Grand scale genome manipulation via chromosome swapping in Escherichia coli programmed by three one megabase chromosomes

In bacterial synthetic biology, whole genome transplantation has been achieved only in mycoplasmas that contain a small genome and are competent for foreign genome uptake. In this study, we developed Escherichia coli strains programmed by three 1-megabase (Mb) chromosomes by splitting the 3-Mb chromosome of a genome-reduced strain. The first split-chromosome retains the original replication origin (oriC) and partitioning (par) system. The second one has an oriC and the par locus from the F plasmid, while the third one has the ori and par locus of the Vibrio tubiashii secondary chromosome. The tripartite-genome cells maintained the rod-shaped form and grew only twice as slowly as their parent, allowing their further genetic engineering. A proportion of these 1-Mb chromosomes were purified as covalently closed supercoiled molecules with a conventional alkaline lysis method and anion exchange columns. Furthermore, the second and third chromosomes could be individually electroporated into competent cells. In contrast, the first split-chromosome was not able to coexist with another chromosome carrying the same origin region. However, it was exchangeable via conjugation between tripartite-genome strains by using different selection markers. We believe that this E. coli-based technology has the potential to greatly accelerate synthetic biology and synthetic genomics.     

Comparison of Optic Disc Morphology of Optic Nerve Atrophy between Compressive Optic Neuropathy and Glaucomatous Optic Neuropathy

Objectives

To compare the optic nerve head (ONH) structure between compressive optic neuropathy (CON) and glaucomatous optic neuropathy (GON), and to determine whether selected ONH quantitative parameters effectively discriminate between GON and CON, especially CON cases presenting with a glaucoma-like disc.

Methods

We prospectively assessed 34 patients with CON, 34 age-matched patients with moderate or severe GON, and 34 age-matched healthy control subjects. The quantitative parameters of ONH structure were compared using the Heidelberg Retina Tomograph 2 (HRT2) and Spectralis optical coherence tomography with an enhanced depth imaging method.

Results

The mean and maximum cup depths of CON were significantly smaller than those with GON (P<0.001 and P<0.001, respectively). The distance between Bruch''s membrane opening and anterior surface of the lamina cribrosa (BMO-anterior LC) of CON was also significantly smaller than that of glaucoma but was similar to that of the healthy group (P<0.001 and P = 0.47, respectively). Based on Moorfields regression analysis of the glaucoma classification of HRT2, 15 eyes with CON were classified with a glaucoma-like disc. The cup/disc area ratio did not differ between cases of CON with a glaucoma-like disc and cases of GON (P = 0.16), but the BMO-anterior LC and mean and maximum cup depths of CON cases with a glaucoma-like disc were smaller than those in GON (P = 0.005, P = 0.003, and P = 0.001, respectively).

Conclusions

Measurements of the cup depths and the LC depth had good ability to differentiate between CON with a glaucoma-like disc and glaucoma. There was no laminar remodeling detected by laminar surface position in the patients with CON compared to those with GON.     

Specific inhibition of hepatitis C virus replication by cyclosporin A

The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection.     

Network analysis of potential migration routes for Oriental White Storks (<Emphasis Type="Italic">Ciconia boyciana</Emphasis>)

From 1998 through to 2000, we satellite-tracked the movements of 13 Oriental White Storks (Ciconia boyciana) on their autumnal migration in order to identify their important stopover sites for preserving links from the Russian Far East breeding sites to the wintering sites in south-eastern China. New analytical methods of satellite tracking data were employed to derive robust information on the locations of stay sites, the number of stopovers made during migration, and the distance traveled without making stopovers. Based on the derived information, we modeled a stay site network as an abstraction of the storks potential migration routes from their breeding sites to wintering sites. Using network analysis techniques, we explored how the loss of stopover sites could affect the connectivity of potential migration routes. The results suggested that if the seashore stopover sites facing Bohai Bay in eastern China were lost, the storks wintering sites along the Yangtze River in south-eastern China would be isolated. Among the seashore stopover sites, Jiantuozhi Gley Mire (39.185°N, 118.627°E), located on the northern seashore of Bohai Bay, was considered particularly important for migrating storks, because it was used every year by the storks we tracked. If conservation needs of this critically located site fail to be addressed, the stay site network of storks can create weak links in the chain of migration and, if broken, storks will have great difficulties in completing their autumnal migration.     

Identification and characterization of nuclear localization signals of CaMKP-N

Calmodulin-dependent protein kinase phosphatase (CaMKP) and CaMKP-N dephosphorylate and regulate multifunctional Ca(2+)/calmodulin-dependent protein kinases. The enzymatic properties of CaMKP-N and CaMKP resemble each other, whereas their localizations are different. CaMKP-N is localized in the nucleus, whereas CaMKP is localized in the cytosol. In the present study, the nuclear localization signals (NLSs) of CaMKP-N were identified and characterized. CaMKP-N contains two NLSs, NLS1 and NLS2, at the C-terminus. A cluster of basic residues in the NLSs is important for their function. NLS1 and NLS2 function independently, but mutagenesis analysis suggests that these NLSs interact with each other.     

Isolation and Diversity of Actinomycetes in the Chesapeake Bay

Chesapeake Bay was investigated as a source of actinomycetes to screen for production of novel bioactive compounds. The presence of relatively large populations of actinoplanetes (chemotype II/D actinomycetes) in Chesapeake Bay sediment samples indicates that it is an eminently suitable ecosystem from which to isolate actinomycetes for screening programs. Actinomycetes were isolated from sediment samples collected in Chesapeake Bay with an isolation medium containing nalidixic acid, which proved to be more effective than heat pretreatment of samples. Actinomycete counts ranged from a high of 1.4 × 10⁵ to a low of 1.8 × 10² CFU/ml of sediment. Actinomycetes constituted 0.15 to 8.63% of the culturable microbial community. The majority of isolates from the eight stations studied were actinoplanetes (i.e., chemotype II/D), and 249 of these isolates were obtained in a total of 298 actinomycete isolates. Antimicrobial activity profiles indicated that diverse populations of actinoplanetes were present at each station. DNA hybridization studies showed considerable diversity among isolates between stations, but indicated that actinoplanete strains making up populations at nearby stations were more similar to each other than to populations sampled at distant stations. The diversity of actinoplanetes and the ease with which these organisms were isolated from Chesapeake Bay sediments make this a useful source of these actinomycetes.     

Exogenous sodium nitroprusside and glutathione alleviate copper toxicity by reducing copper uptake and oxidative damage in rice (Oryza sativa L.) seedlings

Nitric oxide (NO) and glutathione (GSH) regulate a variety of physiological processes and stress responses; however, their involvement in mitigating Cu toxicity in plants has not been extensively studied. This study investigated the interactive effect of exogenous sodium nitroprusside (SNP) and GSH on Cu homeostasis and Cu-induced oxidative damage in rice seedlings. Hydroponically grown 12-day-old seedlings were subjected to 100 μM CuSO₄ alone and in combination with 200 μM SNP (an NO donor) and 200 μM GSH. Cu exposure for 48 h resulted in toxicity symptoms such as stunted growth, chlorosis, and rolling in leaves. Cu toxicity was also manifested by a sharp increase in lipoxygenase (LOX) activity, lipid peroxidation (MDA), hydrogen peroxide (H₂O₂), proline (Pro) content, and rapid reductions in biomass, chlorophyll (Chl), and relative water content (RWC). Cu-caused oxidative stress was evident by overaccumulation of reactive oxygen species (ROS; superoxide (O₂ ^?–) and H₂O₂). Ascorbate (AsA) content decreased while GSH and phytochelatin (PC) content increased significantly in Cu-stressed seedlings. Exogenous SNP, GSH, or SNP?+?GSH decreased toxicity symptoms and diminished a Cu-induced increase in LOX activity, O₂ ^?–, H₂O₂, MDA, and Pro content. They also counteracted a Cu-induced increase in superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and glyoxalase I and glyoxalase II activities, which paralleled changes in ROS and MDA levels. These seedlings also showed a significant increase in catalase (CAT), glutathione peroxidase (GPX), dehydroascorbate reductase (DHAR), glutathione S-transferase (GST) activities, and AsA and PC content compared with the seedlings stressed with Cu alone. Cu analysis revealed that SNP and GSH restricted the accumulation of Cu in the roots and leaves of Cu-stressed seedlings. Our results suggest that Cu exposure provoked an oxidative burden while reduced Cu uptake and modulating the antioxidant defense and glyoxalase systems by adding SNP and GSH play an important role in alleviating Cu toxicity. Furthermore, the protective action of GSH and SNP?+?GSH was more efficient than SNP alone.     

Influence of farming system on the floristic composition of paddy landscapes: a case study in a rural hilly zone in Zhejiang province,China

The effects of two contrasting farming systems on the floristic composition and vegetation structure that make up the Yatoda landscape were investigated. The experimental field was set up at Yatoda in a hilly district in Zhejiang province, China. Vegetation surveys were done in summer 2002 and 2003. Vegetation was divided into four types based on species composition: grassland formerly used for rice cultivation (G), rice paddy (P), ridge way (R), and levee slope (S). In P, R, and S plots, each vegetation type was subdivided according to the different farming systems. Under the traditional system, there were a number of species, and the diversity index values were greater than those under the conventional system. Blyxa echinosperma and Marsilea quadrifolia, which are threatened species in Japan, were seen in the paddy field under the traditional farming system.     

Influence of livestock grazing on vegetation in a saline area in Northeast Thailand

The influence of grazing by water buffalo (Bubalas bufalis) and cattle (Bos taurus) was estimated for vegetation inside and outside cages in a saline area at Khon Kaen, Northeast Thailand. The home range of water buffalo and cattle shifted in response to the period of rice cultivation: during the rice-growing season these animals grazed on roadsides and abandoned places such as the study area; after rice harvest they grazed mainly rice stubble on the paddy. The vegetation in the study area was divided into three types: 1) dominated by the annual grassesChloris barbata andIschaemum rugosum; vegetational cover and plant height in the cage increase due to the increase of these grasses; 2) thorny shrub patch ofMaytenus mekongensis; other species in this patch almost died a year after experimental elimination of this shrub; this salt-tolerant shrub not only protected the co-existing species from grazing, but also suppressed salt accumulation; 3) almost pure stand ofPanicum repens; livestock preferred this perennial grass over others. Electrical conductivity (EC) of surface soil did not increase under the vegetation protected from grazing. Litter and other organic matter in the soil suppressed the upward movement of brine. Grazing led to an increase of bare ground where NaCl accumulated, and modified the heterogeneity of vegetation, which was reflected in the degree of salt accumulation.     

Environmental DNA analysis shows high potential as a tool for estimating intraspecific genetic diversity in a wild fish population

Environmental DNA (eDNA) analysis has recently been used as a new tool for estimating intraspecific diversity. However, whether known haplotypes contained in a sample can be detected correctly using eDNA‐based methods has been examined only by an aquarium experiment. Here, we tested whether the haplotypes of Ayu fish (Plecoglossus altivelis altivelis) detected in a capture survey could also be detected from an eDNA sample derived from the field that contained various haplotypes with low concentrations and foreign substances. A water sample and Ayu specimens collected from a river on the same day were analysed by eDNA analysis and Sanger sequencing, respectively. The 10 L water sample was divided into 20 filters for each of which 15 PCR replications were performed. After high‐throughput sequencing, denoising was performed using two of the most widely used denoising packages, unoise3 and dada2 . Of the 42 haplotypes obtained from the Sanger sequencing of 96 specimens, 38 (unoise3 ) and 41 (dada2 ) haplotypes were detected by eDNA analysis. When dada2 was used, except for one haplotype, haplotypes owned by at least two specimens were detected from all the filter replications. Accordingly, although it is important to note that eDNA‐based method has some limitations and some risk of false positive and false negative, this study showed that the eDNA analysis for evaluating intraspecific genetic diversity provides comparable results for large‐scale capture‐based conventional methods. Our results suggest that eDNA‐based methods could become a more efficient survey method for investigating intraspecific genetic diversity in the field.