Sensitization of Chrysosplenetin against Artemisinin Resistant Plasmodium berghei K173 via Blocking Host ABC Transporters Potentially Mediated by NF-κBB p52 or PXR/CAR Signaling Pathway

This study investigated if artemisinin-chrysosplenetin combination (ART-CHR) improved ART antimalarial efficacy against resistant Plasmodium berghei K173 via depressing host ABC transporter and potential molecular mechanism. Parasitaemia% and inhibition% were calculated and gene/protein expressions of ABC transporters or PXR/CAR/NF-κB p52 were detected by Western-blot and RT-qPCR. In vitro transcription of PXR/CAR was studied by dual-luciferase reporter assay. Our data indicated that ART-CHR improved ART efficacy against resistant parasites. P-gp inhibitor verapamil and CHR showed a stronger effect in killing resistant parasites while vehicle and Bcrp inhibitor novobiocin did not. ART activated intestinal ABCB1/ABCG2 and CHR inhibited them. ART decreased Bcrp protein whereas CHR increased it. ART ascended ABCC1/ABCC4/ABCC5 mRNA but ART-CHR descended them. CHR as well as rifampin (RIF) or 5-fluorouracil (5-FU) increased transcription levels of PXR/CAR while showed a versatile regulation on in vivo hepatic and enternal PXR/CAR in Mdr1a+/+ (WT) or Mdr1a-/- (KO) mice infected with sensitive or resistant parasites. Oppositely, hepatic and enteric N-7κB p52 mRNA was conformably decreased in WT but increased in KO-resistant mice. NF-κB pathway should potentially involved in the mechanism of CHR on inhibiting ABC transporters and ART resistance while PXR/CAR play a more complicated role in this mechanism.

Regulation of Gene Expression of phiEco32-like Bacteriophage 7-11

Salmonella enterica serovar Newport bacteriophage 7-11 shares 41 homologous ORFs with Escherichia coli phage phiEco32 and both phages encode a protein similar to bacterial RNA polymerase promoter specificity  subunit. Here, we investigated the temporal pattern of 7-11 gene expression during the infection and compared it to the previously determined transcription strategy of phiEco32. Using primer extension and in vitro transcription assays we identified eight promoters recognized by host RNA polymerase holoenzyme containing 7-11  subunit SaPh711_gp47. These promoters are characterized by a bipartite consensus GTAAtg-(16)-aCTA and are located upstream of late phage genes. While dissimilar from single-element middle and late promoters of phiEco32 recognized by holoenzyme formed by the phi32_gp36  factor, the 7-11 late promoters are located at genome positions similar to those of phiEco32 middle and late promoters. Two early 7-11 promoters are recognized by RNA polymerase holoenzyme containing host primary σ70 factor. Unlike the case of phiEco32, no shut off of σ70-dependent transcription is observed during 7-11 infection and there are no middle promoters. These differences can be explained by the fact that phage 7-11 does not encode a homologue of phi32_gp79, an inhibitor of host and early phage transcription and an activator of transcription by the phi32_gp36-holoenzyme.

Preparation of Labeled DNA, RNA, and Oligonucleotide Probes

Labeled nucleic acids and oligonucleotides are typically generated by enzymatic methods such as end-labeling, random priming, nick translation, in vitro transcription, and variations of the polymerase chain reaction (PCR). Some of these methods place the label in specific locations within the nucleic acid (e.g., at the 5′ or 3′ terminus); others generate molecules that are labeled internally at multiple sites. Some methods yield labeled single-stranded products, whereas others generate double-stranded nucleic acids. Finally, some generate probes of defined length, whereas others yield a heterogeneous population of labeled molecules. Options available for generating and detecting labeled nucleic acids, as well as advice on designing oligonucleotides for use as probes, is included here.

The human RNA polymerase I structure reveals an HMG-like transcription factor docking domain specific to metazoans

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP-fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This "dock II" domain resembles a truncated HMG-box incapable of DNA-binding which may serve as a downstream-transcription factor binding platform in metazoans. Biochemical analysis and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG-box domain containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

Long noncoding RNAs transcribed downstream of the human β-globin locus regulate β-globin gene expression

The five β-like globin genes (ε, Gγ, Aγ, δ, and β) at the human β-globin gene locus are known to be expressed at specific developmental stages, although details of the underlying mechanism remain to be uncovered. Here we used an in vitro transcription assay to clarify the mechanisms that control this gene expression. We first tested nuclear RNA from HeLa cells using RT-qPCR and discovered a long noncoding RNAs (lncRNAs) within a 5.2-kb region beginning 4.4 kb downstream of the β-globin gene coding region. We investigated nuclear RNA from K562 cells using a primer-extension assay and determined the transcription start sites (TSSs) of these lncRNAs. To clarify their functional role, we performed knockdown (KD) of these lncRNAs in K562 cells. Hydroxyurea, which induces differentiation of K562 cells, increased hemoglobin peptide production, and the effect was enhanced by KD of these lncRNAs, which also enhanced upregulation of the γ-globin expression induced by hydroxyurea. To confirm these results, we performed an in vitro transcription assay. Noncoding single-stranded RNAs inhibited β-globin expression, which was upregulated by GATA1. Furthermore, lncRNAs interacted with GATA1 without sequence specificity and inhibited its binding to its target DNA response element in vitro. Our results suggest that lncRNAs downstream of the β-globin gene locus are key factors regulating globin gene ex pression.

Inexpensive and colorimetric RNA detection by E. coli cell-free protein synthesis platform at room temperature

We report colorimetric detection of SARS-CoV-2 viral RNA by an in vitro transcription/translation assay with crude E. coli extracts at room temperature, with the aid of body heat. Clinically-relevant concentrations of viral RNA (ca. 600 copies/test) were detected from synthetic RNA samples. The activation of cell-free gene expression was achieved by toehold-switch-mediated riboregulatory elements that are specific to viral RNA sequences. The colorimetric output was generated by the α-complementation of β-galactosidase ω-fragment (LacZ-ω) with cell-free expressed LacZ-α, using an X-gal analogue as a substrate. The estimated cost of single reaction is less than 1 euro/test, which may facilitate diagnostic kit accessibility in developing countries.

Protein-Based Systems for Translational Regulation of Synthetic mRNAs in Mammalian Cells

Synthetic mRNAs, which are produced by in vitro transcription, have been recently attracting attention because they can express any transgenes without the risk of insertional mutagenesis. Although current synthetic mRNA medicine is not designed for spatiotemporal or cell-selective regulation, many preclinical studies have developed the systems for the translational regulation of synthetic mRNAs. Such translational regulation systems will cope with high efficacy and low adverse effects by producing the appropriate amount of therapeutic proteins, depending on the context. Protein-based regulation is one of the most promising approaches for the translational regulation of synthetic mRNAs. As synthetic mRNAs can encode not only output proteins but also regulator proteins, all components of protein-based regulation systems can be delivered as synthetic mRNAs. In addition, in the protein-based regulation systems, the output protein can be utilized as the input for the subsequent regulation to construct multi-layered gene circuits, which enable complex and sophisticated regulation. In this review, I introduce what types of proteins have been used for translational regulation, how to combine them, and how to design effective gene circuits.

Digital Twins for Continuous mRNA Production

The global coronavirus pandemic continues to restrict public life worldwide. An effective means of limiting the pandemic is vaccination. Messenger ribonucleic acid (mRNA) vaccines currently available on the market have proven to be a well-tolerated and effective class of vaccine against coronavirus type 2 (CoV2). Accordingly, demand is presently outstripping mRNA vaccine production. One way to increase productivity is to switch from the currently performed batch to continuous in vitro transcription, which has proven to be a crucial material-consuming step. In this article, a physico-chemical model of in vitro mRNA transcription in a tubular reactor is presented and compared to classical batch and continuous in vitro transcription in a stirred tank. The three models are validated based on a distinct and quantitative validation workflow. Statistically significant parameters are identified as part of the parameter determination concept. Monte Carlo simulations showed that the model is precise, with a deviation of less than 1%. The advantages of continuous production are pointed out compared to batchwise in vitro transcription by optimization of the space–time yield. Improvements of a factor of 56 (0.011 µM/min) in the case of the continuously stirred tank reactor (CSTR) and 68 (0.013 µM/min) in the case of the plug flow reactor (PFR) were found.