Aqueous Triple-Phase System in Microwell Array for Generating Uniform-Sized DNA Hydrogel Particles.

DNA hydrogels are notable for their biocompatibility and ability to incorporate DNA information and computing properties into self-assembled micrometric structures. These hydrogels are assembled by the thermal gelation of DNA motifs, a process which requires a high salt concentration and yields polydisperse hydrogel particles, thereby limiting their application and physicochemical characterization. In this study, we demonstrate that single, uniform DNA hydrogel particles can form inside aqueous/aqueous two-phase systems (ATPSs) assembled in a microwell array. In this process, uniform dextran droplets are formed in a microwell array inside a microfluidic device. The dextran droplets, which contain DNA motifs, are isolated from each other by an immiscible PEG solution containing magnesium ions and spermine, which enables the DNA hydrogel to undergo gelation. Upon thermal annealing of the device, we observed the formation of an aqueous triple-phase system in which uniform DNA hydrogel particles (the innermost aqueous phase) resided at the interface of the aqueous two-phase system of dextran and PEG. We expect ATPS microdroplet arrays to be used to manufacture other hydrogel microparticles and DNA/dextran/PEG aqueous triple-phase systems to serve as a highly parallel model for artificial cells and membraneless organelles.

Biosynthesis of a sulfated exopolysaccharide, synechan, and bloom formation in the model cyanobacterium Synechocystis sp. strain PCC 6803.

Extracellularpolysaccharides of bacteria contribute to biofilm formation, stress tolerance, and infectivity. Cyanobacteria, the oxygenic photoautotrophic bacteria, uniquely produce sulfated extracellular polysaccharides among bacteria to support phototrophic biofilms. In addition, sulfated polysaccharides of cyanobacteria and other organisms have been focused as beneficial biomaterial. However, very little is known about their biosynthesis machinery and function in cyanobacteria. Here, we found that the model cyanobacterium, Synechocystis sp. strain PCC 6803, formed bloom-like cell aggregates embedded in sulfated extracellular polysaccharides (designated as synechan) and identified whole set of genes responsible for synechan biosynthesis and its transcriptional regulation, thereby suggesting a model for the synechan biosynthesis apparatus. Because similar genes are found in many cyanobacterial genomes with wide variation, our findings may lead elucidation of various sulfated polysaccharides, their functions, and their potential application in biotechnology.

Bacteria are single-cell microorganisms that can form communities called biofilms, which stick to surfaces such as rocks, plants or animals. Biofilms confer protection to bacteria and allow them to colonize new environments. The physical scaffold of biofilms is a viscous matrix made of several molecules, the main one being polysaccharides, complex carbohydrates formed by many monosaccharides (single sugar molecules) joined together. Cyanobacteria, also known as blue-green algae, are a type of bacteria that produce oxygen and use sunlight as an energy source, just as plants and algae do. Cyanobacteria produce extracellular polysaccharides that contain sulfate groups. These sulfated polysaccharides are also produced by animals and algae but are not common in other bacteria or plants. One possible role of sulfated, extracellular polysaccharides in cyanobacteria is keeping cells together in the floating aggregates found in cyanobacterial blooms. These are visible discolorations of the water caused by an overgrowth of cyanobacteria that occur in lakes, estuaries and coastal waters. However, little is known about how these polysaccharides are synthesized in cyanobacteria and what their natural role is. Maeda et al. found a strain of cyanobacteria that formed bloom-like aggregates that were embedded in sulfated extracellular polysaccharides. Using genetic engineering techniques, the researchers identified a set of genes responsible for producing a sulfated extracellular polysaccharide and regulating its levels. They also found that cell aggregates of cyanobacteria can float without having intracellular gas vesicles, which was previously thought to enable blooms to float. The results of the present study could have applications for human health, since many sulfated polysaccharides have antiviral, antitumor or anti-inflammatory properties, and similar genes are found in many cyanobacteria. In addition, these findings could be useful for controlling toxic cyanobacterial blooms, which are becoming increasingly problematic for society.

Tlr0485 is a cAMP-activated c-di-GMP phosphodiesterase in a cyanobacterium Thermosynechococcus.

Second messenger molecules are crucial components of environmental signaling systems to integrate multiple inputs and elicit physiological responses. Among various kinds of second messengers, cyclic nucleotides cAMP and cyclic di-GMP (c-di-GMP) play pivotal roles in bacterial environmental responses. However, how these signaling systems are interconnected for a concerted regulation of cellular physiology remains elusive. In a thermophilic cyanobacterium Thermosynechococcus vulcanus strain RKN, incident light color is sensed by cyanobacteriochrome photoreceptors to transduce the light information to the levels of c-di-GMP, which induces cellular aggregation probably via cellulose synthase activation. Herein, we identified that Tlr0485, which is composed of a cGMP-specific phosphodiesterases, adenylate cyclases, and FhlA (GAF) domain and an HD-GYP domain, is a cAMP-activated c-di-GMP phosphodiesterase. We also show biochemical evidence that the two class-III nucleotide cyclases, Cya1 and Cya2, are both adenylate cyclases to produce cAMP in T. vulcanus. The prevalence of cAMP-activated c-di-GMP phosphodiesterase genes in cyanobacterial genomes suggests that the direct crosstalk between cAMP and c-di-GMP signaling systems may be crucial for cyanobacterial environmental responses.

Photolithographic shape control of DNA hydrogels by photo-activated self-assembly of DNA nanostructures.

We report a photolithographic method for the shape control of DNA hydrogels based on photo-activated self-assembly of Y-shaped DNA nanostructures (Y-motifs). To date, various methods to control the shape of DNA hydrogels have been developed to enhance the functions of the DNA hydrogel system. However, photolithographic production of shape-controlled DNA hydrogels formed through the self-assembly of DNA nanostructures without the use of radical polymerizations has never been demonstrated, although such a method is expected to be applied for the shape-control of DNA hydrogels encapsulating sensitive biomolecules, such as proteins. In this study, we used a photo-activated linker to initiate the self-assembly of Y-motifs, where the cross-linker DNA was at first inactive but was activated after UV light irradiation, resulting in the formation of shape-controlled DNA hydrogels only at the UV-exposed area produced by photomasks. We believe that this method will be applied for the construction of biohybrid machines, such as molecular robots and artificial cells that contain intelligent biomolecular devices, such as molecular sensors and computers.

Astaxanthin production in a model cyanobacterium Synechocystis sp. PCC 6803.

Heterologous production of a useful carotenoid astaxanthin was achieved in a cyanobacterium Synechocystis sp. PCC 6803 with the aid of marine bacterial genes. Astaxanthin and its intermediates emerged at high levels, whereas ß-carotene and zeaxanthin disappeared in the strain. Total carotenoid accumulation was nearly two fold compared with wild type. The astaxanthin-producing strain was capable of only growing heterotrophically, which was likely due to the absence of ß-carotene. Further enhanced accumulation was pursued by gene overexpression for possible rate-limiting steps in the biosynthesis pathway.

Improved sorbitol production and growth in cyanobacteria using promiscuous haloacid dehalogenase-like hydrolase.

Sorbitol can be produced photosynthetically in the model cyanobacterium Synechocystis sp. PCC 6803 harboring sorbitol-6-phosphate (S6P) dehydrogenase (S6PDH) from apple, representing a promising metabolic engineering strategy for environmentally friendly carbon-based compound production. However, no gene for S6P phosphatase has been reported to date. We previously found that members of the Escherichia coli haloacid dehalogenase-like hydrolase (HAD) superfamily, a group of promiscuous hydrolases, exert phosphatase activity to S6P in vitro. Here, we examined the effects of HADs on sorbitol production in cyanobacterial cells. Overexpression of E. coli HAD1 improved sorbitol production induced by toxic S6PDH, whereas it suppressed growth even without induction. Moreover, overexpression of HAD1 in combination with engineering of other pathways successfully allowed for the constitutive expression of toxic S6PDH. Consequently, the sorbitol production was highly improved, which in turn suppressed the growth suppression effect of promiscuous HAD1. These results provide a good example of a novel metabolic engineering strategy using a combination of a promiscuous enzyme with an abundant supply of one of its substrates.

Glossopharyngeal Neuralgia with Syncope Caused by Recurrence of Esophageal Squamous Cell Carcinoma.

We herein report a case of glossopharyngeal neuralgia with repeated syncope caused by the recurrence of esophageal carcinoma. The typical symptoms of glossopharyngeal neuralgia are paroxysmal, stabbing, electric shock-like pain in the pharynx and/or base of the tongue on swallowing and talking. In addition, syncope can also be caused by glossopharyngeal neuralgia. The diagnosis of glossopharyngeal neuralgia is not always easy because of its rarity. In the present case, we suspected that repeated syncope was caused by glossopharyngeal neuralgia due to the recurrence of esophageal carcinoma. Concurrent chemoradiation therapy was effective in reducing the tumor size, which resulted in the complete resolution of the symptoms.

Sorbitol production and optimization of photosynthetic supply in the cyanobacterium Synechocystis PCC 6803.

Biochemicals production is a major theme in the application of photosynthesis to address global warming and organic-resource problems. Among biochemicals, sugar alcohols have attracted research attention because they are directly derived from two photosynthetic products, sugars and reductants. Here, we produced sorbitol photosynthetically by using cyanobacteria and modified the supply of its substrates through genetic engineering. Expression of an NADPH-dependent enzyme that generates sorbitol-6-phosphate, S6PDH, was highly toxic to cyanobacteria likely due to the sorbitol production, whereas expression of an NADH-dependent enzyme, SrlD2, yielded no sorbitol. The toxicity was partly overcome by introducing a theophylline-inducible riboswitch for S6PDH expression and optimizing induction, but sorbitol production was still low and severely inhibited growth. Co-expression of fructose-1,6-bisphosphatase drastically alleviated the growth inhibition, but did not increase short-term sorbitol production. The NADPH/NADP+ ratio decreased during sorbitol production. Overexpression of a membrane-bound transhydrogenase for NADPH generation from NADH elevated the short-term sorbitol production, but only partly alleviated the growth inhibition. Notably, a strain overexpressing all three enzymes exhibited sustainable sorbitol production at 312 mg/L, which was nearly 27-fold higher than the yield of the initial S6PDH-overexpressing strain. We discuss these results in relation to the optimization of photosynthetic supply for sorbitol production in cyanobacteria.

Genetic identification of factors for extracellular cellulose accumulation in the thermophilic cyanobacterium Thermosynechococcus vulcanus: proposal of a novel tripartite secretion system.

Cells of the thermophilic cyanobacterium Thermosynechococcus vulcanus strain RKN (NIES-2134) aggregate and produce extracellular cellulose under induced conditions of blue light and low temperature, and both aggregation and cellulose production require the cellulose synthase Tll0007 (XcsA) and photosensory diguanylate cyclases. However, overexpression of both the cellulose synthase and a constitutively active diguanylate cyclase was not sufficient to induce cellulose-mediated cell aggregation under normal growth conditions. Synteny analysis and gene knockout revealed that two putative genes, hlyD-like tlr0903 (xcsB) and endoglucanase-like tlr1902 (xcsC), are linked to tll0007, although they are located apart from tll0007 in the T. vulcanus genome. Gene knockdown revealed that tlr1605 (tolC) was essential for the cellulose-mediated cell aggregation. Low temperature induced marked upregulation of tlr0903, and overexpression of both tlr0903 (but not tlr1902) and diguanylate cyclase resulted in the strong cell aggregation and cellulose accumulation under normal conditions. Based on these and phylogenetic analysis, we propose that the cyanobacterial extracellular cellulose production is due to a novel variant of the bacterial tripartite secretion system.

Tlr1612 is the major repressor of cell aggregation in the light-color-dependent c-di-GMP signaling network of Thermosynechococcus vulcanus.

Cyclic diguanylate (c-di-GMP) is a bacterial second messenger involved in sessile/motile lifestyle transitions. We previously reported that c-di-GMP is a crucial inducer of cell aggregation of the cyanobacterium Thermosynechococcus vulcanus. The three cooperating cyanobacteriochrome photoreceptors (SesA/B/C) regulate cell aggregation in a light color-dependent manner by synthesizing/degrading c-di-GMP. Although a variety of c-di-GMP signaling proteins are encoded in cyanobacterial genomes, how c-di-GMP signaling networks are organized remains elusive. Here we experimentally demonstrate that the cellulose synthase Tll0007, which is essential for cell aggregation, binds c-di-GMP although the affinity is low (Kd = 63.9 ± 5.1 µM). We also show that SesA-the main trigger of cell aggregation-is subject to strict product feedback inhibition (IC50 = 1.07 ± 0.13 µM). These results suggest that SesA-produced c-di-GMP may not directly bind to Tll0007. We therefore systematically analyzed all 10 of the genes encoding proteins containing a c-di-GMP synthesis/degradation domain. We identified Tlr1612, harboring both domains, as the major repressor of cell aggregation under the repressing teal-green light irradiation. tlr1612 acts downstream of sesA and is not regulated transcriptionally by light color, suggesting that Tlr1612 may be involved in c-di-GMP amplification in the signaling cascade. Post-transcriptional control is likely crucial for the light-regulated c-di-GMP signaling.

Wnt signaling as a possible promoting factor of cell differentiation in pleomorphic adenomas.

There are well known that Wnt signaling was some roles of cell differentiation at the development tissues, especially the oral and maxillofacial regions of some developmental stages. Therefore, to determine Wnt signaling in the pleomorphic adenoma tissues, we examined. The expression of Wnt1 and ß-catenin as well as the distribution of various cytoskeletal proteins CK7 and CK13 was examined in 30 cases of pleomorphic adenoma by immunohistochemistry. Wnt1 was detected in almost all tumor cells. The peripheral columnar cells in squamous metaplasia and small cuboidal cells in duct-like structures were strongly positive to Wnt1. Although ß-catenin was clearly localized on the cell membrane of tumor cells, nuclear translocation was observed in small cuboidal cells and in some basaloid cells. The immunofluorescent staining pattern of Wnt1 and CK7 as well as Wnt1 and CK13 was consistent with IHC results. Thus, in pleomorphic adenoma, Wnt is involved in tumor cell differentiation of peripheral columnar cells forming solid nests and small peripheral columnar cells forming duct-like structures. Moreover, among the three currently known Wnt pathways, ß-catenin is the suggested pathway working during cell differentiation. Furthermore, peripheral columnar cells in solid tumor nests and in squamous metaplasia are governed by another Wnt pathway other than ß-catenin. Therefore, Wnt signaling through ß-catenin pathway may be involved in the 'mixed' differentiation characteristic of pleomorphic adenoma although another pathway may also be possibly working in other parts of the tumor tissue.

Engineering of cyanobacteria for the photosynthetic production of limonene from CO2.

Isoprenoids, major secondary metabolites in many organisms, are utilized in various applications. We constructed a model photosynthetic production system for limonene, a volatile isoprenoid, using a unicellular cyanobacterium that expresses the plant limonene synthase. This system produces limonene photosynthetically at a nearly constant rate and that can be efficiently recovered using a gas-stripping method. This production does not affect the growth of the cyanobacteria and is markedly enhanced by overexpression of three enzymes in the intrinsic pathway to provide the precursor of limonene, geranyl pyrophosphate. The photosynthetic production of limonene in our system is more or less sustained from the linear to stationary phase of cyanobacterial growth for up to 1 month.

ULBP4/RAET1E is highly polymorphic in the Old World monkey.

Natural-killer group 2 member D (NKG2D) is an activating receptor that plays an important role in the immune response mediated by NK cells, γδ(+) T cells, and CD8(+) T cells. In humans, MHC class I chain-related genes and UL-16 binding protein (ULBP)/retinoic acid early transcript 1 (REAT1) gene family encode ligands for NKG2D. The rhesus and crab-eating macaques, which belong to the Old World monkeys, are widely used as non-human primate models in medical researches on the immunological process. In the present study, we investigated the polymorphisms of ULBP4/RAET1E, a member of the ULBP/RAET1 family, and found 25 and 14 alleles from the rhesus and crab-eating macaques, respectively, of which diversities were far more extended than in humans. A phylogenetic study suggested that the allelic diversification of ULBP4/RAET1E predated the divergence of rhesus and crab-eating macaques.

[A case of glycogen storage disease type I with hepatocellular carcinoma].

The patient was a 55-year-old female. In 1997, she was diagnosed as type-I glycogen storage disease (von Gierke disease). In March 2002, abdominal ultrasound tomography revealed an early enhanced lesion at liver S2, which suspected to a well differentiated hepatocellular carcinoma (HCC) with super-paramagnetic iron oxide (SPIO) enhanced magnetic resonance imaging (MRI). From 2002 to 2006, she received three times trans-arterial chemo-embolization (TACE) at enhanced lesions. But abdominal computer tomography (CT) revealed a 3.6 cm-in diameter early enhanced lesion near this tumor at S2, which was suspected to a diagnosis of recurrent HCC in December 2008. Therefore, she received a partial hepatectomy at S2. This resected specimen was diagnosed as poorly differentiated HCC. This patient is still alive with no recurrence after 6 months from operation. In conclusion, it might be effective for an early detection of recurrent HCC to see the doctor for a long regular checkup, because the growth of HCC with glycogen storage disease would be very slow.