p53, known as a tumor suppressor, is a DNA binding protein that regulates cell cycle, activates DNA repair proteins, and triggers apoptosis in multicellular animals. More than 50% of human cancers contain a mutation or deletion of the p53 gene, and p53R175 is one of the hot spots of p53 mutation. Nucleic acid aptamers are short singlestranded oligonucleotides that are able to bind various targets, and they are typically isolated from an experimental procedure called systematic evolution of ligand exponential enrichment (SELEX). Using a previously unidentified strategy of contrast screening with SELEX, we have isolated an RNA aptamer targeting p53R175H. This RNA aptamer (p53R175H-APT) has a significantly stronger affinity to p53R175H than to the wild-type p53 in both in vitro and in vivo assays. p53R175H-APT decreased the growth rate, weakened the migration capability, and triggered apoptosis in human lung cancer cells harboring p53R175H. Further analysis actually indicated that p53R175H-APT might partially rescue or correct the p53R175H to function more like the wild-type p53. In situ injections of p53R175H-APT to the tumor xenografts confirmed the effects of this RNA aptamer on p53R175H mutation in mice.p53 | RNA aptamer | contrast screening | SELEX | tumor N ucleic acid aptamers, as single-stranded DNA or RNA oligonucleotides that are able to bind various targets with high specificity, were first isolated from a pool of random sequences with a process called systematic evolution of ligand exponential enrichment (SELEX) in 1990 by two laboratories (1, 2). Over the years, an array of methods have been invented to facilitate SELEX screening, and specific aptamers binding to partners ranging from small molecules to large proteins have been isolated. However, an RNA aptamer that can distinguish a protein with a single amino acid mutation from the wild-type (WT) protein remains absent (3-11).Protein with a single amino acid substitution is the cause of a plethora of human diseases (12)(13)(14). A well-known example is sickle-cell anemia, which is caused by a point mutation in the β-globin chain of hemoglobin (15). Also, point mutations in multiple tumor suppressor proteins cause cancer (16-18). The protein p53 is a tumor suppressor and functions as a transcription factor to regulate the expression of genes involved in DNA repair, cell cycle, and apoptosis. A mutation within one allele of this gene can result in inactivation of the remaining WT allele in a dominant-negative manner, and mutations from six mutation hot spots located in the DNA-binding surface of p53 are frequently found in almost all cancer types (19). Actually, more than half of human cancer cases relate to mutations in p53, and the single amino acid substitution p53R175H is one of the mutations at the p53R175 hot spot (20,21). R175H mutation abolishes the p53 WT functions in both MEF cells and thymocytes (22). p53R175H possesses a marked anti-apoptotic gain-of-function in lung cancer cells (23). Also, p53R175H cooperates better than any other mutant ...

Previous studies have shown that metformin not only is a hypoglycemic agent but also has neuroprotective effects. However, the mechanism of action of metformin in ischemic stroke is unclear. Oxidative stress is an important factor in the pathogenesis of cerebral ischemia-reperfusion injury. It has been reported that metformin is associated with stroke risk in the clinical population. This study is aimed at investigating the effect and mechanism of metformin in an experimental model of oxidative stress induced by ischemia/reperfusion (I/R) in vivo and oxygen glucose deprivation/reperfusion (OGD/R) in vitro. Metformin (100, 200, and 300 mg/kg) was administered intraperitoneally immediately after induction of cerebral ischemia. The indicators of oxidative stress selected were antioxidant enzyme activities of catalase, malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), and glutathione peroxidation enzyme (GSHPx). First, we demonstrated that metformin can significantly alleviate acute and chronic cerebral I/R injury and it has a strong regulatory effect on stroke-induced oxidative stress. It can reduce the elevated activities of MDA and NO and increase the levels of GSHPx and SOD in the cerebrum of mice and N2a cells exposed to I/R. Furthermore, real-time PCR and western blot were used to detect the expression of long noncoding RNA H19 (lncRNA-H19), microRNA-148a-3p (miR-148a-3p), and Rho-associated protein kinase 2 (Rock2). The direct interaction of lncRNA-H19, miR-148a-3p, and Rock2 was tested using a dual luciferase reporter assay. lncRNA-H19 altered OGD/R-induced oxidative stress by modulating miR-148a-3p to increase Rock2 expression. The expression of lncRNA-H19 and Rock2 could be downregulated with metformin in vivo and in vitro. In conclusion, our study confirmed that metformin exerts neuroprotective effects by regulating ischemic stroke-induced oxidative stress injury via the lncRNA-H19/miR-148a-3p/Rock2 axis. These results provide new evidence that metformin may represent a potential treatment for stroke-related brain injury.

Ethylene is widely used in the petrochemical industry and has traditionally been produced via the steam cracking of petroleum-based feedstock. The exploration of sustainable and carbon-neutral methods of producing ethylene from the renewable feedstock seems promising. The direct photosynthetic production of ethylene after the recycling of carbon dioxide shows great potential. In this study, continuous and stable ethylene production was achieved in Synechocystis sp. PCC 6803 by introducing a codonoptimized ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. sesami and using 2-oxoglutarate (2-OG) as the substrate. Based on diverse promoter screening, PcpcB was proved to be a highly efficient promoter for ethylene production in cyanobacteria. The genes encoding 2-OG decarboxylase (OGDC) and succinic semialdehyde dehydrogenase (SSADH) in the tricarboxylic acid (TCA) cycle in Synechocystis sp. PCC 6803 were identified, and the TCA cycle was genetically modified by blocking these two enzymes with the simultaneous overexpression of EFE. Meanwhile, a gene encoding 2-OG permease (KgtP) from E. coli was introduced into the phaAB loci to increase the 2-OG supply. A peak volumetric production rate of 9.7 mL L −1 h −1 for ethylene was eventually achieved in the Synechocystis recombinant (XX110), with the genetic modification of the TCA cycle and heterologous expression of 2-oxoglutarate permease by the modified semi-continuous cultivation.

). Hypothetically, certain factors induced during preconditioning are involved in acquisition of chill-light tolerance. In this study, Rbp1 (RNA-binding protein 1) rather than Rbp2 was found to be accumulated during preconditioning, and the accumulation of Rbp1 was correlated with the increase of chill-light tolerance. Inactivation of its encoding gene rbp1 led to a great reduction in the acquired chill-light tolerance, while ectopic expression of rbp1 enabled the cyanobacterium to survive the chill-light stress without preconditioning. Microarray analyses suggested that the Rbp1-dependent chill-light tolerance may not be based on its influence on mRNA abundance of certain genes. Similarly to that in Synechocystis, the Rbp1 homologue(s) can be accumulated in Microcystis cells collected from a subtropic lake in low-temperature seasons. Rbp1 is the first factor shown to be both accumulated early during preconditioning and directly involved in development of chill-light tolerance in Synechocystis. Its accumulation may greatly enhance the overwintering capability in certain groups of cyanobacteria.

Cyanobacteria can synthesize alkanes and alkenes, which are considered to be infrastructure-compatible biofuels. In terms of physiological function, cyanobacterial hydrocarbons are thought to be essential for membrane flexibility for cell division, size, and growth. The genetic basis for the biosynthesis of terminal olefins (1-alkenes) is a modular type I polyketide synthase (PKS) termed olefin synthase (Ols). The modular architectures of Ols and structural characteristics of alkenes have been investigated only in a few species of the small percentage (approximately 10%) of cyanobacteria that harbor putative Ols pathways. In this study, investigations of the domains, modular architectures, and phylogenies of Ols in 28 cyanobacterial strains suggested distinctive pathway evolution. Structural feature analyses revealed 1-alkenes with three carbon chain lengths (C, C, and C). In addition, the total cellular fatty acid profile revealed the diversity of the carbon chain lengths, while the fatty acid feeding assay indicated substrate carbon chain length specificity of cyanobacterial Ols enzymes. Finally, analyses suggested that the N terminus of the modular Ols enzyme exhibited characteristics typical of a fatty acyl-adenylate ligase (FAAL), suggesting a mechanism of fatty acid activation via the formation of acyl-adenylates. Our results shed new light on the diversity of cyanobacterial terminal olefins and a mechanism for substrate activation in the biosynthesis of these olefins. Cyanobacterial terminal olefins are hydrocarbons with promising applications as advanced biofuels. Despite the basic understanding of the genetic basis of olefin biosynthesis, the structural diversity and phylogeny of the key modular olefin synthase (Ols) have been poorly explored. An overview of the chemical structural traits of terminal olefins in cyanobacteria is provided in this study. In addition, we demonstrated by fatty acid feeding assays that cyanobacterial Ols enzymes might exhibit substrate carbon chain length specificity. Furthermore, by performing bioinformatic analyses, we observed that the substrate activation domain of Ols exhibited features typical of a fatty acyl-adenylate ligase (FAAL), which activates fatty acids by converting them to fatty acyl-adenylates. Our results provide further insight into the chemical structures of terminal olefins and further elucidate the mechanism of substrate activation for terminal olefin biosynthesis in cyanobacteria.

The majority of bacteria and archaea possess an RNA-guided adaptive and inheritable immune system against viruses and other foreign genetic elements that consists of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPRassociated (Cas) proteins. In most CRISPR-Cas systems, the maturation of CRISPRderived small RNAs (crRNAs) is essential for functionality. In some bacteria, multiple instances of cas gene-free (orphan) repeat-spacer arrays exist, while additional instances of arrays that are linked to cas gene cassettes are present elsewhere in the genome.In the cyanobacterium Anabaena sp. PCC 7120, ten CRISPR-Cas repeat-spacer arrays are present, but only two cas gene cassettes plus a Tn7-associated eleventh array are observed. In this study, we deleted the two cas6 genes alr1482 (Type III-D) or alr1566 (Type I-D) and tested the specificities of the two corresponding enzymes in the resulting mutant strains, as recombinant proteins and in a cell-free transcriptiontranslation system. The results assign the direct repeats (DRs) to three different groups. While Alr1566 is specific for one group, Alr1482 has a higher preference for the DRs of the second group but can also cleave those of the first group. We found that this cross-recognition limits crRNA accumulation for the Type I-D system in vivo.We also show that the DR of the cas gene-free CRISPR array of cyanophage N-1 is processed by these enzymes, suggesting that it is fully competent in association with host-encoded Cas proteins. The data support a strong tendency for array fragmentation in multicellular cyanobacteria and disfavor other possibilities, such as the nonfunctionality of these orphan repeat-spacer arrays. Our data demonstrate the functional coordination of Cas6 endonucleases with both neighboring and remote repeat-spacer arrays in the CRISPR-Cas system of cyanobacteria..

Significance and Impact of the Study: This study established the efficacy of the dual fluorescence method in evaluating the viability of cyanobacteria under chill-light stress. The results provided the direct evidence for acquired chill-light tolerance and the viability of overwintering Microcystis colonies. Such information can be useful in prediction of cyanobacterial blooms. AbstractChill in the light is the major environmental stress that cyanobacteria encounter in winter. Cyanobacterial cells may acquire chill-light tolerance upon exposure to low temperature in autumn and early winter. We sought to establish the efficacy of the dual fluorescence method in detecting the viability of overwintering cyanobacteria and to provide further evidence for the chilllight tolerance of preconditioned cyanobacteria. Synechocystis sp. PCC 6803 and Microcystis aeruginosa PCC 7806 were exposed to chill (5°C)-light stress with or without pretreatment at 15°C and stained with SYTO 9 and propidium iodide. Live and dead cells were observed under a fluorescence microscope, and the percentage of viable cells was quantified on a microplate reader. The dual fluorescence method showed consistent results with tests of the ability to reinitiate growth. Cell viability was quantitatively correlated with ratio of SYTO 9/propidium iodide fluorescence. Previously, Microcystis colonies in Lake Taihu had been found to accumulate RNA-binding protein 1 in autumn and winter. Use of this method directly showed the viability of such Microcystis colonies throughout the winter.

Baeocytous cyanobacteria (Pleurocapsales/Subsection II) can thrive in a wide range of habitats on Earth but, compared to other cyanobacterial lineages, they remain poorly studied at genomic level. In this study, we sequenced the first genome from a member of the Hyella genus-H. patelloides LEGE 07179, a recently described species isolated from the Portuguese foreshore. This genome is the largest of the thirteen baeocyteforming cyanobacterial genomes sequenced so far, and diverges from the most closely related strains. Comparative analysis revealed strain-specific genes and horizontal gene transfer events between H. patelloides and its closest relatives. Moreover, H. patelloides genome is distinctive by the number and diversity of natural product biosynthetic gene clusters (BGCs). The majority of these clusters are strain-specific BGCs with a high probability of synthesizing novel natural products. One BGC was identified as being putatively involved in the production of terminal olefin. Our results showed that, H. patelloides produces hydrocarbon with C 15 chain length, and synthesizes C 14 , C 16 , and C 18 fatty acids exceeding 4% of the dry cell weight. Overall, our data contributed to increase the information on baeocytous cyanobacteria, and shed light on H. patelloides evolution, phylogeny and natural product biosynthetic potential.