Characterization of double-negative T cells in colorectal cancers and their corresponding lymph nodes.

TCRαß+ CD4- CD8- double-negative T (DNT) cells are minor populations in peripheral blood, and their roles have mostly been discussed in inflammation and autoimmunity. However, the functions of DNT cells in tumor microenvironment remain to be elucidated. We investigated their characteristics, possible origins and functions in colorectal cancer tissues as well as their corresponding tumor-draining lymph nodes. We found a significant enrichment of DNT cells in tumor tissues compared with their corresponding lymph nodes, especially in tumors with lower T cell infiltration. T cell receptor (TCR) sequence analysis of CD4+ T, CD8+ T and DNT cells indicated that TCR sequences detected in DNT cells were found in CD8+ T cells, but rarely in CD4+ T cells, suggesting that a part of DNT cells was likely to be originated from CD8+ T cells. Through a single-cell transcriptomic analysis of DNT cells, we found that a DNT cell cluster, which showed similar phenotypes to central memory CD8+ T cells with low expression of effector and exhaustion markers, revealed some specific gene expression patterns, including higher GZMK expression. Moreover, in flow cytometry analysis, we found that DNT cells lost production of cytotoxic mediators. These findings imply that DNT cells might function as negative regulators of anti-tumor immune responses in tumor microenvironment.

Dosimetric impact of MLC positional errors on dose distribution in IMRT.

Optimizing the positional accuracy of multileaf collimators (MLC) for radiotherapy is important for dose accuracy and for reducing doses delivered to normal tissues. This study investigates dose sensitivity variations and complexity metrics of MLC positional error in volumetric modulated arc therapy and determines the acceptable ranges of MLC positional accuracy in several clinical situations. Treatment plans were generated for four treatment sites (prostate cancer, lung cancer, spinal, and brain metastases) using different treatment planning systems (TPSs) and fraction sizes. Each treatment plan introduced 0.25-2.0 mm systematic or random MLC leaf bank errors. The generalized equivalent uniform dose (gEUD) sensitivity and complexity metrics (MU/Gy and plan irregularity) were calculated, and the correlation coefficients were assessed. Furthermore, the required tolerances for MLC positional accuracy control were calculated. The gEUD sensitivity showed the highest dependence of systematic positional error on the treatment site, followed by TPS and fraction size. The gEUD sensitivities were 6.7, 4.5, 2.5, and 1.7%/mm for Monaco and 8.9, 6.2, 3.4, and 2.3%/mm (spinal metastasis, lung cancer, prostate cancer, and brain metastasis, respectively) for RayStation. The gEUD sensitivity was strongly correlated with the complexity metrics (r = 0.88-0.93). The minimum allowable positional error for MLC was 0.63, 0.34, 1.02, and 0.28 mm (prostate, lung, brain, and spinal metastasis, respectively). The acceptable range of MLC positional accuracy depends on the treatment site, and an appropriate tolerance should be set for each treatment site with reference to the complexity metric. It is expected to enable easier and more detailed MLC positional accuracy control than before by reducing dose errors to patients at the treatment planning stage and by controlling MLC quality based on complexity metrics, such as MU/Gy.

Identification of T Cell Receptors Targeting a Neoantigen Derived from Recurrently Mutated FGFR3.

Immunotherapies, including immune checkpoint blockades, play a critically important role in cancer treatments. For immunotherapies, neoantigens, which are generated by somatic mutations in cancer cells, are thought to be good targets due to their tumor specificity. Because neoantigens are unique in individual cancers, it is challenging to develop personalized immunotherapy targeting neoantigens. In this study, we screened "shared neoantigens", which are specific types of neoantigens derived from mutations observed commonly in a subset of cancer patients. Using exome sequencing data in the Cancer Genome Atlas (TCGA), we predicted shared neoantigen peptides and performed in vitro screening of shared neoantigen-reactive CD8+ T cells using peripheral blood from healthy donors. We examined the functional activity of neoantigen-specific T cell receptors (TCRs) by generating TCR-engineered T cells. Among the predicted shared neoantigens from TCGA data, we found that the mutated FGFR3Y373C peptide induced antigen-specific CD8+ T cells from the donor with HLA-A*02:06 via an ELISPOT assay. Subsequently, we obtained FGFR3Y373C-specific CD8+ T cell clones and identified two different sets of TCRs specifically reactive to FGFR3Y373C. We found that the TCR-engineered T cells expressing FGFR3Y373C-specific TCRs recognized the mutated FGFR3Y373C peptide but not the corresponding wild-type peptide. These two FGFR3Y373C-specific TCR-engineered T cells showed cytotoxic activity against mutated FGFR3Y373C-loaded cells. These results imply the possibility of strategies of immunotherapies targeting shared neoantigens, including cancer vaccines and TCR-engineered T cell therapies.

Anticancer Activities of Novel Nicotinamide Phosphoribosyltransferase Inhibitors in Hematological Malignancies.

Targeting cancer cells that are highly dependent on the nicotinamide adenine dinucleotide (NAD+) metabolite is a promising therapeutic strategy. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme catalyzing NAD+ production. Despite the high efficacy of several developed NAMPT inhibitors (i.e., FK866 (APO866)) in preclinical studies, their clinical activity was proven to be limited. Here, we report the synthesis of new NAMPT Inhibitors, JJ08, FEI191 and FEI199, which exhibit a broad anticancer activity in vitro. Results show that these compounds are potent NAMPT inhibitors that deplete NAD+ and NADP(H) after 24 h of drug treatment, followed by an increase in reactive oxygen species (ROS) accumulation. The latter event leads to ATP loss and mitochondrial depolarization with induction of apoptosis and necrosis. Supplementation with exogenous NAD+ precursors or catalase (ROS scavenger) abrogates the cell death induced by the new compounds. Finally, in vivo administration of the new NAMPT inhibitors in a mouse xenograft model of human Burkitt lymphoma delays tumor growth and significantly prolongs mouse survival. The most promising results are collected with JJ08, which completely eradicates tumor growth. Collectively, our findings demonstrate the efficient anticancer activity of the new NAMPT inhibitor JJ08 and highlight a strong interest for further evaluation of this compound in hematological malignancies.

Nicotinaldehyde, a Novel Precursor of NAD Biosynthesis, Abrogates the Anti-Cancer Activity of an NAD-Lowering Agent in Leukemia.

Targeting NAD depletion in cancer cells has emerged as an attractive therapeutic strategy for cancer treatment, based on the higher reliance of malignant vs. healthy cells on NAD to sustain their aberrant proliferation and altered metabolism. NAD depletion is exquisitely observed when NAMPT, a key enzyme for the biosynthesis of NAD, is inhibited. Growing evidence suggests that alternative NAD sources present in a tumor environment can bypass NAMPT and render its inhibition ineffective. Here, we report the identification of nicotinaldehyde as a novel precursor that can be used for NAD biosynthesis by human leukemia cells. Nicotinaldehyde supplementation replenishes the intracellular NAD level in leukemia cells treated with NAMPT inhibitor APO866 and prevents APO866-induced oxidative stress, mitochondrial dysfunction and ATP depletion. We show here that NAD biosynthesis from nicotinaldehyde depends on NAPRT and occurs via the Preiss-Handler pathway. The availability of nicotinaldehyde in a tumor environment fully blunts the antitumor activity of APO866 in vitro and in vivo. This is the first study to report the role of nicotinaldehyde in the NAD-targeted anti-cancer treatment, highlighting the importance of the tumor metabolic environment in modulating the efficacy of NAD-lowering cancer therapy.

DNA methylation is regulated by both the stability and topology of G-quadruplex.

Since DNA methylation alters the chromatin state and regulates gene expression, elucidating the regulatory mechanisms of DNA methylation in response to environmental changes in the cell is crucial and urgent in understanding and regulating DNA methylation. G-quadruplex (G4) regulates transcription, translation and replication. Although it has recently been suggested that G4 regulates methylation, the detailed regulatory mechanism remains unclear. Here, we systematically analysed the effect of G4 formation on DNA methylation using G4s with various stabilities and topologies. The methylation efficiency decreased as the stability of G4 increased. Moreover, the degree of methylation suppression can be also controlled by G4 topology. Furthermore, our results showed the possibility of regulating methylation by modulating G4 stability and topology.

Volumetric Strategy for Quantitatively Elucidating a Local Hydration Network around a G-Quadruplex.

Hydration around nucleic acids, such as DNA and RNA, is an important factor not only for the stability of nucleic acids but also for their interaction with binding molecules. Thus, it is necessary to quantitatively elucidate the hydration properties of nucleic acids around a certain structure. In this study, volumetric changes in G-quadruplex (G4) RNA formation were investigated by systematically changing the number of G-quartet stacks under high pressure. The volumetric contribution at the level of each G4 structural unit revealed that the core G4 helix was significantly more dehydrated than the other parts, including the edges of G-quartets and loops. These findings will help in predicting the binding of G4 ligands on the surface of G4, depending on the chemical structure of the ligand and solution environment. Therefore, the preset volumetric parameter provides information that can predict molecular interactions in G4 formations during molecular crowding in cells.

Gut microbiota severely hampers the efficacy of NAD-lowering therapy in leukemia.

Most cancer cells have high need for nicotinamide adenine dinucleotide (NAD+) to sustain their survival. This led to the development of inhibitors of nicotinamide (NAM) phosphoribosyltransferase (NAMPT), the rate-limiting NAD+ biosynthesis enzyme from NAM. Such inhibitors kill cancer cells in preclinical studies but failed in clinical ones. To identify parameters that could negatively affect the therapeutic efficacy of NAMPT inhibitors and propose therapeutic strategies to circumvent such failure, we performed metabolomics analyses in tumor environment and explored the effect of the interaction between microbiota and cancer cells. Here we show that tumor environment enriched in vitamin B3 (NAM) or nicotinic acid (NA) significantly lowers the anti-tumor efficacy of APO866, a prototypic NAMPT inhibitor. Additionally, bacteria (from the gut, or in the medium) can convert NAM into NA and thus fuel an alternative NAD synthesis pathway through NA. This leads to the rescue from NAD depletion, prevents reactive oxygen species production, preserves mitochondrial integrity, blunts ATP depletion, and protects cancer cells from death.Our data in an in vivo preclinical model reveal that antibiotic therapy down-modulating gut microbiota can restore the anti-cancer efficacy of APO866. Alternatively, NAphosphoribosyltransferase inhibition may restore anti-cancer activity of NAMPT inhibitors in the presence of gut microbiota and of NAM in the diet.

Dielectricity of a molecularly crowded solution accelerates NTP misincorporation during RNA-dependent RNA polymerization by T7 RNA polymerase.

In biological systems, the synthesis of nucleic acids, such as DNA and RNA, is catalyzed by enzymes in various aqueous solutions. However, substrate specificity is derived from the chemical properties of the residues, which implies that perturbations of the solution environment may cause changes in the fidelity of the reaction. Here, we investigated non-promoter-based synthesis of RNA using T7 RNA polymerase (T7 RNAP) directed by an RNA template in the presence of polyethylene glycol (PEG) of various molecular weights, which can affect polymerization fidelity by altering the solution properties. We found that the mismatch extensions of RNA propagated downstream polymerization. Furthermore, PEG promoted the polymerization of non-complementary ribonucleoside triphosphates, mainly due to the decrease in the dielectric constant of the solution. These results indicate that the mismatch extension of RNA-dependent RNA polymerization by T7 RNAP is driven by the stacking interaction of bases of the primer end and the incorporated nucleotide triphosphates (NTP) rather than base pairing between them. Thus, proteinaceous RNA polymerase may display different substrate specificity with changes in dielectricity caused by molecular crowding conditions, which can result in increased genetic diversity without proteinaceous modification.

Image quality and radiologists' subjective acceptance using model-based iterative and deep learning reconstructions as adjuncts to ultrahigh-resolution CT in low-dose contrast-enhanced abdominopelvic CT: phantom and clinical pilot studies.

PURPOSE: In contrast-enhanced abdominopelvic CT (CE-APCT) for oncologic follow-up, ultrahigh-resolution CT (UHRCT) may improve depiction of fine lesions and low-dose scans are desirable for minimizing the potential adverse effects by ionizing radiation. We compared image quality and radiologists' acceptance of model-based iterative (MBIR) and deep learning (DLR) reconstructions of low-dose CE-APCT by UHRCT. METHODS: Using our high-resolution (matrix size: 1024) and low-dose (tube voltage 100 kV; noise index: 20-40 HU) protocol, we scanned phantoms to compare the modulation transfer function and noise power spectrum between MBIR and DLR and assessed findings in 36 consecutive patients who underwent CE-APCT (noise index: 35 HU; mean CTDIvol: 4.2 ± 1.6 mGy) by UHRCT. We used paired t-test to compare objective noise and contrast-to-noise ratio (CNR) and Wilcoxon signed-rank test to compare radiologists' subjective acceptance regarding noise, image texture and appearance, and diagnostic confidence between MBIR and DLR using our routine protocol (matrix size: 512; tube voltage: 120 kV; noise index: 15 HU) for reference. RESULTS: Phantom studies demonstrated higher spatial resolution and lower low-frequency noise by DLR than MBIR at equal doses. Clinical studies indicated significantly worse objective noise, CNR, and subjective noise by DLR than MBIR, but other subjective characteristics were better (P < 0.001 for all). Compared with the routine protocol, subjective noise was similar or better by DLR, and other subjective characteristics were similar or worse by MBIR. CONCLUSION: Image quality, except regarding noise characteristics, and acceptance by radiologists were better by DLR than MBIR in low-dose CE-APCT by UHRCT.

Improved production of human hemoglobin in yeast by engineering hemoglobin degradation.

With the increasing demand for blood transfusions, the production of human hemoglobin (Hb) from sustainable sources is increasingly studied. Microbial production is an attractive option, as it may provide a cheap, safe, and reliable source of this protein. To increase the production of human hemoglobin by the yeast Saccharomyces cerevisiae, the degradation of Hb was reduced through several approaches. The deletion of the genes HMX1 (encoding heme oxygenase), VPS10 (encoding receptor for vacuolar proteases), PEP4 (encoding vacuolar proteinase A), ROX1 (encoding heme-dependent repressor of hypoxic genes) and the overexpression of the HEM3 (encoding porphobilinogen deaminase) and the AHSP (encoding human alpha-hemoglobin-stabilizing protein) genes - these changes reduced heme and Hb degradation and improved heme and Hb production. The reduced hemoglobin degradation was validated by a bilirubin biosensor. During glucose fermentation, the engineered strains produced 18% of intracellular Hb relative to the total yeast protein, which is the highest production of human hemoglobin reported in yeast. This increased hemoglobin production was accompanied with an increased oxygen consumption rate and an increased glycerol yield, which (we speculate) is the yeast's response to rebalance its NADH levels under conditions of oxygen limitation and increased protein-production.

New Insights into the Functions of Nucleic Acids Controlled by Cellular Microenvironments.

The right-handed double-helical B-form structure (B-form duplex) has been widely recognized as the canonical structure of nucleic acids since it was first proposed by James Watson and Francis Crick in 1953. This B-form duplex model has a monochronic and static structure and codes genetic information within a sequence. Interestingly, DNA and RNA can form various non-canonical structures, such as hairpin loops, left-handed helices, triplexes, tetraplexes of G-quadruplex and i-motif, and branched junctions, in addition to the canonical structure. The formation of non-canonical structures depends not only on sequence but also on the surrounding environment. Importantly, these non-canonical structures may exhibit a wide variety of biological roles by changing their structures and stabilities in response to the surrounding environments, which undergo vast changes at specific locations and at specific times in cells. Here, we review recent progress regarding the interesting behaviors and functions of nucleic acids controlled by molecularly crowded cellular conditions. New insights gained from recent studies suggest that nucleic acids not only code genetic information in sequences but also have unknown functions regarding their structures and stabilities through drastic structural changes in cellular environments.

Effect of DNA modifications on the transition between canonical and non-canonical DNA structures in CpG islands during senescence.

Patterns and levels of DNA modifications play important roles in senescence. Two major epigenetic modifications of DNA, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), target CpG sites. Importantly, CpG concentrated regions, known as CpG islands, contain GC-rich sequences, which have the potential to fold into non-canonical DNA structures such as i-motifs and G-quadruplexes. In this study, we investigated the effect of 5mC and 5hmC modifications on the transition between a duplex, and i-motif and G-quadruplexes. To examine the transition, we firstly investigated the stability and structure of the i-motif and G-quadruplexes, considering the molecular environment in senescent cells. Analyses of their stability showed that the modifications did not drastically affect the stability. However, noteworthily, the modification can weaken the (de)stabilisation effect on G-quadruplexes caused by cosolute(s) and cations. Circular dichroism analyses indicated that the surrounding environments, including the molecular crowding and the type of cations such as K+ and Na+, regulate the topology of G-quadruplexes, while neither 5mC nor 5hmC had a drastic effect. On the other hand, the modifications changed the transition between duplexes and quadruplexes. Unmodified DNA preferred to fold into quadruplexes, whereas DNA with 5mC and 5hmC preferred to fold into duplexes in the absence of PEG200; on the other hand, DNA with or without modifications tended to fold into i-motifs under crowded conditions. Furthermore, an investigation of quadruplexes forming sequences in CpG islands, which are hyper- or hypomethylated during senescence, followed by gene ontology enrichment analysis for each gene group classified by the presence of quadruplexes, showed a difference in function between genes with and without quadruplexes in the CpG region. These results indicate that it is important to consider the effects of patterns and levels of DNA modifications on the transition between canonical and non-canonical DNA structures to understand gene regulation by epigenetic modification during senescence.

Enhancement of fatty acid biosynthesis by exogenous acetyl-CoA carboxylase and pantothenate kinase in Escherichia coli.

OBJECTIVES: To establish a technique for efficient fatty acid production through enhancement of coenzyme A (CoA) biosynthesis and malonyl-CoA supply by introducing exogenous pantothenate kinase (coaA) and acetyl-CoA carboxylase (acc) in Escherichia coli. RESULTS: The expression of acc, obtained from Corynebacterium glutamicum, accumulated 2.2-fold more fatty acids in E. coli. The addition of coaA from Pseudomonas putaida or fatty acid synthase (fasA) from C. glutamicum resulted in a 3.1- and 3.6-fold increase in fatty acid synthesis in E. coli cells, which expressed acc and coaA, or acc and fasA, respectively. The transformants, simultaneously possessing all three genes, produced 5.6-fold more fatty acids. The strain possessing acc, coaA, and fasA stored 691 mg/L of fatty acids, primarily as phospholipids, inside the inner membrane after 72-h cultivation. In addition, 19% of the total CoA pool was occupied by malonyl-CoA. CONCLUSIONS: Increased malonyl-CoA significantly contributed to fatty acid production, and the effect was boosted by the expanded total CoA pool. Manipulation of the intracellular CoA species is effective for fatty acid production in E. coli.

SARS-CoV-2 genomic variations associated with mortality rate of COVID-19.

The coronavirus disease 2019 (COVID-19) outbreak, caused by SARS-CoV-2, has rapidly expanded to a global pandemic. However, numbers of infected cases, deaths, and mortality rates related to COVID-19 vary from country to country. Although many studies were conducted, the reasons of these differences have not been clarified. In this study, we comprehensively investigated 12,343 SARS-CoV-2 genome sequences isolated from patients/individuals in six geographic areas and identified a total of 1234 mutations by comparing with the reference SARS-CoV-2 sequence. Through a hierarchical clustering based on the mutant frequencies, we classified the 28 countries into three clusters showing different fatality rates of COVID-19. In correlation analyses, we identified that ORF1ab 4715L and S protein 614G variants, which are in a strong linkage disequilibrium, showed significant positive correlations with fatality rates (r = 0.41, P = 0.029 and r = 0.43, P = 0.022, respectively). We found that BCG-vaccination status significantly associated with the fatality rates as well as number of infected cases. In BCG-vaccinated countries, the frequency of the S 614G variant had a trend of association with the higher fatality rate. We also found that the frequency of several HLA alleles, including HLA-A*11:01, were significantly associated with the fatality rates, although these factors were associated with number of infected cases and not an independent factor to affect fatality rate in each country. Our findings suggest that SARS-CoV-2 mutations as well as BCG-vaccination status and a host genetic factor, HLA genotypes might affect the susceptibility to SARS-CoV-2 infection or severity of COVID-19.

Effect of Molecular Crowding on the Stability of RNA G-Quadruplexes with Various Numbers of Quartets and Lengths of Loops.

G-Quadruplexes are noncanonical structures formed by guanine-rich regions of not only DNA but also RNA. RNA G-quadruplexes are widely present in the transcriptome as mRNAs and noncoding RNAs and take part in various essential functions in cells. Furthermore, stable RNA G-quadruplexes control the extent of biological functions, such as mRNA translation and antigen presentation. To understand and regulate the functions controlled by RNA G-quadruplexes in cellular environments, which are molecularly crowded, we would be required to investigate the stability of G-quadruplexes in molecular crowding. Here, we systematically investigated the thermodynamic stability of RNA G-quadruplexes with different numbers of G-quartets and lengths of loops. The molecular crowding conditions of polyethylene glycol with an average molecular weight of 200 (PEG200) were found to stabilize RNA G-quadruplexes with three and four G-quartets, while G-quadruplexes with two G-quartets did not exhibit any stabilization upon addition of PEG200. On the other hand, no difference in stabilization by PEG200 was observed among the G-quadruplexes with different loop lengths. Thermodynamic analysis of the RNA G-quadruplexes revealed more appropriate motifs for identifying G-quadruplex-forming sequences. The informatics analysis with new motifs demonstrated that the distributions of G-quadruplexes in human noncoding RNAs differed depending on the number of G-quartets. Therefore, RNA G-quadruplexes with different numbers of G-quartets may play different roles in response to environmental changes in cells.

Tyr-Trp administration facilitates brain norepinephrine metabolism and ameliorates a short-term memory deficit in a mouse model of Alzheimer's disease.

The physiological actions of orally ingested peptides on the brain remain poorly understood. This study examined the effects of 39 orally administered synthetic Tyr-containing dipeptides on the enhancement of brain norepinephrine metabolism in mice by comparing the concentration of 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG). Although Tyr-Tyr administration increased blood and cerebral cortex (Cx) Tyr concentrations the most, Tyr-Trp increased Cx MHPG concentration the most. The oral administration of Tyr-Trp ameliorated a short-term memory deficit of a mouse model of cognitive dysfunction induced by amyloid beta peptide 25-35. Gene expression profiling of mouse brain using a microarray indicated that Tyr-Trp administration led to a wide variety of changes in mRNA levels, including the upregulation of genes encoding molecules involved in catecholamine metabolism. A comparative metabolome analysis of the Cx of mice given Tyr-Trp or Tyr-Tyr demonstrated that Tyr-Trp administration yielded higher concentrations of Trp and kynurenine pathway metabolites than Tyr-Tyr administration, as well as higher L-dopa levels, which is the initial product of catecholamine metabolism. Catecholamines were not significantly increased in the Cx of the Tyr-Tyr group compared with the Tyr-Trp group, despite a marked increase in Tyr. Presumably, Tyr-Trp administration enhances catecholamine synthesis and metabolism via the upregulation of genes involved in Tyr and Trp metabolism as well as metabolites of Tyr and Trp. These findings strongly suggest that orally ingested Tyr-Trp modulates the brain metabolome involved in catecholamine metabolism and contributes to higher brain function.

Hydroxyl groups in cosolutes regulate the G-quadruplex topology of telomeric DNA.

Telomeric G-quadruplex topology has the ability to regulate telomerase activity, which counteracts the shortening of telomere with successive cell divisions, thereby causing genomic longevity. However, the detailed mechanism of G-quadruplexes topologies formed by telomeric sequences requires further investigation. In this study, we quantitatively investigated the effect of cosolutes, particularly the varying number of hydroxyl groups, on the structural transition between hybrid type and parallel G-quadruplexes formed by telomeric DNA sequences. Cosolutes with one or no hydroxyl groups in the vicinal position more efficiently induced the transition to parallel G-quadruplex from hybrid G-quadruplex than those with more hydroxyl groups. We also examined the effect of cosolute structures on the hydration of G-quadruplex formation; the results indicated that cosolutes with fewer hydroxyl groups lead to the release of greater amount of water during G-quadruplex formation. Molecular dynamics results showed that the parallel G-quadruplex was more dehydrated than the hybrid type G-quadruplex. Generally, a dehydrated structure is favored under crowding condition. Thus, depending on the surrounding cosolutes, the G-quadruplex topology can be controlled by the G-quadruplex hydration state.

Small synthetic molecule-stabilized RNA pseudoknot as an activator for -1 ribosomal frameshifting.

Programmed -1 ribosomal frameshifting (-1PRF) is a recoding mechanism to make alternative proteins from a single mRNA transcript. -1PRF is stimulated by cis-acting signals in mRNA, a seven-nucleotide slippery sequence and a downstream secondary structure element, which is often a pseudoknot. In this study we engineered the frameshifting pseudoknot from the mouse mammary tumor virus to respond to a rationally designed small molecule naphthyridine carbamate tetramer (NCTn). We demonstrate that NCTn can stabilize the pseudoknot structure in mRNA and activate -1PRF both in vitro and in human cells. The results illustrate how NCTn-inducible -1PRF may serve as an important component of the synthetic biology toolbox for the precise control of gene expression using small synthetic molecules.

Validation of a calibration method using the cross-calibration factor and system planar sensitivity in quantitative single-photon emission computed tomography imaging.

The present study aimed to validate the absolute quantitative accuracy of a calibration method for single-photon emission computed tomography (SPECT) using cross-calibration factor (CCF)- and system sensitivity-based calibration methods. The CCF obtained with different reconstruction parameters was evaluated using a cylindrical phantom (diameter 20 cm, height 20 cm). SPECT images were acquired with a positron emission tomography/computed tomography (CT) phantom. Subsequently, they were reconstructed by using ordered subset expectation maximization with resolution recovery, scatter, and CT-based attenuation correction. All reconstructed SPECT counts were converted to activity concentrations based on the CCF and system planar sensitivity. We placed 12 circular regions of interest, 37 mm in diameter, on the phantom background, and the converted activity concentration and relative measurement error were assessed. The CCF obtained using a cylindrical phantom was affected by the iterative update number and post-smoothing filter function. The activity concentration calibrated using the CCF showed over- and underestimation. However, the activity concentration obtained from the system planar sensitivity was similar to that gained using the phantom. The values obtained using the system planar sensitivity were within 10% of the activity concentrations obtained with the phantom. These findings demonstrated that the calibration method using system planar sensitivity provides accurate quantification within 10% of the true activity concentration. Further clinical examination is required to validate the present results.