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Technologies for visualizing and tracking RNA are essential in molecular biology, including in disease-related fields. In this study, we propose a novel probe set (DAt-probe and T-probe) that simultaneously detects two mutations in the same RNA using fluorescence resonance energy transfer (FRET). The DAt-probe carrying the fluorophore Atto488 and the quencher Dabcyl were used to detect a cancer mutation (exon19del), and the T-probe carrying the fluorophore Tamra was used to detect drug resistance mutations (T790M) in epidermal growth factor receptor (EGFR) mRNA. These probes were designed to induce FRET when both mutations were present in the mRNA. Gel electrophoresis confirmed that the two probes could efficiently bind to the mutant mRNA. We measured the FRET ratios using wild-type and double-mutant RNAs and found a significant difference between them. Even in living cells, the FRET probe could visualize mutant RNA. As a result, we conclude that this probe set provides a method for detecting two mutations in the single EGFR mRNA via FRET.

A 66-year-old Japanese woman developed pulseless electrical activity following an acute pulmonary embolism and was treated with thrombolytic therapy. She remained hemodynamically unstable and therefore underwent extracorporeal membrane oxygenation (ECMO). While receiving treatment with ECMO, blood clots induced by endobronchial hemorrhage caused tracheobronchial airway obstruction, leading to ventilatory defect. Furthermore, her cardiac function improved, resulting in cerebral hypoxemia progression. Therefore, the blood clots were removed with a Fogarty balloon catheter and endobronchial urokinase administration, resulting in improvement in her respiratory condition. Finally, ECMO was decannulated, and the patient was discharged from our hospital without difficulties in her activities of daily living.

Iatrogenic left main coronary artery (LMCA) dissection is a complication inadvertently caused by the interventional cardiologist and can have significant consequences. A 38-year-old man presented to hospital with non-ST-elevation myocardial infarction. Coronary angiography (CAG) revealed an obstructed proximal left circumflex artery (LCx) that was successfully treated with revascularization using a drug-eluting stent (DES). However, CAG after recanalization of the LCx demonstrated a spiral dissection of the left coronary artery from the mid-LMCA to the left anterior descending (LAD) artery and LCx. The dissection was classified as National Heart, Lung and Blood Institute type D in LAD and type F in LCx. Immediate exclusion stenting of the dissection flap by another DES and thrombolysis in myocardial infarction 3 flow were achieved in the LAD and LCx. The patient achieved hemodynamic stability with improvement in symptoms, despite residual dissection in the LAD. We, therefore, preferred careful observation over revascularization. The false lumen remained visible with a double-barrel appearance in the LAD on 6-month follow-up CAG, which disappeared at the 2-year follow-up. We report a rare case of a large double-barrel dissection that spontaneously occluded over time without any aggressive interventions.

Liquid biopsies have been receiving tremendous attentions as easy, rapid, and non-invasive tools for cancer diagnosis. Liquid biopsy can be performed repeatedly for disease monitoring and is expected to overcome the limitations of tissue biopsies. With the advancement of next generation sequencing technologies, it is now possible to detect minute amount of tumor-derived circulation tumor DNA (ctDNA) from blood samples. Importantly, ctDNA detection could be complementary to tissue biopsies or tumor biomarkers particularly in cases of which tumor biopsy is clinically difficult to obtain. Here, we introduce the up-to-date technologies used in cfDNA-based liquid biopsy and review the clinical utilities of ctDNA in cancer screening, detection of minimal residual diseases, selection of molecular-targeted drugs, as well as monitoring of treatment responsiveness. We also discuss the challenges and future perspectives of liquid biopsy implementation in clinical setting.

The A3B-type Lactosome comprised of poly(sarcosine)3-block-poly(l-lactic acid), a biocompatible and biodegradable polymeric nanomicelle, was reported to accumulate in tumors in vivo via the enhanced permeability and retention (EPR) effect. Recently, the cellular uptake of Lactosome particles was enhanced through the incorporation of a cell-penetrating peptide (CPP), L7EB1. However, the ability of Lactosome as a drug delivery carrier has not been established. Herein, we have developed a method to conjugate the A3B-type Lactosome with ATP-binding cassette transporter G2 (ABCG2) siRNA for inducing in vitro apoptosis in the cancer cell lines PANC-1 and NCI-H226. The L7EB1 peptide facilitates the cellular uptake efficiency of Lactosome but does not deliver siRNA into cytosol. To establish the photoinduced cytosolic dispersion of siRNA, a photosensitizer loaded L7EB1-Lactosome was prepared, and the photosensitizer 5,10,15,20-tetra-kis(pentafluorophenyl)porphyrin (TPFPP) showed superiority in photoinduced cytosolic dispersion. We exploited the combined effects of enhanced cellular uptake by L7EB1 and photoinduced endosomal escape by TPFPP to efficiently deliver ABCG2 siRNA into the cytosol for gene silencing. Moreover, the silencing of ABCG2, a protoporphyrin IX (PpIX) transporter, also mediated photoinduced cell death via 5-aminolevulinic acid (ALA)-mediated PpIX accumulated photodynamic therapy (PDT). The synergistic capability of the L7EB1/TPFPP/siRNA-Lactosome complex enabled both gene silencing and PDT.

To investigate RNA degradation in live cells, detection methods that do not require RNA extraction from cells are necessary. In this study, we examined the utility of fluorescence lifetime measurements using a probe attached to the end of an RNA molecule for detecting RNA degradation. We optimized a short fluorescein-labeled RNA sequence whose fluorescence lifetime varied significantly before and after degradation. The selected HHG-fluorescein sequence (H = U, C, or A) is a promising RNA labeling unit (fluorescence lifetime probe) for live cell imaging of RNA degradation.

Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of peptides and proteins, but CPP fusion peptides and proteins are often transported by endocytosis and trapped in endosomes. Photochemical internalization (PCI) is a method for the endosomal escape of the trapped peptide or protein and release into the cytosol using light and photosensitizers. In PCI, endosomal membranes are thought to be destabilized by singlet oxygen (1O2) photogenerated from photosensitizers localized in endosomes. We previously developed CPP-cargo-photosensitizer (PS) conjugates able to photodependently enter the cytosol via the PCI mechanism. For example, TatU1A-PS (a covalent complex of Tat [CPP], U1A RNA-binding protein [cargo], and PS) can photodependently deliver RNAs into the cytosol, and TatBim-PS (a covalent complex of Tat, Bim [cargo], and PS) can photoinduce apoptosis in mammalian cells. However, for many newly created conjugates, the induction of PCI has been insufficient. We hypothesized that the amino acid linker sequence (XX) adjacent to the photosensitizer is an important determinant of PCI efficiency. In this study, using CPP-cargo-XX-PS platforms, we examined the relationship between PCI efficiency and the linker amino acid sequence near the photosensitizer. We found that hydrophobic FF and LL linkers enhanced the PCI efficiencies of both TatBim-XX-PS and TatU1A-XX-PS. The effectiveness of the linker depended, in part, on both the cargo moiety and the photosensitizer. These results may guide the design of CPP-cargo-PS conjugates conferring broad target functions for PCI and photodynamic therapy.

BACKGROUND The slow-flow/no-reflow phenomenon and impaired ST segment resolution (STR) following primary percutaneous coronary intervention (PCI) in ST-elevation myocardial infarction (STEMI) predict unfavorable prognosis and are characterized by obstruction of the coronary microvascular. Several predictors of slow-flow/no-reflow have been revealed, but few studies have investigated predictors of slow-flow/no-reflow and STR exclusively in acute myocardial infarction patients with initial Thrombolysis in Myocardial Infarction (TIMI) Grade 0.Methods and Results:In all, 279 STEMI patients with initial TIMI Grade 0 were enrolled in the study. Slow-flow/no-reflow was defined as TIMI Grade

Methods to spatially induce apoptosis are useful for cancer therapy. To control the induction of apoptosis, methods using light, such as photochemical internalization (PCI), have been developed. We hypothesized that photoinduced delivery of microRNAs (miRNAs) that regulate apoptosis could spatially induce apoptosis. In this study, we identified pre-miR-664a as a novel apoptosis-inducing miRNA via mitochondrial apoptotic pathway. Further, we demonstrated the utility of photoinduced cytosolic dispersion of RNA (PCDR), which is an intracellular RNA delivery method based on PCI. Indeed, apoptosis is spatially regulated by pre-miR-664a and PCDR. In addition, we found that apoptosis induced by pre-miR-664a delivered by PCDR was more rapid than that by lipofection. These results suggest that pre-miR-664a is a nucleic acid drug candidate for cancer therapy and PCDR and pre-miR-664a-based strategies have potential therapeutic uses for diseases affecting various cell types.

The inefficient endosomal escape of drugs or macromolecules is a major obstacle to achieving successful delivery to therapeutic targets. An efficient approach to circumvent this barrier is photochemical internalization (PCI), which uses light and photosensitizers for endosomal escape of the delivered macromolecules. The PCI mechanism is related to photogenerated singlet oxygen, but the mechanism is still unclear. In this study, we examined the relation of PCI to heat, pH and Ca2+ ions using cell penetrating peptide (CPP)-cargo-photosensitizer (Alexa546 or Alexa633) conjugates. A cell temperature changing experiment demonstrated that heat (thermal mechanism) does not significantly contribute to the photoinduced endosomal escape. Inhibition of V-ATPase proton pump activity and endosomal pH upregulation indicated that PCI-mediated endosomal escape needs endosomal acidification prior to photoirradiation. Imaging of the CPP-cargo-photosensitizer and Ca2+ ions during photostimulation showed that intracellular calcium increase is not the cause of the endosomal escape of the complex. The increment is mainly due to Ca2+ influx. These findings show the importance of extra- and intracellular milieu conditions in the PCI mechanism and enrich our understanding of PCI-related changes in cell.

Background Since a displaced bronchus related to the left upper lobe is an uncommon anatomical anomaly, it has a risk of being accidentally resected during left upper lobe resection unless they are identified preoperatively. A case of video-assisted thoracic surgery (VATS) segmentectomy that was safely performed under preoperative identification of a displaced subsegmental bronchus and anomalous pulmonary vessels is presented. Case presentation A 48-year-old woman visited our hospital because of an abnormal shadow on a radiograph on a health check. The chest computed tomography (CT) showed a multicystic mass with a diameter of 35 mm on dorsal interlobar parenchyma between the S1+2 and S6 segments in the left lung. The three-dimensional (3D) CT with multiplanar reconstruction showed that B1+2b+c passed to the dorsal side of the left main pulmonary artery (PA), which was considered a displaced bronchus. The branch of A6 arose from the left main PA at the level of the branches of A3 and A1+2, more proximal than the normal anatomy, and passed to the dorsal side of a displaced B1+2b+c. The branch of V1+2 passed between B6 and the bronchus to the basal segment and joined V6 at the dorsal side of the pulmonary hilum. Intraoperative findings of the anatomy of the bronchi and pulmonary vessels were exactly the same as the preoperative 3D CT findings, so segmentectomy of S1+2b+c and S6 by VATS was performed safely. Then there were accessory fissures between S1+2 and S3 and between S6 and the basal segment. The pathological diagnosis was a left lung abscess. Conclusions A preoperative 3D CT may be helpful for identifying anatomical anomalies. An anatomical anomaly should be suspected if accessory fissure is found during surgery.

Tat-U1A-rose bengal conjugate (TatU1A-RB) was prepared as an ultrasound-sensitive RNA carrier molecule. This molecule consists of Tat cell-penetrating peptide, U1A RNA-binding protein, and rose bengal as a sonosensitizer. We demonstrated that TatU1A-RB delivered RNA via the endocytosis pathway, which was followed by ultrasound-dependent endosomal escape and cytosolic dispersion of the RNA. A short hairpin RNA (shRNA) delivered by TatU1A-RB mediated RNA interference (RNAi) ultrasound-dependently. Even by ultrasound irradiation through blood cells, RNAi could be induced with TatU1A-RB and the shRNA. This ultrasound-dependent cytosolic RNA delivery method will serve as the basis for a new approach to nucleic acid therapeutics.

Stress resistance mechanisms include upregulation of heat shock proteins (HSPs) and formation of granules. Stress-induced granules are classified into stress granules and nuclear stress bodies (nSBs). The present study examined the involvement of nSB formation in thermal resistance. We used chemical compounds that inhibit heat shock transcription factor 1 (HSF1) and scaffold attachment factor B (SAFB) granule formation and determined their effect on granule formation and HSP expression in HeLa cells. We found that formation of HSF1 and SAFB granules was inhibited by 2,5-hexanediol. We also found that suppression of HSF1 and SAFB granule formation enhanced heat stress-induced apoptosis. In addition, the upregulation of HSP27 and HSP70 during heat stress recovery was suppressed by 2,5-hexanediol. Our results suggested that the formation of HSF1 and SAFB granules was likely to be involved in the upregulation of HSP27 and HSP70 during heat stress recovery. Thus, the formation of HSF1 and SAFB granules was involved in thermal resistance.

Painful left bundle branch block (LBBB) syndrome is a rare disease that presents as simultaneous chest pain and transient LBBB without myocardial ischemia. We diagnosed a 72-year-old Japanese man with painful LBBB syndrome complicated by iron-overload cardiomyopathy. Phlebotomy was initially performed to improve myocardial iron deposition and conductive disturbance. Ironically, his chest pain was fully improved by the completion of incessant LBBB and walk-through phenomenon. However, this case demonstrates a clinically significant therapeutic strategy for cardiomyopathy-induced painful LBBB syndrome. Due to the lack of treatment guidelines, individualized treatment is required for each case of painful LBBB.

Background: In diabetes mellitus-related cardiomyopathy (DMCMP), hyperglycemia causes endothelial dysfunction, fibrosis, and myocardial injury, which result in left ventricular (LV) dysfunction. Treatment with sodium–glucose co-transporter 2 inhibitor (SGLT2i) reduces the risk of exacerbation of heart failure (HF). The beneficial effects of SGLT2i on HF depend not only on indirect actions such as osmotic diuresis but also direct actions on the myocardium leading to improvements in LV function. However, it remains unclear whether SGLT2i treatment is equally effective in any phase of DMCMP. The aim of this observational study was to compare the efficacy of SGLT2i treatment on LV dysfunction between early and advanced DMCMP.Methods: Thirty-five symptomatic non-ischemic HF patients with LV ejection fraction (EF) greater than 40% and type 2 diabetes mellitus (T2DM) treated with administration of empagliflozin (10 mg/day) were enrolled. According to the myocardial extracellular volume fraction (ECV), a reliable marker of cardiac fibrosis quantified by cardiac magnetic resonance, the patients were divided into the early DMCMP group (n = 16, ECV ≤ 30%) and advanced DMCMP group (n = 19, ECV > 30%) and followed-up prospectively. Echocardiography was performed at baseline and after 12 months. LV systolic function assessed as LV global longitudinal strain (GLS) and diastolic function assessed as the ratio of early diastolic mitral inflow velocity to early diastolic mitral annular velocity (E/e’) were compared.Results: ECV was strongly correlated with T2DM duration (r2 = 0.65, p < 0.001). At baseline, both groups had similar backgrounds (LVGLS: 7.9 ± 2.4% vs. 6.7 ± 3.0%, p = 0.207, and E/e’: 13.2 ± 6.1 cm/s vs. 12.6 ± 3.8 cm/s, p = 0.694). After 12 months, the early DMCMP group showed greater improvement in LVGLS (ΔLVGLS: 4.6 ± 1.5% vs. 1.6 ± 3.3%, p = 0.003) and E/e’ (ΔE/e’: -3.4 ± 5.5 cm/s vs. -0.1 ± 3.5 cm/s, p = 0.043) than in the advanced DMCMP group.Conclusion: The positive effects of empagliflozin on LV dysfunction were more remarkable in DMCMP with mild cardiac fibrosis than with advanced fibrosis. Early intervention of SGLT2i for DMCMP is preferable.

Background Diabetes mellitus-related cardiomyopathy (DMCMP), defined as left ventricular (LV) dysfunction caused by hyperglycemia in the absence of coronary artery disease, leads to heart failure (HF). Previous studies have shown that treatment with sodium-glucose co-transporter 2 inhibitor (SGLT2i) reduces the risk of exacerbation of HF. The beneficial effects of SGLT2i on HF depend not only on indirect actions such as osmotic diuresis but also on direct actions on the myocardium, leading to improvements in LV function. However, it remains unclear whether SGLT2i treatment is equally effective in any phase of DMCMP. The aim of this observational study was to compare the efficacy of SGLT2i treatment on LV dysfunction between early and advanced DMCMP. Methods Thirty-five symptomatic non-ischemic HF patients with LV ejection fraction > 40% and type 2 diabetes mellitus (T2DM) treated with empagliflozin (EMPA group) and 20 controls treated without SGLT2i were enrolled. According to the myocardial extracellular volume fraction (ECV), a reliable marker of cardiac fibrosis quantified by cardiac magnetic resonance, the EMPA group was further divided into early DMCMP (n = 16, ECV ≤ 30%) and advanced DMCMP (n = 19, ECV > 30%) groups and followed up prospectively. Echocardiography was performed at baseline and after 12 months. LV function assessed as LV global longitudinal strain (LVGLS) and the ratio of early diastolic mitral inflow velocity to early diastolic mitral annular velocity (E/e′) were compared. Results ECV was strongly correlated with T2DM duration (r2 = 0.65, p p = 0.005, and 4.6 ± 1.5% (early DMCMP) vs. 1.6 ± 3.3% (advanced DMCMP), p = 0.003) and E/e′ (ΔE/e′: − 1.5 ± 4.7 vs. − 0.3 ± 3.0, p = 0.253, and − 3.4 ± 5.5 vs. − 0.1 ± 3.5, p = 0.043). Conclusions The positive effects of empagliflozin on LV dysfunction were more remarkable in early than in advanced DMCMP. Early intervention of SGLT2i for DMCMP may be preferable.

Hemorrhagic myocardial infarction (HMI) is a complication associated with percutaneous coronary intervention (PCI) for acute myocardial infarction (AMI). We carried out a successful PCI for a 59-year old Japanese man presenting with chest pain due to AMI over 5 h. The onset to balloon time was 363 min. The next morning, he suffered cardiogenic shock, even with an auxiliary circulating device, which eventually resulted in death. An autopsy revealed extensive HMI. The necrotic myocardium showed not only coagulation necrosis but also contraction band necrosis which suggests myocardial injury due to late reperfusion. Although the intramyocardial hemorrhage was confined to the necrotic area, it was beyond the perfusion area of the culprit artery. Here, we describe a case of death with severe HMI. HMI can be a serious complication and worsen prognosis.

Murine membrane-bound phospholipase A2receptor 1 (PLA2R) is shed and released into plasma in a soluble form that retains all of the extracellular domains. Relatively little is known about human PLA2R. This study examined whether human soluble PLA2R has biological functions and whether soluble PLA2R exists in human plasma. Here, we showed that human recombinant soluble PLA2R (rsPLA2R) bound to collagen-I and inhibited interaction of collagen-I with the extracellular domain of integrin β1 on the cell surface of human embryonic kidney 293 (HEK293) cells. As a result, rsPLA2R suppressed integrin β1-mediated migratory responses of HEK293 cells to collagen-I in Boyden chamber experiments. Inhibition of phosphorylation of FAK Tyr397 was also observed. Similar results were obtained with experiments using soluble PLA2R released from HEK293 cells transfected with a construct encoding human soluble PLA2R. rsPLA2R lacking the fibronectin-like type II (FNII) domain had no inhibitory effects on cell responses to collagen-I, suggesting an important role of the FNII domain in the interaction of rsPLA2R with collagen-I. In addition, rsPLA2R suppressed the migratory response to collagen-IV and binding of collagen-IV to the cell surface of human podocytes that endogenously express membrane-bound, full-length PLA2R. Immunoprecipitation and Western blotting showed the existence of immunoreactive PLA2R in human plasma. In conclusion, human recombinant soluble PLA2R inhibits integrin β1-mediated cell responses to collagens. Further studies are warranted to elucidate whether immunoreactive PLA2R in human plasma has the same properties as rsPLA2R.

Many cellular events are thought to be controlled by the temporal upregulation of multiple RNAs; the timing of the upregulation of these RNAs is not always the same. In this study, we first show that our light-directed intracellular RNA delivery method induced high concentrations of RNA in a short period. This effect was beneficial for the temporal control of cellular events by functional RNAs. Next, we stimulated the short-term upregulation of two different RNAs at different time points. Cytosolic delivery of a first RNA was induced by red light; thereafter, cytosolic delivery of a second RNA was induced by near-infrared light. The time difference between the introduction of the first and second RNA can be short (0.5-4 h) or long (>8 h). This strategy shows the potential for future applications of the deliberate control of time-dependent RNA concentration to guide various cellular functions by multiple RNAs.

MicroRNAs (miRNAs) belong to a class of small noncoding RNAs that play important roles in the translational regulation of gene expression. A number of miRNAs are known to act as key regulators of diverse processes such as neuronal differentiation. In this study, we have attempted to identify novel miRNAs related to neuronal differentiation via microarray analysis in the human neuronal differentiation model neuroblastoma SH-SY5Y cells. We identified 15 up-regulated and eight down-regulated miRNAs in SH-SY5Y cells treated with all-trans retinoic acid to induce differentiation. We further showed that one of the up-regulated miRNAs, miR-664a-5p, promoted neuronal differentiation of SH-SY5Y cells. These findings enhance our understanding of the miRNAs involved in the process of neurogenesis and, in particular, highlight an important role of miR-664a-5p in SH-SY5Y cell neuronal differentiation. Further studies will be required to confirm the function of miR-664-5p in neuronal development and disease and to identify its relevant target genes.

A method to induce cytoplasmic peptide delivery, using ultrasound, was demonstrated using a molecular conjugate of a cell-penetrating peptide (CPP), a functional peptide, and a sonosensitizer. As a model of such molecular conjugates, TatBim-RB, consisting of the Tat CPP, the Bim apoptosis inducing peptide, and the sonosensitizer rose bengal was synthesized. CPPs have been widely used for intracellular delivery of various cargos; however, CPP-fused molecules tend to become entrapped in endosomes, as was observed for TatBim-RB molecules applied to cells. To promote escape of the entrapped TatBim-RB molecules, cells were irradiated with ultrasound, which successfully induced endosomal escape and cytoplasmic dispersion of TatBim-RB, and subsequently apoptosis. Our results suggest that this peptide-sonosensitizer conjugate strategy may facilitate numerous kinds of medicinal chemistry studies, and furthermore, this specific conjugate may exhibit potential as a novel therapeutic agent for the promotion of apoptosis.

The use of molecular beacons is a versatile method to detect RNAs. Typically, a single-stranded region of RNA is selected as a target sequence for molecular beacons. Therefore, the detection of highly structured short RNAs, such as tRNAs, seems to be difficult. In this study, as an example of highly structured target RNA, we used human tRNALys3, which is known to have functions in protein synthesis and the reverse transcription of human immunodeficiency virus (HIV)-1. No long single-stranded region more than 8 nt is present in this tRNA, which is much shorter than the standard target length of molecular beacons (∼20 nt). This study showed that sensitive detection of highly structured RNAs using molecular beacons was much more difficult than that of unstructured or less structured RNAs. However, efficient detection of the tRNALys3 was achieved by selecting the best target region, i.e., the region around the D arm, probably due to the ease of unfolding of this arm. Accordingly, our findings suggested that molecular beacons may have applications in the detection of highly structured RNAs related to various biological functions and diseases.

Pro-apoptotic peptides may be promising agents for cancer therapy owing to their ability to induce apoptosis in cancer cells. TatBim, a fusion peptide of Tat cell-penetrating peptide (CPP) and the BH3 domain derived from Bim apoptosis-inducing protein, is a pro-apoptotic peptide. In this study, based on the TatBim sequence, we attempted to minimize the CPP-Bim peptide while retaining apoptosis-inducing activity. The CPP and Bim parts were systematically shortened, and the pro-apoptotic activities of the shortened peptides were examined. We obtained TatBim-N1C2 and R8Bim-N1C2 as minimized peptides with efficient apoptotic activity. These peptides may have potential applications in future biomedical studies, such as cancer therapeutics.

Endosomal escape in cell-penetrating peptide (CPP)-based drug/macromolecule delivery systems is frequently insufficient. The CPP-fused molecules tend to remain trapped inside endosomes and end up being degraded rather than delivered into the cytosol. One of the methods for endosomal escape of CPP-fused molecules is photochemical internalization (PCI), which is based on the use of light and a photosensitizer and relies on photoinduced endosomal membrane destabilization to release the cargo molecule. Currently, it remains unclear how this delivery strategy behaves after photostimulation. Recent findings, including our studies using CPP-cargo-photosensitizer conjugates, have shed light on the photoinduced endosomal escape mechanism. In this review, we discuss the structural design of CPP-photosensitizer and CPP-cargo-photosensitizer conjugates, and the PCI mechanism underlying their application.

The possibility of spatiotemporally photocontrolling translation holds considerable promise for studies on the biological roles of local translation in cells and tissues. Here we report caged aminoacyl-tRNAs (aa-tRNAs) synthesized using a (7-diethylaminocoumarin-4-yl)methoxycarbonyl (DEACM)-cage compound. DEACM-caged aa-tRNA does not spontaneously deacylate for at least 4 h in neutral aqueous solution, and does not bind to the elongation factor Tu. On irradiation at ∼405 nm at 125 mW cm−2, DEACM-aa-tRNA is converted into active aa-tRNA with a half-life of 19 s. Notably, this rapid uncaging induced by visible light does not impair the translation system. Translation is photoinduced when DEACM-aa-tRNA carrying a CCCG or a CUA anticodon is uncaged in the presence of mRNAs harbouring a CGGG four-base codon or a UAG amber codon, respectively. Protein synthesis is phototriggered in several model systems, including an in vitro translation system, an agarose gel, in liposomes and in mammalian cells., Spatiotemporal regulation of protein synthesis would advance studies into the consequences of localised protein translation in cells and tissues. Here, Ohtsuki et al. improve on an earlier caged-tRNA design to provide caged aminoacyl-tRNAs that are rapidly uncaged by visible light.

A novel molecule, TatBim-Alexa, consisting of the HIV1 Tat cell-penetrating peptide, the Bim apoptosis-inducing peptide, and Alexa Fluor 546 was synthesized for photoinducion of apoptosis. The Alexa Fluor 546 was used as a photosensitizer and covalently attached at the C-terminus of TatBim peptide by the thiol-maleimide reaction. Photo-dependent cytosolic internalization of TatBim-Alexa and photo-dependent apoptosis using TatBim-Alexa were demonstrated in several kinds of mammalian cells including human cancer cell lines.