) to that without a polarizer (I0) reflected the orientation information of AuNRs. Furthermore, the light scattering signal ratio between the parallel polarization (Ip) and that without a polarizer (I0) was closely related with the aspect ratio of AuNRs, which could not be affected by external conditions. To verify this concept, a highly sensitive and selective assay of the alkaline phosphatase activity in human serum was successfully developed based on the chemical etching of AuNRs, resulting in a lower aspect ratio and a lesser Ip/I0. This result holds great promise for polarization-dependent colorimetric nanomaterials and single-particle tracers in living cells.

Case report: Double L611S/V617L JAK2 mutation in a patient with polycythemia vera originally diagnosed with essential thrombocythemia.

Double JAK2 mutations have rarely been described in myeloproliferative neoplasms (MPNs) and are demonstrated to be associated with the polycythemia vera (PV) phenotype. Here, we first report a case of a PV patient with a de novo double L611S/V617L in cis mutation of JAK2. A 40-year-old woman was admitted to the hospital with massive splenomegaly, multiple splenic infarcts, and abdominal pain. She had a 4-year history of erythrocytosis with an antecedent 10-year history of thrombocytosis before coming to our hospital. She was diagnosed with JAK2 L611S/V617L double-mutant PV after a detailed medical examination in 2017. According to the literature, IFNα therapy can induce clinical, hematological, histopathological, and occasionally molecular remission in individuals with MPNs. Our report demonstrates that combination therapy with ruxolitinib and IFNα can lead to a substantial reduction in JAK2 L611S/V617L double-mutant allele burden.

Establishment of a human cell line with a surface display system for screening and optimizing Na+-taurocholate cotransporting polypeptide-binding peptides.

One of the most desirable targets for HBV medications is the sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for the hepatitis B virus (HBV). N-myristoylated preS1 2-48 (Myrcludex B or Hepcludex), an NTCP-binding peptide from the large surface protein of HBV, has been developed as the first-in-class entry inhibitor. However, its relatively large molecular weight contributes to increased immunogenicity and antibody production. As a result, it is preferable to look for an NTCP-binding peptide with a smaller size. To do this, we developed a human cell surface display strategy and screened peptides based on preS1-21. PreS1-21 (genotype D) was extended by 7 random amino acids and fused with mCherry and FasL transmembrane domain. The pooled constructs were transfected into HEK293 cells by using the transposon/transposase system to create a library displaying various peptides on the cell surface with red fluorescence. On the other hand, we expressed NTCP protein fused with EGFP on HEK293 and used the membrane lysate containing NTCP-GFP as the bait protein to select peptides with increased NTCP affinity. After 7 cycles of selection, the deep sequencing results revealed that some polypeptides were more than 1,000 times enriched. Further screening of the mostly enriched 10 peptides yields the peptide preS1-21-pep3. Replacing the preS1-21 sequence of preS1-21-pep3 with those from different genotypes demonstrated that the consensus sequence of genotype A-F had the best performance. The peptide (Myr-preS1-21-pep3) was synthesized and tested on the HepG2-NTCP cell model. The results showed that Myr-preS1-21-pep3 is approximately 10 times more potent than the initial peptide Myr-preS1-21 in preventing HBV infection. In conclusion, we developed a new strategy for screening peptides binding to membrane proteins and identified a new NTCP-binding peptide with a much smaller size than Hepcludex.

MIB2: metal ion-binding site prediction and modeling server.

MOTIVATION: MIB2 (metal ion-binding) attempts to overcome the limitation of structure-based prediction approaches, with many proteins lacking a solved structure. MIB2 also offers more accurate prediction performance and more metal ion types. RESULTS: MIB2 utilizes both the (PS)2 method and the AlphaFold Protein Structure Database to acquire predicted structures to perform metal ion docking and predict binding residues. MIB2 offers marked improvements over MIB by collecting more MIB residue templates and using the metal ion type-specific scoring function. It offers a total of 18 types of metal ions for binding site predictions. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://bioinfo.cmu.edu.tw/MIB2/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Nicotinamide N-Methyltransferase inhibits HBV replication by suppressing NR5A1 expression invitro.

Chronic hepatitis B virus (HBV) infection can lead to fibrosis, liver cirrhosis, and primary hepatocellular carcinoma. Investigating host factors that regulate HBV replication helps to identify antiviral targets. In the current study, we identified Nicotinamide N-Methyltransferase gene (NNMT) as a novel factor that regulates HBV transcription. NNMT is up-regulated at both the mRNA and protein levels in HepG2.2.15 cells compared to HepG2 cells. Overexpression of NNMT reduces HBV replication in several cell models, while knockdown of NNMT enhances HBV DNA levels. Mechanistically, NNMT suppresses HBV DNA replication by inhibiting HBV RNA transcription. The region required for the inhibitory effect of NNMT was narrowed to nt 1672-1708 in enhancer II by luciferase assays. On the other hand, ChIP assays and EMSA results showed that NNMT does not bind to this region substantially, either directly or indirectly. Next, a collection of hepatic nuclear receptor transcription factors was screened to determine whether they were affected by NNMT overexpression. NR5A1, a positive regulator of HBV replication, decreased significantly after NNMT overexpression. Collectively, the findings of this study shed light on the regulation of HBV transcription.

Recognition of a Novel Gene Signature for Human Glioblastoma.

Glioblastoma (GBM) is one of the most common malignant and incurable brain tumors. The identification of a gene signature for GBM may be helpful for its diagnosis, treatment, prediction of prognosis and even the development of treatments. In this study, we used the GSE108474 database to perform GSEA and machine learning analysis, and identified a 33-gene signature of GBM by examining astrocytoma or non-GBM glioma differential gene expression. The 33 identified signature genes included the overexpressed genes COL6A2, ABCC3, COL8A1, FAM20A, ADM, CTHRC1, PDPN, IBSP, MIR210HG, GPX8, MYL9 and PDLIM4, as well as the underexpressed genes CHST9, CSDC2, ENHO, FERMT1, IGFN1, LINC00836, MGAT4C, SHANK2 and VIPR2. Protein functional analysis by CELLO2GO implied that these signature genes might be involved in regulating various aspects of biological function, including anatomical structure development, cell proliferation and adhesion, signaling transduction and many of the genes were annotated in response to stress. Of these 33 signature genes, 23 have previously been reported to be functionally correlated with GBM; the roles of the remaining 10 genes in glioma development remain unknown. Our results were the first to reveal that GBM exhibited the overexpressed GPX8 gene and underexpressed signature genes including CHST9, CSDC2, ENHO, FERMT1, IGFN1, LINC00836, MGAT4C and SHANK2, which might play crucial roles in the tumorigenesis of different gliomas.

The restructure of Au@Ag nanorods for cell imaging with dark-field microscope.

The facile and noninjurious image of cells with high resolution and low toxicity is essential since imaging can offer rich and direct information and insights into metabolic activities, clinical diagnosis, drug delivery and cancer therapy. In this contribution, a smart imaging probe was employed as a contrast agent for dark-field cell imaging. Au core/Ag shell nanorods (Au@Ag NRs) that characterized by X-ray diffraction and X-ray photoelectron spectroscopy, formed Au@Ag@AgI NRs when exposed to iodine, which greatly enhanced the light scattering of nanorods. Herein, the silver shell acted as the response element for iodine as well as the protective agent for Au core. When conjugated with folate, the nanorods can be used to image human cervical cancer cells (HeLa cells) under a dark-field microscope. Nanorods were demonstrated with excellent tumor cellular uptake ability without obvious cytotoxicity, making them ideal candidates in biosensing and bioimaging applications.

Predicting Anticancer Drug Resistance Mediated by Mutations.

Cancer drug resistance presents a challenge for precision medicine. Drug-resistant mutations are always emerging. In this study, we explored the relationship between drug-resistant mutations and drug resistance from the perspective of protein structure. By combining data from previously identified drug-resistant mutations and information of protein structure and function, we used machine learning-based methods to build models to predict cancer drug resistance mutations. The performance of our combined model achieved an accuracy of 86%, a Matthews correlation coefficient score of 0.57, and an F1 score of 0.66. We have constructed a fast, reliable method that predicts and investigates cancer drug resistance in a protein structure. Nonetheless, more information is needed concerning drug resistance and, in particular, clarification is needed about the relationships between the drug and the drug resistance mutations in proteins. Highly accurate predictions regarding drug resistance mutations can be helpful for developing new strategies with personalized cancer treatments. Our novel concept, which combines protein structure information, has the potential to elucidate physiological mechanisms of cancer drug resistance.

Upregulation of TRPM8 can promote the colon cancer liver metastasis through mediating Akt/GSK-3 signal pathway.

This study aims to clarify the function of transient receptor potential melastatin 8 (TRPM8) in colon cancer liver metastasis. First, TRPM8 expression was determined by Western blotting in colon cancer patients with/without liver metastasis. Second, colon cancer cells were grouped into Mock, siCON, and siTRPM8 groups. Then, a series of in vitro experiments were conducted. Last, CT26 cells were used to construct colon cancer liver metastasis models on mice in vivo, followed by comparison of liver metastasis and determination of AKT/glycogen synthase kinase-3ß (GSK-3ß) pathway. Consequently, TRPM8 was upregulated in both colon cancer patients with/without liver metastasis, especially in those with metastasis. Compared with Mock and siCON groups, cells in siTRPM8 group demonstrated significant decreases in clone numbers, cell invasion, and migration; and obvious downregulations of p-AKT/AKT, p-GSK3ß/GSK3ß, Snail, and Vimentin, with an upregulation of E-cadherin. For in vivo experiments, a sharp decrease was observed in metastatic liver of mice in siTRPM8 group, with significant downregulations of p-AKT/AKT, p-GSK3ß/GSK3ß, Snail, and Vimentin and an upregulation of E-cadherin, as compared with Mock and siCON groups. Thus, TRPM8 was upregulated in colon cancer patients with liver metastasis, and silencing TRPM8 may suppress the progression and epithelial-mesenchymal transition of colon cancer cells to block its liver metastasis possibly by inhibiting AKT/GSK-3ß pathway.

The structure-based cancer-related single amino acid variation prediction.

Single amino acid variation (SAV) is an amino acid substitution of the protein sequence that can potentially influence the entire protein structure or function, as well as its binding affinity. Protein destabilization is related to diseases, including several cancers, although using traditional experiments to clarify the relationship between SAVs and cancer uses much time and resources. Some SAV prediction methods use computational approaches, with most predicting SAV-induced changes in protein stability. In this investigation, all SAV characteristics generated from protein sequences, structures and the microenvironment were converted into feature vectors and fed into an integrated predicting system using a support vector machine and genetic algorithm. Critical features were used to estimate the relationship between their properties and cancers caused by SAVs. We describe how we developed a prediction system based on protein sequences and structure that is capable of distinguishing if the SAV is related to cancer or not. The five-fold cross-validation performance of our system is 89.73% for the accuracy, 0.74 for the Matthews correlation coefficient, and 0.81 for the F1 score. We have built an online prediction server, CanSavPre ( http://bioinfo.cmu.edu.tw/CanSavPre/ ), which is expected to become a useful, practical tool for cancer research and precision medicine.

A Regulatory Loop Involving Notch and Wnt Signaling Maintains Leukemia Stem Cells in T-Cell Acute Lymphoblastic Leukemia.

Leukemia-initiating cells play critical role in relapse, resistance to therapies and metastases but the mechanism remains largely elusive. We report that ß-catenin is over-expressed in almost all T-ALL patients and flow sorted ß-cateninhigh fractions are highly resistant to therapy, leading to liver metastases in nude mice as well as dysregulated lncRNAs. Pharmacological inhibition through XAV-939 as well as si-RNA mediated inhibition of ß-catenin is initially effective in re-sensitization to therapy, however, prolonged inhibition shifts dependency from ß-catenin to Notch signaling, with particularly high levels of receptors Notch 1 and Notch 2. The results are verifiable in a cohort of T-ALL patients comprising of responders vs. those who have progressed, with ß-catenin, Notch 1 and Notch 2 elevated in progressed patients. Further, in patients-derived cells, silencing of Notch 1 or Notch 2 does not counter resistance to ß-catenin inhibition, rather pharmacological pan-Notch inhibition is needed to overcome resistance and its effect on in vitro tumor sphere formations as well as in vivo liver metastases. Thus, wnt and Notch signaling are part of a regulatory loop mutually compensating for each other in T-ALL, while ensuring the maintenance of stem cell phenotype.

The depletion of Circ-PRKDC enhances autophagy and apoptosis in T-cell acute lymphoblastic leukemia via microRNA-653-5p/Reelin mediation of the PI3K/AKT/mTOR signaling pathway.

A range of circular (Circ) RNAs have been demonstrated to be of therapeutic significance for the treatment of acute lymphoblastic leukemia (ALL). Here, we investigated the mechanisms underlying the action of Circ-PRKDC and the microRNA-653-5p/Reelin (miR-653-5p/RELN) axis in T-cell ALL (T-ALL).Clinical specimens were obtained from patients with T-ALL (n = 39) and healthy controls (n = 30). In each specimen, we determined the expression levels of Circ-PRKDC, miR-653-5p, and RELN. Human T-ALL cells (Jurkat) were transfected with Circ-PRKDC- or miR-653-5p-related sequences to investigate cell proliferation, apoptosis, and autophagy. We also determined the levels of Circ-PRKDC, miR-653-5p, RELN, and signaling proteins related to phosphoinositide 3-kinase (PI3K), AKT, and mammalian target of rapamycin (mTOR). Finally, we decoded the interactions between Circ-PRKDC, miR-653-5p, and RELN. The expression levels of Circ-PRKDC and RELN were upregulated in T-ALL tissues and cells while the levels of miR-653-5p were downregulated. Thereafter, then silencing of Circ-PRKDC, or the enforced expression of miR-653-5p, repressed the expression of RELN and the activation of the PI3K/AKT/mTOR signaling pathway, thus enhancing cell autophagy and apoptosis, and disrupting cell proliferation. Circ-PRKDC acted a sponge for miR-653-5p while miR-653-5p targeted RELN. The knockdown of miR-653-5p abrogated the silencing of Circ-PRKDC-induced effects in T-ALL cells. The depletion of Circ-PRKDC elevated miR-653-5p to silence RELN-mediated PI3K/AKT/mTOR signaling activation, thereby enhancing autophagy and apoptosis in T-ALL cells.

Size-Dependent Plasmonic Resonance Scattering Characteristics of Gold Nanorods for Highly Sensitive Detection of microRNA-27a.

MicroRNAs, as one kind of significant biomarkers, play indispensable roles in the diagnosis and treatment of cancers. Yet, owing to low expression and high sequence homology, high sensitivity and specificity for microRNA detection are greatly challenging. Herein, a sensitive sensing platform with high specificity was developed for microRNA-27a (miRNA-27a) based on the miRNA-27a-triggered chemical etching of gold nanorods to a smaller size, which was accompanied by a significant blue shift and a large decrease of intensity in the localized surface plasmon resonance (LSPR) scattering and remarkable color variability from red to green. When combined with strand displacement reactions as well as liposome signal amplification and transduction, the proposed bioassay presented high selectivity toward miRNA-27a in a dynamic range from 100 fM to 3 pM and a low limit of detection of 16.5 fM (3σ/k) by dark-field microscopy. Additionally, the remarkable discrimination of single nucleotide difference suggested superior selectivity and was able to detect miRNA-27a extracted from breast cancer cells. The strategy put forward is universal, presenting amusing application prospects in the early diagnosis of various cancers by adapting the corresponding nucleotide sequences.

Catalytic hairpin assembly mediated liposome-encoded magnetic beads for signal amplification of peroxide test strip based point-of-care testing of ricin.

Herein, we propose a new peroxide test strip (PTS) based point-of-care testing (POCT) method to detect ricin B-chain qualitatively and quantitatively by using catalytic hairpin assembly (CHA) mediated liposome-encoded magnetic beads for signal amplification. The sensitivity of this PTS based POCT method was improved significantly because it combined CHA signal amplification and liposome-based signal amplification.

The synergistic effect enhanced chemical etching of gold nanorods for the rapid and sensitive detection of biomarks.

The rapid and sensitive assay is the goal for advancing analytical methods and techniques to save time and achieve the early diagnosis, which are of crucial importance in clinic. The concept of synergistic effects for fastening reaction and improving sensitivity was constructed by introducing iodide (I-) to the typical Fenton's reaction system as a model reaction, which was calculated theoretically and demonstrated experimentally. In an acid medium, the simultaneous presence of Fe2+ and I- could significantly improve the oxidation capability and rate of H2O2 to etch gold nanorods (AuNRs) because of the synergistic effect, which was further successfully applied to the rapid and sensitive assay of cholesterol, glucose and uric acid in human serum. In this work, the synergistic effect greatly improved the reaction rate and sensitivity, which showed potential applications in biological sensing and clinic diagnosis.