Human Transcription Research Articles

Abstract 2446 Evaluation of DNA-binding preferences of human SIX family transcription factors using DAP-seq

Abstract 2446 Evaluation of DNA-binding preferences of human SIX family transcription factors using DAP-seq

DNA binding redistributes activation domain ensemble and accessibility in pioneer factor Sox2

More than 1600 human transcription factors orchestrate the transcriptional machinery to control gene expression and cell fate. Their function is conveyed through intrinsically disordered regions (IDRs) containing activation or repression domains but lacking quantitative structural ensemble models prevents their mechanistic decoding. Here we integrate single-molecule FRET and NMR spectroscopy with molecular simulations showing that DNA binding can lead to complex changes in the IDR ensemble and accessibility. The C-terminal IDR of pioneer factor Sox2 is highly disordered but its conformational dynamics are guided by weak and dynamic charge interactions with the folded DNA binding domain. Both DNA and nucleosome binding induce major rearrangements in the IDR ensemble without affecting DNA binding affinity. Remarkably, interdomain interactions are redistributed in complex with DNA leading to variable exposure of two activation domains critical for transcription. Charged intramolecular interactions allowing for dynamic redistributions may be common in transcription factors and necessary for sensitive tuning of structural ensembles.

Abstract B015: Tobacco-derived miRNAs and their crosstalk with human transcripts towards carcinogenesis: A cross kingdom approach

Abstract There are more than 70 different species of Nicotiana reported around the world; however, Nicotiana tabacum (common tobacco) and Nicotiana rustica (aztec tobacco) are widely used for snuffing, chewing, smoking, etc., worldwide. Early reports have described that tobacco causes cancer and is associated with many neurological and cardiac disorders. The current study was an effort to understand the paradoxical regulatory mechanism of tobacco-derived miRNAs on human transcripts at 3’ untranslated regions (UTRs) through a computational and experimental genomics approach. This is the first attempt at cross-kingdom analysis where tobacco-specific miRNAs were predicted from different available sources like express sequence tags (EST), genome-wide approaches, and deep sequencing. The miRNA-derived plant targets were identified using stringent bioinformatics methods. A total of 164 mature reported miRNAs, 38 miRNAs from EST datasets, and 21 miRNAs from the genome were studied in detail for cross-kingdom regulation. The potential predicted and reported tobacco-derived miRNAs were analysed against human transcripts. Interestingly, we found tobacco-derived miRNAs such as miR156, miR403, miR7997, miR8011, and miR8036 target tumour suppressor genes and have a putative role in pathways in cancer. Collectively, we have identified a comprehensive set of miRNAs from small RNA sequencing of N. rustica and revealed 842 new miRNAs that have not been reported yet and have putative targets in cancer pathways like AMPK and WNT signalling pathways. Conclusively, this study is the first report of intra- and interspecies relationships using N. tabacum miRNAs with Human and has created a clear road map to focus on specific cancer targets that are modulated by tobacco-derived miRNAs for future studies. Citation Format: Saumya Patel, Manasi Bhavsar, Rakesh Rawal. Tobacco-derived miRNAs and their crosstalk with human transcripts towards carcinogenesis: A cross kingdom approach [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B015.

Cirscan: a shiny application to identify differentially active sponge mechanisms and visualize circRNA–miRNA–mRNA networks

BackgroundNon-coding RNAs represent a large part of the human transcriptome and have been shown to play an important role in disease such as cancer. However, their biological functions are still incompletely understood. Among non-coding RNAs, circular RNAs (circRNAs) have recently been identified for their microRNA (miRNA) sponge function which allows them to modulate the expression of miRNA target genes by taking on the role of competitive endogenous RNAs (ce-circRNAs). Today, most computational tools are not adapted to the search for ce-circRNAs or have not been developed for the search for ce-circRNAs from user’s transcriptomic data.ResultsIn this study, we present Cirscan (CIRcular RNA Sponge CANdidates), an interactive Shiny application that automatically infers circRNA–miRNA–mRNA networks from human multi-level transcript expression data from two biological conditions (e.g. tumor versus normal conditions in the case of cancer study) in order to identify on a large scale, potential sponge mechanisms active in a specific condition. Cirscan ranks each circRNA–miRNA–mRNA subnetwork according to a sponge score that integrates multiple criteria based on interaction reliability and expression level. Finally, the top ranked sponge mechanisms can be visualized as networks and an enrichment analysis is performed to help its biological interpretation. We showed on two real case studies that Cirscan is capable of retrieving sponge mechanisms previously described, as well as identifying potential novel circRNA sponge candidates.ConclusionsCirscan can be considered as a companion tool for biologists, facilitating their ability to prioritize sponge mechanisms for experimental validations and identifying potential therapeutic targets. Cirscan is implemented in R, released under the license GPL-3 and accessible on GitLab (https://gitlab.com/geobioinfo/cirscan_Rshiny). The scripts used in this paper are also provided on Gitlab (https://gitlab.com/geobioinfo/cirscan_paper).

Detection of new pioneer transcription factors as cell-type-specific nucleosome binders.

Wrapping of DNA into nucleosomes restricts accessibility to DNA and may affect the recognition of binding motifs by transcription factors. A certain class of transcription factors, the pioneer transcription factors, can specifically recognize their DNA binding sites on nucleosomes, initiate local chromatin opening, and facilitate the binding of co-factors in a cell-type-specific manner. For the majority of human pioneer transcription factors, the locations of their binding sites, mechanisms of binding, and regulation remain unknown. We have developed a computational method to predict the cell-type-specific ability of transcription factors to bind nucleosomes by integrating ChIP-seq, MNase-seq, and DNase-seq data with details of nucleosome structure. We have demonstrated the ability of our approach in discriminating pioneer from canonical transcription factors and predicted new potential pioneer transcription factors in H1, K562, HepG2, and HeLa-S3 cell lines. Last, we systematically analyzed the interaction modes between various pioneer transcription factors and detected several clusters of distinctive binding sites on nucleosomal DNA.

Interaction network of human early embryonic transcription factors

Embryonic genome activation (EGA) occurs during preimplantation development and is characterized by the initiation of de novo transcription from the embryonic genome. Despite its importance, the regulation of EGA and the transcription factors involved in this process are poorly understood. Paired-like homeobox (PRDL) family proteins are implicated as potential transcriptional regulators of EGA, yet the PRDL-mediated gene regulatory networks remain uncharacterized. To investigate the function of PRDL proteins, we are identifying the molecular interactions and the functions of a subset family of the Eutherian Totipotent Cell Homeobox (ETCHbox) proteins, seven PRDL family proteins and six other transcription factors (TFs), all suggested to participate in transcriptional regulation during preimplantation. Using mass spectrometry-based interactomics methods, AP-MS and proximity-dependent biotin labeling, and chromatin immunoprecipitation sequencing we derive the comprehensive regulatory networks of these preimplantation TFs. By these interactomics tools we identify more than a thousand high-confidence interactions for the 21 studied bait proteins with more than 300 interacting proteins. We also establish that TPRX2, currently assigned as pseudogene, is a transcriptional activator.

Detection of new pioneer transcription factors as cell-type-specific nucleosome binders

Wrapping of DNA into nucleosomes restricts accessibility to DNA and may affect the recognition of binding motifs by transcription factors. A certain class of transcription factors, the pioneer transcription factors, can specifically recognize their DNA binding sites on nucleosomes, initiate local chromatin opening, and facilitate the binding of co-factors in a cell-type-specific manner. For the majority of human pioneer transcription factors, the locations of their binding sites, mechanisms of binding, and regulation remain unknown. We have developed a computational method to predict the cell-type-specific ability of transcription factors to bind nucleosomes by integrating ChIP-seq, MNase-seq, and DNase-seq data with details of nucleosome structure. We have demonstrated the ability of our approach in discriminating pioneer from canonical transcription factors and predicted new potential pioneer transcription factors in H1, K562, HepG2, and HeLa-S3 cell lines. Last, we systematically analyzed the interaction modes between various pioneer transcription factors and detected several clusters of distinctive binding sites on nucleosomal DNA.

Lnc RNA LOC102163816 Promotes Proliferation of Porcine Follicular Granulosa Cells Via miR-455-3p/PTK2B/PI3K/AKT Pathway.

The proliferation and differentiation of granulosa cells (GCs) is a crucial process in follicular development. However, the molecular regulatory mechanism of follicular proliferation and differentiation of GCs needs further research. Studies have reported that follicular fluid exosomes are involved in regulation of proliferation of GCs, but the specific mechanism is unclear. This study demonstrated that LOC102163816 is upregulated in porcine GCs treated with follicular fluid exosomes. Further study defined LOC102163816 to be a novel long noncoding RNA that is highly homologous to human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and enriched in porcine follicular fluid exosomes. We have speculated that LOC102163816 might have a cell-proliferative effect similar to that of MALAT1. We found that overexpression of LOC102163816 promoted transition from the G1 phase to the S phase of the cell cycle, thereby promoting proliferation of GCs. To explore the specific mechanism underlying this promotion of proliferation, miRNA sequencing was performed after overexpression of LOC102163816. Our results showed that LOC102163816 sponged miR-455-3p, promoting expression of protein tyrosine kinase 2 beta (PTK2B), thereby activating the PI3K/AKT signaling pathway to regulate proliferation of porcine follicular GCs. These findings provide useful insights into follicular development.

Impact of Disease-Associated Mutations on the Deaminase Activity of ADAR1.

The innate immune system relies on molecular sensors to detect distinctive molecular patterns, including viral double-stranded RNA (dsRNA), which triggers responses resulting in apoptosis and immune infiltration. Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I), serving as a mechanism to distinguish self from non-self RNA and prevent aberrant immune activation. Loss-of-function mutations in the ADAR1 gene are one cause of Aicardi Goutières Syndrome (AGS), a severe autoimmune disorder in children. Although seven out of the eight AGS-associated mutations in ADAR1 occur within the catalytic domain of the ADAR1 protein, their specific effects on the catalysis of adenosine deamination remain poorly understood. In this study, we carried out a biochemical investigation of four AGS-causing mutations (G1007R, R892H, K999N, and Y1112F) in ADAR1 p110 and truncated variants. These studies included adenosine deamination rate measurements with two different RNA substrates derived from human transcripts known to be edited by ADAR1 p110 (glioma-associated oncogene homologue 1 (hGli1), 5-hydroxytryptamine receptor 2C (5-HT2cR)). Our results indicate that AGS-associated mutations at two amino acid positions directly involved in stabilizing the base-flipped conformation of the ADAR-RNA complex (G1007R and R892H) had the most detrimental impact on catalysis. The K999N mutation, positioned near the RNA binding interface, altered catalysis contextually. Finally, the Y1112F mutation had small effects in each of the assays described here. These findings shed light on the differential effects of disease-associated mutations on adenosine deamination by ADAR1, thereby advancing our structural and functional understanding of ADAR1-mediated RNA editing.

Specific RNA structures in the 5' untranslated region of the human cytomegalovirus major immediate early transcript are critical for efficient virus replication.

Human cytomegalovirus (HCMV) requires the robust expression of two immediate early proteins, IE1 and IE2, immediately upon infection to suppress the antiviral response and promote viral gene expression. While transcriptional control of IE1 and IE2 has been extensively studied, the role of post-transcriptional regulation of IE1 and IE2 expression is relatively unexplored. We previously found that the shared major immediate early 5' untranslated region (MIE 5' UTR) of the mature IE1 and IE2 transcripts plays a critical role in facilitating the translation of the IE1 and IE2 mRNAs. As RNA secondary structure in 5' UTRs can regulate mRNA translation efficiency, we used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to identify RNA structures in the shared MIE 5' UTR. We found that the MIE 5' UTR contains three stable stem loop structures. Using a series of recombinant viruses to investigate the role of each stem loop in IE1 and IE2 protein synthesis, we found that the stem loop closest to the 5' end of the MIE 5' UTR (SL1) is both necessary and sufficient for efficient IE1 and IE2 mRNA translation and HCMV replication. The positive effect of SL1 on mRNA translation and virus replication was dependent on its location within the 5' UTR. Surprisingly, a synthetic stem loop with the same free energy as SL1 in its native location also supported wild type levels of IE1 and IE2 mRNA translation and virus replication, suggesting that the presence of RNA structure at a specific location in the 5' UTR, rather than the primary sequence of the RNA, is critical for efficient IE1 and IE2 protein synthesis. These data reveal a novel post-transcriptional regulatory mechanism controlling IE1 and IE2 expression and reinforce the critical role of RNA structure in regulating HCMV protein synthesis and replication.IMPORTANCEThese results reveal a new aspect of immediate early gene regulation controlled by non-coding RNA structures in viral mRNAs. Previous studies have largely focused on understanding viral gene expression at the level of transcriptional control. Our results show that a complete understanding of the control of viral gene expression must include an understanding of viral mRNA translation, which is driven in part by RNA structure(s) in the 5' UTR of viral mRNAs. Our results illustrate the importance of these additional layers of regulation by defining specific 5' UTR RNA structures regulating immediate early gene expression in the context of infection and identify important features of RNA structure that govern viral mRNA translation efficiency. These results may therefore broadly impact current thinking on how viral gene expression is regulated for human cytomegalovirus and other DNA viruses.

Silencing AREG Enhances Sensitivity to Irradiation by Suppressing the PI3K/AKT Signaling Pathway in Colorectal Cancer Cells.

It has been established that Spalt-Like Transcription Factor 4 (SALL4) promotes Colorectal Cancer (CRC) cell proliferation. Furthermore, Amphiregulin (AREG) is crucially involved in cancer cell proliferation and therapeutic resistance regulation. In this regard, this study aimed to establish whether SALL4 affects the radiosensitization of CRC cells via AREG expression regulation. Transcriptome sequencing and the Human Transcription Factor Database (HumanTFDB) were used to identify the potential SALL4 targets. The dual-luciferase reporter analysis was used to confirm the SALL4-induced AREG activation. Western Blot (WB) and Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) assays were used to examine the effect of X-ray irradiation on SALL4 and AREG expression. The AREG-KD (Knockdown) stable cell lines were created through lentiviral infection. Cell proliferation was tracked using Cell Counting Kit 8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU)-incorporation assays. Cell cycle and apoptosis were examined through flow cytometry. The cells were exposed to a controlled X-ray radiation dose (6 Gy) for imaging purposes. SALL4 could bound to the AREG promoter, enhancing AREG expression.Furthermore, irradiation upregulated SALL4 and AREG in CRC cells. Additionally, AREG knockdown in CRC cells led to reduced DNA replication efficiency, suppressed cell proliferation, increased DNA damage, and enhanced G1 phase arrest and apoptosis following irradiation. On the other hand, AREG overexpression reversed the inhibitory effects of SALL4 downregulation on AREG expression. In CRC cells, SALL4 downregulation suppressed AREG expression, regulating CRC cell radiosensitivity via the PI3K-AKT pathway, thus presenting a potential therapeutic pathway for CRC treatment using Radiotherapy (RT).

SUMO Sites Prediction in Human Transcription Factors Involved in Hypoxia induced Cardiac Illnesses

Protein SUMOylation is a reversible and well knownpost-translational modificationprocess of the cells. It may change a protein's cellular location, interactions, and possible structural shape before it develops to carry out its basic functions.Also, it decides the binding of transcription factors and DNA binding proteins tochromatin in addition to various cis and trans regulatory factors. Alterations in protein SUMOylation have been linked with a variety of disorders and developmental anomalies.Tentative approaches to identify SUMO binding sites are challenging due todynamic nature of the SUMOylation processand various critical lab experimentswhich are involved very high cost.Therefore, the computational methodologies may guide the experimental identification of SUMOylation sites and provide insights for improving comprehensionofSUMOylation mechanism in the cells.In this study, we identify the SUMO binding sites in transcription factors that are actively involved and have crucial roles in cardiac development andpathophysiology of the heart.A list of important transcription factors was preparedfrom thehuman transcription factor database.The GPS-SUMO, SUMO plot, and JASSA web serverswere used for the prediction of SUMO binding sites in cardiac transcription factors.We identified the SUMOylation of several novel, previously uncharacterized SUMO targetsthat are actively involved in thecardiovascular system.Thus, the present study may help to uncoverthe significance ofSUMO modificationin cardiac development and illnesses which creates a fresh avenue for future studies ontarget-specific SUMOylation for identification of novel therapeutic targets andmanagement strategies forhypoxia-induced cardiovascular disorders.

Validation of nuclear receptor RORγ isoform 1 as a novel host-directed antiviral target based on the modulation of cholesterol levels

Currently, the clinically approved repertoire of antiviral drugs predominantly comprises direct-acting antivirals (DAAs). However, the use of DAAs is frequently limited by adverse effects, restriction to individual virus species, or the induction of viral drug resistance. These issues will likely be resolved by the introduction of host-directed antivirals (HDAs) targeting cellular proteins crucial for viral replication. However, experiences with the development of antiviral HDAs and clinical applications are still in their infancy. With the present study, we explored the human nuclear receptor and transcription factor RORγ isoform 1 (RORγ1), a member of the retinoic acid receptor-related orphan receptor (ROR) family, as a putative target of antiviral HDAs. To this end, cell culture models were used to investigate major viral human pathogens, i.e. the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human cytomegalovirus (HCMV), varicella zoster virus (VZV) and human immunodeficiency virus 1 (HIV-1). Our results demonstrated (i) an antiviral activity of the clinically relevant RORγ modulators cedirogant and others, (ii) that isoform RORγ1 acts as the responsible determinant and drug target in the analyzed cell culture-based models, (iii) a selectivity of the antiviral effect for RORγ1 over related receptors RORα and RORβ, (iv) a late-phase inhibition exerted by cedirogant in HCMV replication and (v) a mechanistic link to the cellular cholesterol biosynthesis. Combined, the data highlight this novel RORγ-specific antiviral targeting concept and the developmental potential of RORγ-directed small molecules.

Comprehensive gene profiling of the metabolic landscape of humanized livers in mice

The human liver transcriptome is complex and highly dynamic, e.g. one gene may produce multiple distinct transcripts, each with distinct posttranscriptional modifications. Direct knowledge of transcriptome dynamics, however, is largely obscured by the inaccessibility of the human liver to treatments and the insufficient annotation of the human liver transcriptome at transcript and RNA modification levels. We generated mice that carry humanized livers of identical genetic background and subjected them to representative metabolic treatments. We then analyzed the humanized livers with nanopore single-molecule direct RNA sequencing to determine the expression level, m6A modification and poly(A) tail length of all RNA transcript isoforms. Our system allows for the de novo annotation of human liver transcriptomes to reflect metabolic responses and for the study of transcriptome dynamics in parallel. Our analysis uncovered a vast number of novel genes and transcripts. Our transcript-level analysis of human liver transcriptomes also identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional short-read RNA sequencing. We revealed for the first time the dynamic changes in m6A and poly(A) tail length of human liver transcripts, many of which are transcribed from key metabolic genes. Furthermore, we performed comparative analyses of gene regulation between humans and mice, and between two individuals using the liver-specific humanized mice, revealing that transcriptome dynamics are highly species- and genetic background-dependent. Our work revealed a complex metabolic response landscape of the human liver transcriptome and provides a novel resource to understand transcriptome dynamics of the human liver in response to physiologically relevant metabolic stimuli (https://caolab.shinyapps.io/human_hepatocyte_landscape/). Direct knowledge of the human liver transcriptome is currently very limited, hindering the overall understanding of human liver pathophysiology. We combined a liver-specific humanized mouse model and long-read direct RNA sequencing technology to establish a de novo annotation of the human liver transcriptome and identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional technologies. The extensive regulatory information on human genes we provided could enable basic scientists to infer the pathological relevance of their genes of interest and physician scientists to better pinpoint the changes in metabolic networks underlying a specific pathophysiology.

PP063 Tobacco-derived miRNAs and their crosstalk with human transcripts towards carcinogenesis: A cross-kingdom approach

PP063 Tobacco-derived miRNAs and their crosstalk with human transcripts towards carcinogenesis: A cross-kingdom approach

Abstract B012: Transcriptional activation of mouse LINE-1 promoter by transcription factor YY1

Abstract Long interspersed element type 1 (LINE-1) are highly abundant non-LTR retrotransposons in mammalian genomes. LINE-1 hypomethylation and overexpression has been considered as a hallmark of many human cancers. Moreover, active movement of LINE-1 results in insertional mutagenesis and may contribute to cancer initiation or progression. Transcription is the initial and critical regulatory step for active LINE-1 movement. Therefore, investigating LINE-1 transcriptional regulation is important for our understanding of LINE-1’s role in cancer. In human LINE-1, an intact YY1 transcription factor binding motif is required for accurate transcription initiation. Two subfamilies of mouse LINE-1, Tf_I and Gf_I, have predicted YY1 binding motifs in their promoter region but their role in mouse LINE-1 transcription has not been characterized. In this study, we aim to investigate the role of these putative YY1 motifs in Tf_I and Gf_I subfamilies. Promoter sequences carrying the wild-type or mutant YY1 motifs were cloned into a dual-luciferase reporter system. Reporter assays showed transcription inactivation upon mutation of the YY1 motif for the Tf_I subfamily. The electrophoretic mobility shift assay (EMSA) demonstrated specific interaction of the Tf_I promoter sequence with the YY1 protein. Knockdown of YY1 protein level in cells reduced Tf_I promoter activity by 2-fold compared to the wild-type control. These results indicate that YY1 is a transcriptional activator of mouse LINE-1 Tf_I subfamily. On the other hand, our data suggest that the predicted YY1 binding site in the promoter of Gf_I subfamily is not functional due to one nucleotide deviation from the consensus YY1 motif. Indeed, restoring it to the conserved YY1 motif sequence enhanced the promoter activity by 6-fold. In EMSA, YY1 protein did not interact with the putative YY1 motif of the Gf_I subfamily but did so when the motif was restored to consensus YY1 sequence. Findings from our study increase our understanding of the transcriptional regulation of different mouse LINE-1 subfamilies, highlight the differing roles of YY1 in regulating human and mouse L1 transcription, and have important implications in the role of LINE-1s during cancer development. Citation Format: Karani Saha, Grace Nielsen, Wenfeng An. Transcriptional activation of mouse LINE-1 promoter by transcription factor YY1 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B012.

Challenges of Assessing Exon 53 Skipping of the Human DMD Transcript with Locked Nucleic Acid-Modified Antisense Oligonucleotides in a Mouse Model for Duchenne Muscular Dystrophy.

Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy (DMD) patients to restore dystrophin expression by reframing the disrupted open reading frame of the DMD transcript. However, the treatment efficacy of the already conditionally approved AONs remains low. Aiming to optimize AON efficiency, we assessed exon 53 skipping of the DMD transcript with different chemically modified AONs, all with a phosphorothioate backbone: 2'-O-methyl (2'OMe), locked nucleic acid (LNA)-2'OMe, 2'-fluoro (FRNA), LNA-FRNA, αLNA-FRNA, and FANA-LNA-FRNA. Efficient exon 53 skipping was observed with the FRNA, LNA-FRNA, and LNA-2'OMe AONs in human control myoblast cultures. Weekly subcutaneous injections (50 mg/kg AON) for a duration of 6 weeks were well tolerated by hDMDdel52/mdx males. Treatment with the LNA-FRNA and LNA-2'OMe AONs resulted in pronounced exon 53 skip levels in skeletal muscles and heart up to 90%, but no dystrophin restoration was observed. This discrepancy was mainly ascribed to the strong binding nature of LNA modifications to RNA, thereby interfering with the amplification of the unskipped product resulting in artificial overamplification of the exon 53 skip product. Our study highlights that treatment effect on RNA and protein level should both be considered when assessing AON efficiency.

Hepatitis B Virus Modulated Transcriptional Regulatory Map of Hepatic Cellular MicroRNAs.

Hepatitis B virus (HBV) is an enveloped, hepatotropic, noncytopathic virus with a partially double-stranded DNA genome. It infects hepatocytes and is associated with progression to liver fibrosis and cirrhosis, culminating in hepatocellular carcinoma (HCC), accounting for 55% of total HCC cases. MicroRNAs (miRNAs) regulated by HBV play an important role in these pathologies. Mapping the miRNAs responsive to HBV and HBV-specific proteins, including HBV X protein (HBx) that harbor the majority of HBV-human protein interactions, could aid accelerate the diagnostics and therapeutics innovation against the infection and associated diseases. With this in mind, we used a unique annotation strategy whereby we first amassed 362 mature HBV responsive-human Differentially Expressed miRNAs (HBV-hDEmiRs). The core experimentally-validated messenger RNA targets of the HBV-hDEmiRs were mostly associated with viral infections and hepatic inflammation processes. Moreover, our annotation strategy enabled the characterization of HBx-dependent/independent HBV-hDEmiRs as a tool for evaluation of the impact of HBx as a therapeutic target. Bioinformatics analysis of the HBV-human protein-protein interactome revealed new insights into the transcriptional regulatory network of the HBV-hDEmiRs. We performed a comparative analysis of data on miRNAs gathered from HBV infected cell line studies and from tissue studies of fibrosis, cirrhosis, and HCC. Accordingly, we propose hsa-miR-15a-5p that is downregulated by multiple HBV proteins, including HBx, as a potential biomarker of HBV infection, and its progression to HCC. In all, this study underscores (1) the complexity of miRNA regulation in response to HBV infection and its progression into other liver pathologies and (2) provides a regulatory map of HBV-hDEmiRs and the underlying mechanisms modulating their expression through a cross talk between HBV viral proteins and human transcription factors.

An efficient synthesis, structural analysis, and computational studies of benzothiazole derivatives activated by formic acid under solvent-free conditions

In the present work, formic acid was used as a low-cost and eco-friendly catalyst to synthesize 2-(2-hydroxyphenyl)benzothiazole (HB) through a cycloaddition process in a solvent-free reaction medium. Imine molecule was synthesized with the help of acetic acid (B). The ring closure and formation of the thiazole moiety give acetic acid distinctiveness as a catalyst. The cyclization of fused HB failed without an acid catalyst. The chemical structures were characterized using microanalysis techniques such as IR, 1H-NMR, and 13C-NMR. The obtained FT-IR spectra were compared to theoretical data using the B3LYP/6-31G* level of theory. The electronic characteristics of molecules can be predicted utilizing wave function-dependent features, such as Average localized ionization energy (ALIE), Localized orbital locator (LOL), Electron localized function (ELF), Reduced density gradient (RDG), and Noncovalent interactions NCI studies, which have provided a wealth of knowledge. The molecular docking study was investigated to explore binding energy and interaction type. Docking analysis displayed binding energies (kcal/mol) of HB with values of -8.29, -6.80, -6.68, -7.43, and -6.69 of acetylcholinesterase (AChE) and butylcholinesterase (BuChE)), SARS-CoV-2 main protease (Mpro), human immunodeficiency virus reverse transcription (HIV RT)) and cytochrome (P450), respectively, which was the most active compound against all receptors. The dramatic improvement of the proposed approach over the existing methods can be used to derive an efficient and successful route for the synthesis of HB. This process provides a fresh and high-yield method for the synthesis of benzimidazoles.

CRISPR Activation Screen to Optimize Chimeric Antigen Receptor (CAR) T Cell Immunophenotype

Background Adoptive cellular therapies (ACT) offer potential cures for a wide range of human diseases. Immune cells genetically engineered with chimeric antigen receptors (CAR) have a clear role in B cell malignancies, and broader applications to other malignancies are under investigation. Significant obstacles to the long-term durability and potency of these therapies remain. As CAR T-cells remain persistently activated and exposed to the tumor microenvironment, they may become functionally impaired. This exhaustion can result in reduced production of cytokines, cytotoxic molecules, and other factors necessary for effective tumor cell killing. While CAR T-cell exhaustion remains a challenge, ongoing research and clinical trials aim to further understand the underlying mechanisms and develop strategies to overcome this limitation. Clinical studies have demonstrated that sustained in vivo persistence and function of CAR T-cells is correlated with infusion product phenotype. Persistence is associated with a less differentiated, memory-like phenotype. Naive (T N), Central Memory (T CM) and Stem Cell Memory (T SCM) are related to good proliferative potential and durability. CRISPR technologies offer powerful, functional genomic screens that permit the inference and modification of gene regulatory networks to optimize CAR T-cell persistence and function. Genome-wide CRISPR knock out screens have identified the CD2-CD58 axis as critical to T-cell potency and the knock-out of the mediator kinase module as beneficial to potency. Here we report on in vitro CRISPR activation screening of human CAR T-cells for gene expression that enhances T cell phenotypes associated with durable function. Method We designed 9,983 single guide RNA (sgRNA) to target the transcription start sites (TSS) of all known human transcription factors as well as a select set of genes with critical functions in T-cells. The vast majority of genes was targeted with 5 different sgRNA. To this we added a pool of 1,000 non-targeting sgRNA. We lentivirally transduced the sgRNA in tandem to a second-generation anti-CD20 CAR into primary T cells from healthy human donors. In a second lentiviral transduction we integrated catalytically dead Cas9 (dCas9) fused to the strong transcription activator, VP64 and in tandem to a puromycin resistance gene. After puromycin selection, we cultured the CRISPR activated CAR T-cells either alone or together with Raji or K562 cells for nine days before immunophenotypically sorting the CAR T-cells into CD8 + Naive, CD8 + Transitional Memory, CD8 + Central Memory, CD8 + Stem Cell Memory, CD8 + Transitional Effector, CD8 + Effector Memory, CD4 + Naive and CD4 + Memory T-cell populations. We extracted DNA from the sorted cells and prepared sequencing libraries targeting the sgRNA. We carried out two-way ANOVA analysis on the experimental conditions (trivial, Raji or K562 co-culture versus addition of CD2/CD28 activation beads) and T-cell subset to determine which genes targeted by CRISPRa resulted in favorable phenotypes. Result Across the different culture conditions, sgRNA activating IL23A and KLF4 were enriched in Transitional Memory versus Stem Cell Memory T-cells. Across the different CAR T-cell immunophenotypes, sgRNA targeting the TGFß (including KLF10), TNFα (including NFATC4) and FOXO signaling pathways were enriched in CAR T-cells cultured alone compared to CAR T-cells activated with CD2/CD28. Conclusion In this study we have investigated genes whose activation results in CAR T-cell immunophenotypes that are associated with more potent and durable CAR T-cell therapies. Our experiment suggests that the activation of genes, such as IL23 and KLF4, enriches for a Transitional Memory over Stem Cell Memory phenotype in a manner that is largely independent of environment (culture conditions). The activation of other genes, such as KLF10 and NFATC4, results in enrichment of CAR T-cells across all T-cell subsets, but in a manner that is dependent on environment. Gene expression requires coordinated control of gene-proximal and -distal cis-regulatory elements (CREs), whose state varies as cells differentiate or respond to environmental signals. Abrogating or adding CRE may result in more durable and potent adoptive cellular therapies independent of environment.