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The cryptophyte algae, Guillardia theta, possesses 46 genes that are homologous to microbial rhodopsins. Five of them are functionally light-gated cation channelrhodopsins (GtCCR1-5) that are phylogenetically distinct from chlorophyte channelrhodopsins (ChRs) such as ChR2 from Chlamydomonas reinhardtii. In this study, we report the ion channel properties of these five CCRs and compared them with ChR2 and other ChRs widely used in optogenetics. We revealed that light sensitivity varied among GtCCR1-5, in which GtCCR1-3 exhibited an apparent EC 50 of 0.21-1.16 mW/mm 2 , similar to that of ChR2, whereas GtCCR4 and GtCCR5 possess two EC50s, one of which is significantly small (0.025 and 0.032 mW/mm 2 ). GtCCR4 is able to trigger action potentials in high temporal resolution, similar to ChR2, but requires lower light power, when expressed in cortical neurons. Moreover, a high light-sensitive response was observed when GtCCR4 was introduced into blind retina ganglion cells of rd1, a mouse model of retinitis pigmentosa. Thus, GtCCR4 provides optogenetic neuronal activation with high light sensitivity and temporal precision., (© 2023. The Author(s).)

Energy transfer from light-harvesting ketocarotenoids to the light-driven proton pump xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium 1 and a terrestrial cyanobacterium 2 . Attempts to find carotenoids that bind and transfer energy to abundant rhodopsin proton pumps 3 from marine photoheterotrophs have thus far failed 4-6 . Here we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins using functional metagenomics combined with chromophore extraction from the environment. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet- or blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas may have a substantial effect on rhodopsin phototrophy in the world's lakes, seas and oceans. However, the functional implications of our findings are yet to be discovered., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

In pancreatic cancer, methyltransferase-like 3 (METTL3), a N(6)-methyladenosine (m6A) methyltransferase, has a favorable effect on tumors and is a risk factor for patients' prognosis. However, the details of what genes are regulated by METTL3 remain unknown. Several RNAs are methylated, and what genes are favored in pancreatic cancer remains unclear. By epitranscriptomic analysis, we report that polo-like kinase 1 (PLK1) is an important hub gene defining patient prognosis in pancreatic cancer and that RNA methylation is involved in regulating its cell cycle-specific expression. We found that insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) binds to m6A of PLK1 3' untranslated region and is involved in upregulating PLK1 expression and that demethylation of this site activates the ataxia telangiectasia and Rad3-related protein pathway by replicating stress and increasing mitotic catastrophe, resulting in increased radiosensitivity. This suggests that PLK1 methylation is essential for cell cycle maintenance in pancreatic cancer and is a new therapeutic target., (© 2022. The Author(s).)

DNA alterations, such as base modifications and mutations, are closely related to the activity of transcription factors and the corresponding cell functions; therefore, detection of DNA alterations is important for understanding their relationships. Particularly, DNA alterations caused by exposure to exogenous molecules, such as nucleic acid analogues for cancer therapy and the corresponding changes in cell functions, are of interest in medicine for drug development and diagnosis purposes. However, detection of comprehensive direct evidence for the relationship of DNA modifications/mutations in genes, their effect on transcription factors, and the corresponding cell functions have been limited. In this study, we utilized a single-molecule electrical detection method for the direct observation of DNA alterations on transcription factor binding motifs upon exposure to a nucleic acid analogue, trifluridine (FTD), and evaluated the effects of the DNA alteration on transcriptional activity in cancer cell line cells. We found ~ 10% FTD incorporation at the transcription factor p53 binding regions in cancer cells exposed to FTD for 5 months. Additionally, through single-molecule analysis of p53-enriched DNA, we found that the FTD incorporation at the p53 DNA binding regions led to less binding, likely due to weaken the binding of p53. This work suggests that single-molecule detection of DNA sequence alterations is a useful methodology for understanding DNA sequence alterations., (© 2022. The Author(s).)

Epitranscriptomics is the study of RNA base modifications involving functionally relevant changes to the transcriptome. In recent years, epitranscriptomics has been an active area of research. However, a major issue has been the development of sequencing methods to map transcriptome-wide RNA base modifications. We have proposed a single-molecule quantum sequencer for mapping RNA base modifications in microRNAs (miRNAs), such as N6-methyladenosine (m6A) or 5-methylcytidine (5mC), which are related to cancer cell propagation and suppression. Here, we investigated 5mC and m6A in hsa-miR-200c-5p extracted from colorectal cancer cells and determined their methylation sites and rates; the data were comparable to those determined by mass spectrometry. Furthermore, we evaluated the methylation ratio of cytidine and adenosine at each site in the sequences and its relationship. These results suggest that the methylation ratio of cytidine and adenosine is facilitated by the presence of vicinal methylation. Our work provides a robust new tool for sequencing various types of RNA base modifications in their RNA context., (© 2021. The Author(s).)

Halogen-modified nucleic acid molecules, such as trifluorothymidine (FTD) and 5-fluorouracil, are widely used in medical science and clinical site. These compounds have a very similar nucleobase structure. It is reported that both of these compounds could be incorporated into DNA. The incorporation of FTD produces highly anti-tumor effect. However, it is not known whether to occur a significant effect by the incorporation of 5-fluorouracil. Nobody knows why such a difference will occur. To understand the reason why there is large differences between trifluorothymidine and 5-fluorouracil, we have performed the molecular dynamics simulations and molecular orbital calculations. Although the active interaction energy between Halogen-modified nucleic acids or and complementary adenine was increased, in only FTD incorporated DNA, more strongly dispersion force interactions with an adjacent base were detected in many thermodynamic DNA conformations. As the results, the conformational changes occur even if it is in internal body temperature. Then the break of hydrogen bonding between FTD and complementary adenine base occur more frequently. The double helix structural destabilization of DNA with FTD is resulted from autoagglutination caused by the bonding via halogen orbitals such as halogen bonding and the general van der Waals interactions such as CH-[Formula: see text], lone pair (LP)-[Formula: see text], and [Formula: see text]-[Formula: see text] interactions. Therefore, it is strongly speculated that such structural changes caused by trifluoromethyl group is important for the anti-tumor effect of FTD alone.

Cancer stem cells (CSCs) are involved in metastatic colorectal cancer recurrence, but no effective therapy targeting these cells is currently available. Because trifluridine (FTD)/tipiracil therapy is used for refractory colorectal cancer, we sought to determine whether FTD is effective against CSC-like cells. CD44 + CD133 + high-expressing and other populations of human DLD-1 colon cancer cells were separately isolated through fluorescence-activated cell sorting. The sphere-forming activity of each population and the anti-sphere-forming effects of FTD and fluorouracil (5-FU) on CD44 + CD133 + cells were then measured. CD44 + CD133 + DLD-1 cells formed substantially more spheres than other cells. Moreover, treating CD44 + CD133 + DLD-1 cells with subtoxic concentrations of FTD (1 µM) inhibited sphere formation, and this was superior to the effect of subtoxic concentrations (1 µM) of 5-FU. The associated inhibition rates for FTD and 5-FU were 58.2% and 26.1%, respectively. Further, CD44 + CD133 + DLD-1 cells expressed higher levels of thymidine kinase 1, which is responsible for FTD phosphorylation, than DLD-1 cells, and FTD was incorporated into the DNA of CD44 + CD133 + DLD-1 cells. Thus, our data show that FTD treatment is effective against CSC-like cells and might be applied as CSC-targeting chemotherapy for tumor subtypes with high CD44 and CD133 expression.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics 1 . They are widely present in bacteria, archaea, algae and algal viruses 2,3 . Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the β-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

Identifying positions at which anticancer drug molecules incorporate into DNA is essential to define mechanisms underlying their activity, but current methodologies cannot yet achieve this. The thymidine fluorine substitution product trifluridine (FTD) is a DNA-damaging anticancer agent thought to incorporate into thymine positions in DNA. This mechanism, however, has not been directly confirmed. Here, we report a means to detect FTD in a single-stranded oligonucleotide using a method to distinguish single molecules by differences in electrical conductance. Entire sequences of 21-base single-stranded DNAs with and without incorporated drug were determined based on single-molecule conductances of the drug and four deoxynucleosides, the first direct observation of its kind. This methodology may foster rapid development of more effective anticancer drugs.

Previous studies highlighted that aminopeptidase N (APN)/CD13 acts as a scavenger in the survival of hepatocellular carcinoma (HCC) stem cells by reducing reactive oxygen species (ROS) levels. Hence, it has been proposed that APN/CD13 inhibition can increase cellular ROS levels and sensitize cells to chemotherapeutic agents. Although ubenimex, also known as bestatin, competitively inhibits proteases such as APN/CD13 on the cellular membrane and it is clinically used for patients with acute myeloid leukemia and lymphedema, research has demonstrated that higher concentrations of the agent induce the death of APN/CD13 + HCC stem cells. In this study, we developed a poly(ethylene glycol)-poly(lysine) block copolymer-ubenimex conjugate (PEG-b-PLys(Ube)) to increase the efficacy of reagents in APN/CD13 + cancer stem cells. Exposure to PEG-b-PLys(Ube) increased the intracellular ROS concentration by inhibiting APN enzyme activity, permitting the induction of apoptosis and attenuation of HCC cell proliferation. In addition, PEG-b-PLys(Ube) exhibited a relatively stronger antitumor effect in mice than PEG-b-PLys alone or phosphate-buffered saline. Moreover, an isobologram analysis revealed that combinations of fluorouracil, cisplatin, or doxorubicin with PEG-b-PLys(Ube) exhibited synergistic effects. This study demonstrated that PEG-b-PLys(Ube) does not impair the properties of ubenimex and exerts a potent antitumor effect.

Cancer stem cells (CSCs) exhibit tumorigenic potential and can generate resistance to chemotherapy and radiotherapy. A labeled ornithine decarboxylase (ODC, a rate-limiting enzyme involved in polyamine [PA] biosynthesis) degradation motif (degron) system allows visualization of a fraction of CSC-like cells in heterogeneous tumor populations. A labeled ODC degradation motif system allowed visualization of a fraction of CSC-like cells in heterogeneous tumor populations. Using this system, analysis of polyamine flux indicated that polyamine metabolism is active in CSCs. The results showed that intracellular polyamines inhibited the activity of histone lysine 4 demethylase enzymes, including lysine-specific demethylase-1 (LSD1). Chromatin immunoprecipitation with Pol II antibody followed by massively parallel DNA sequencing, revealed the global enrichment of Pol II in transcription start sites in CSCs. Increase of polyamines within cells resulted in an enhancement of ID1 gene expression. The results of this study reveal details of metabolic pathways that drive epigenetic control of cancer cell stemness and determine effective therapeutic targets in CSCs., Competing Interests: Institutional endowments were received from Taiho Pharmaceutical Co., Ltd. (Tokyo, Japan), Evidence Based Medical Research Center Inc. (Osaka, Japan), UNITECH Co., Ltd. (Chiba, Japan), IDEA Consultants, Inc. (Tokyo, Japan), and Kinshu-kai Medical Corporation (Osaka, Japan).

Many organisms capture or sense sunlight using rhodopsin pigments 1,2 , which are integral membrane proteins that bind retinal chromophores. Rhodopsins comprise two distinct protein families 1 , type-1 (microbial rhodopsins) and type-2 (animal rhodopsins). The two families share similar topologies and contain seven transmembrane helices that form a pocket in which retinal is linked covalently as a protonated Schiff base to a lysine at the seventh transmembrane helix 2,3 . Type-1 and type-2 rhodopsins show little or no sequence similarity to each other, as a consequence of extensive divergence from a common ancestor or convergent evolution of similar structures 1 . Here we report a previously unknown and diverse family of rhodopsins-which we term the heliorhodopsins-that we identified using functional metagenomics and that are distantly related to type-1 rhodopsins. Heliorhodopsins are embedded in the membrane with their N termini facing the cell cytoplasm, an orientation that is opposite to that of type-1 or type-2 rhodopsins. Heliorhodopsins show photocycles that are longer than one second, which is suggestive of light-sensory activity. Heliorhodopsin photocycles accompany retinal isomerization and proton transfer, as in type-1 and type-2 rhodopsins, but protons are never released from the protein, even transiently. Heliorhodopsins are abundant and distributed globally; we detected them in Archaea, Bacteria, Eukarya and their viruses. Our findings reveal a previously unknown family of light-sensing rhodopsins that are widespread in the microbial world.

Oligomeric assembly is a common feature of membrane proteins and often relevant to their physiological functions. Determining the stoichiometry and the oligomeric state of membrane proteins in a lipid bilayer is generally challenging because of their large size, complexity, and structural alterations under experimental conditions. Here, we use high-speed atomic force microscopy (HS-AFM) to directly observe the oligomeric states in the lipid membrane of various microbial rhodopsins found within eubacteria to archaea. HS-AFM images show that eubacterial rhodopsins predominantly exist as pentamer forms, while archaeal rhodopsins are trimers in the lipid membrane. In addition, circular dichroism (CD) spectroscopy reveals that pentameric rhodopsins display inverted CD couplets compared to those of trimeric rhodopsins, indicating different types of exciton coupling of the retinal chromophore in each oligomer. The results clearly demonstrate that the stoichiometry of the fundamental oligomer of microbial rhodopsins strongly correlate with the phylogenetic tree, providing a new insight into the relationship between the oligomeric structure and function-structural evolution of microbial rhodopsins.

We investigated the relationship between methylomic [5-methylation on deoxycytosine to form 5-methylcytosine (5mC)] and transcriptomic information in response to chemotherapeutic 5-fluorouracil (5-FU) exposure and cisplatin (CDDP) administration using the ornithine decarboxylase (ODC) degron-positive cancer stem cell model of gastrointestinal tumour. The quantification of 5mC methylation revealed various alterations in the size distribution and intensity of genomic loci for each patient. To summarise these alterations, we transformed all large volume data into a smooth function and treated the area as a representative value of 5mC methylation. The present computational approach made the methylomic data more accessible to each transcriptional unit and allowed to identify candidate genes, including the tumour necrosis factor receptor-associated factor 4 (TRAF4), as novel therapeutic targets with a strong response to anti-tumour agents, such as 5-FU and CDDP, and whose significance has been confirmed in a mouse model in vivo. The present study showed that 5mC methylation levels are inversely correlated with gene expression in a chemotherapy-resistant stem cell model of gastrointestinal cancer. This mathematical method can be used to simultaneously quantify and identify chemoresistant potential targets in gastrointestinal cancer stem cells.

The significance of mitochondrial metabolism in cancer cells has recently been gaining attention. Among other findings, One-carbon folate metabolism has been reported to be closely associated with cellular characteristics in cancer. To study molecular targets for efficient cancer therapy, we investigated the association between the expressions of genes that code enzymes involved in one-carbon metabolism and survival rate of patients with adenocarcinomas of the colorectum and lung. Patients with high expression of genes that control the metabolic cycle of tetrahydrofolate (THF) in mitochondria, SHMT2, MTHFD2, and ALDH1L2, have a shorter overall survival rate compared with patients with low expression of these genes. Our results revealed that these genes could be novel and more promising anticancer targets than dihydrofolate reductase (DHFR), the current target of drug therapy linked with folate metabolism, suggesting the rationale of drug discovery in cancer medicine.

Increased glutamine uptake toward the elevated glutaminolysis is one of the hallmarks of tumour cells. This aberrant glutamine metabolism has recently attracted considerable attention as a diagnostic and therapeutic target. Herein, we developed glutamine-functionalized polymer to achieve a selective high affinity to tumour cells overexpressing glutaminolysis-related transporter ASCT2. In in vitro study, our developed polymer exhibited faster and higher cellular uptake in tumour cells than that in normal cells. Uptake inhibition study revealed the dominant contribution of ASCT2 to the polymer-cell interaction. Furthermore, the binding affinity of the polymer to tumour cells was estimated to be comparable to that of the potent ligand molecules reported in the literature. In in vivo study, the polymer showed prolonged retention at tumour site after intratumoral injection. This study offers a novel approach for designing tumour cell-binding synthetic polymers through the recognition of dense transporters related to tumour-associated metabolism.

Tumor cells respond to their microenvironment, which can include hypoxia and malnutrition, and adapt their metabolism to survive and grow. Some oncogenes are associated with cancer metabolism via regulation of the related enzymes or transporters. However, the importance of metabolism and precise metabolic effects of oncogenes in colorectal cancer remain unclear. We found that colorectal cancer cells survived under the condition of glucose depletion, and their resistance to such conditions depended on genomic alterations rather than on KRAS mutation alone. Metabolomic analysis demonstrated that those cells maintained tricarboxylic acid cycle activity and ATP production under such conditions. Furthermore, we identified pivotal roles of GLUD1 and SLC25A13 in nutritional stress. GLUD1 and SLC25A13 were associated with tumor aggressiveness and poorer prognosis of colorectal cancer. In conclusion, GLUD1 and SLC25A13 may serve as new targets in treating refractory colorectal cancer which survive in malnutritional microenvironments., Competing Interests: Institutional endowments were partly received from Taiho Pharmaceutical Co., Ltd. (http://www.taiho.co.jp/english/), the Evidence Based Medical (EBM) Research Center (http://ebmrce.co.jp/index.html), Chugai Co., Ltd. (http://www.chugai-pharm.co.jp/english/index.html), Yakult Honsha Co., Ltd. (http://www.yakult.co.jp/english/index.html), and Merck Co., Ltd. (http://www.merck.co.jp/en/index.html). Those funders had no role in the main experimental equipment, supply expenses, study design, data collection and analysis, decision to publish, or preparation of the manuscript for this work.

Deranged metabolism is a hallmark of cancer, playing a significant role in driving the disease process. One such example is the induction of carcinogenesis by the oncometabolite D-2 hydroxyglutarate (D-2HG), which is produced by the mutated enzyme isocitrate dehydrogenase (IDH) occurring in subsets of leukaemias and brain tumours. The oncogenic property of D-2HG appears to stem from its ability to interfere with the activities of α-ketoglutarate-dependent dioxygenases, including the Jumonji family histone demethylases. Here, we find in colorectal cancer cells that even in the absence of IDH mutation, the levels of D-2HG and its enantiomer L-2HG were elevated through glutamine anaplerosis. D-2HG, but not L-2HG, increased the trimethylation of histone H3 lysine 4 of the promoter region of ZEB1, a master regulator of epithelial-mesenchymal transition (EMT), and increased the expression of the ZEB1 gene to directly induce EMT in colorectal cancer cells. EMT promotes the ability of cancer cells to invade the local tissue and enter into the bloodstream, leading to distant organ metastasis. D-2HG levels were elevated in colorectal cancer specimens, particularly in those associated with distant metastasis, supporting the observations in vitro and implicating the contribution of D-2HG in metastasis, the major cause of death in this disease.

Bioinformatics and computational modelling are expected to offer innovative approaches in human medical science. In the present study, we performed computational analyses and made predictions using transcriptome and metabolome datasets obtained from fluorescence-based visualisations of chemotherapy-resistant cancer stem cells (CSCs) in the human oesophagus. This approach revealed an uncharacterized role for the ornithine metabolic pathway in the survival of chemotherapy-resistant CSCs. The present study fastens this rationale for further characterisation that may lead to the discovery of innovative drugs against robust CSCs.

Although BRAF(V600E) mutation is associated with adverse clinical outcomes in patients with colorectal cancer (CRC), response and resistance mechanisms for therapeutic BRAF(V600E) inhibitors remains poorly understood. In the present study, we demonstrate that selective BRAF(V600E) inhibition activates AMP-activated protein kinase (AMPK), which induces autophagy as a mechanism of therapeutic resistance in human cancers. The present data show AMPK-dependent cytoprotective roles of autophagy under conditions of therapeutic BRAF(V600E) inhibition, and AMPK was negatively correlated with BRAF(V600E)-dependent activation of MEK-ERK-RSK signaling and positively correlated with unc-51-like kinase 1 (ULK1), a key initiator of autophagy. Furthermore, selective BRAF(V600E) inhibition and concomitant suppression of autophagy led to the induction of apoptosis. Taken together, present experiments indicate that AMPK plays a role in the survival of BRAF(V600E) CRC cells by selective inhibition and suggest that the control of autophagy contributes to overcome the chemoresistance of BRAF(V600E) CRC cells.

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

Strains of Streptococcus pyogenes isolated from pediatric patients with acute infections which were resistant to one or more of the antibiotics, tetracycline (TC), chloramphenicol (CP), macrolide antibiotics (erythromycin, kitasamycin, oleandomycin, josamycin), lincomycin (LCM) and clindamycin (CLM), were used for transduction of drug resistance. These drug-resistant strains were treated with mitomycin C to induce phages and transduction of drug resistance was attempted by means of phages so induced. It was found that transduction of resistance to the above antibiotics was possible. The transductants obtained on TC-containing selective agar plate were resistant to TC alone while those produced on CP- or erythromycin (EM)-containing selective agar plate were resistant to CP, macrolide antibiotics (Mac), LCM and CLM. From this finding, it was inferred that transduction of resistance to TC, CP, Mac, LCM and CLM via phages occurred in two different patterns, i.e., transfer of resistance to TC alone and that of resistance to CP, Mac, LCM and CLM. All of the transductants obtained were found to belong to group A. In T-typing, they were of the same T-12 type as the donor and recipient strains in a majority of cases though some were not typable.