The open reading frame 3 gene of hepatitis E virus contains a cis-reactive element and encodes a protein required for infection of macaques

An infectious cDNA clone of hepatitis E virus was mutated in order to prevent synthesis of either open reading frame 2 (ORF2) protein or ORF3 protein. HuH-7 cells transfected with an ORF2-null mutant produced ORF3, and those transfected with an ORF3-null mutant produced ORF2. Silent mutations introduced into a highly conserved nucleotide sequence in the ORF3 coding region eliminated the synthesis of both ORF2 and ORF3 proteins, suggesting that it comprised a cis-reactive element. A mutant that was not able to produce ORF3 protein did not produce a detectable infection in rhesus macaques. However, a mutant that encoded an ORF3 protein lacking a phosphorylation site reported to be critical for function was able to replicate its genome in cell culture and to induce viremia and seroconversion in rhesus monkeys, suggesting that phosphorylation of ORF3 protein was not necessary for genome replication or for production of infectious virions.     

Regulation of the Phosphate Stress Response in Rhizobium meliloti by PhoB

Alkaline phosphatase activity and phosphate transport rates in Rhizobium meliloti increased significantly when medium phosphate levels decreased to approximately 10 (mu)M. Both responses were abolished in a Tn5:: phoB mutant, but the mutant could be complemented by a plasmid that contained cloned R. meliloti phoB. The PhoB(sup-) mutant had a normal symbiosis phenotype under growth conditions that supplied either limiting or nonlimiting levels of phosphate to the host plant Medicago sativa, suggesting that induction of genes by PhoB was not required for normal symbiotic function.     

Drosophila fasciclin I is a novel homophilic adhesion molecule that along with fasciclin III can mediate cell sorting

Fasciclin I is a membrane-associated glycoprotein that is regionally expressed on a subset of fasciculating axons during neuronal development in insects; it is expressed on apposing cell surfaces, suggesting a role in specific cell adhesion. In this paper we show that Drosophila fasciclin I is a novel homophilic cell adhesion molecule. When the nonadhesive Drosophila S2 cells are transfected with the fasciclin I cDNA, they form aggregates that are blocked by antisera against fasciclin I. When cells expressing fasciclin I are mixed with cells expressing fasciclin III, another Drosophila homophilic adhesion molecule, the mixture sorts into aggregates homogeneous for either fasciclin I- or fasciclin III-expressing cells. The ability of these two novel adhesion molecules to mediate cell sorting in vitro suggests that they might play a similar role during neuronal development.     

Rapid and inexpensive species differentiation using a multiplex real-time polymerase chain reaction high-resolution melt assay

We demonstrate a method for developing real-time polymerase chain reaction (PCR) high-resolution melt (HRM) assays to identify multiple species present in a mixture simultaneously using LCGreen Plus and melt temperatures. Highly specific PCR primers are designed to yield amplicons with different melt temperatures for simple routine species identification compared with differentiating melt curve kinetics traces or difference plots. This method is robust and automatable, and it leads to savings in time and reagent costs, is easily modified to probe any species of interest, eliminates the need for post-PCR gel or capillary electrophoresis in routine assays, and requires no expensive dye-labeled primers.     

Acetylation of fluoroquinolone antimicrobial agents by an Escherichia coli strain isolated from a municipal wastewater treatment plant

Aims:  To isolate environmental bacteria capable of transforming fluoroquinolones to inactive molecules.
Methods and Results:  Bacteria were isolated from the aerobic liquor of a wastewater treatment plant on a medium containing norfloxacin (100 mg l⁻¹). Twenty-two isolates were highly resistant (minimal inhibitory concentration: 6·25−200 μg ml⁻¹) to five fluoroquinolones and six of them were positive by PCR amplification for the aminoglycoside resistance gene aac(6')-Ib. Of these, only Escherichia coli strain LR09 had the ciprofloxacin-acetylating variant gene aac(6')-Ib-cr ; HPLC and mass spectrometry showed that this strain transformed both ciprofloxacin and norfloxacin by N -acetylation. This bacterium also had mutations in the quinolone-resistance determining regions of the gyrA and parC genes.
Conclusions:  An E. coli isolate from wastewater, which possessed at least two distinct fluoroquinolone resistance mechanisms, inactivated ciprofloxacin and norfloxacin by N -acetylation.
Significance and Impact of the Study:  This is the first report of N -acetylation of fluoroquinolones by an aac(6')-Ib-cr -containing bacterium from an environmental source.     

Complete Genome Sequence of the Cellulolytic Thermophile Caldicellulosiruptor obsidiansis OB47T

Caldicellulosiruptor obsidiansis OB47^T (ATCC BAA-2073, JCM 16842) is an extremely thermophilic, anaerobic bacterium capable of hydrolyzing plant-derived polymers through the expression of multidomain/multifunctional hydrolases. The complete genome sequence reveals a diverse set of carbohydrate-active enzymes and provides further insight into lignocellulosic biomass hydrolysis at high temperatures.Members of the genus Caldicellulosiruptor within the order Clostridiales can solubilize cellulose at extremely thermophilic growth temperatures (65 to 80°C). Caldicellulosiruptor obsidiansis OB47^T was isolated from Obsidian Pool, Yellowstone National Park, in enrichment cultures containing dilute acid-pretreated switchgrass as the primary carbon and energy source for cultivation (5). High-temperature saccharification can promote higher hydrolysis rates while reducing cooling costs following biomass pretreatment and suppressing contamination in reactors (9). Given the organism''s rapid growth on cellulosic substrates and ability to use a wide range of plant-derived sugars, a complete genome sequence was determined using a sequencing-by-synthesis approach.The genome of C. obsidiansis OB47^T was sequenced by the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) using a combination of Illumina (1) and 454 technologies (8). All of the general aspects of library construction and sequencing performed at the JGI can be found at http://www.jgi.doe.gov/. Illumina sequencing data were assembled with VELVET (10), and the consensus sequences were shredded into 1.5-kbp overlapped fake reads and assembled together with the 454 data. The initial Newbler assembly contained 64 contigs in two scaffolds. The initial 454 assembly was converted into a Phrap assembly by making fake reads from the consensus and collecting the read pairs in the 454 paired-end library. The Phred/Phrap/Consed software package was used for sequence assembly and quality assessment (2-4) in the following finishing process. Illumina data were used to correct potential base errors and increase consensus quality using the Polisher software developed at the JGI (Alla Lapidus, unpublished data). After the shotgun stage, reads were assembled with parallel Phrap (High Performance Software, LLC). Possible misassemblies were corrected with gapResolution (Cliff Han, unpublished data), Dupfinisher (6), or sequencing of cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR, and by Bubble PCR primer walks. A total of 773 additional reactions and seven shatter libraries were necessary to close gaps and to raise the quality of the finished sequence. The genome was annotated at Oak Ridge National Laboratory using the automated annotation pipeline, which is driven by the gene prediction algorithm Prodigal (7). Annotation quality was verified by the JGI.Although many well-characterized bacteria and fungi can use cellulose, C. obsidiansis was selected and isolated specifically for its ability to deconstruct potential bioenergy feedstocks (e.g., pretreated switchgrass or Populus sp.). Through high-throughput sequencing of novel strains relevant to different aspects of renewable energy production, genome-enabled technologies can be used to discover important cellular properties (such as the secretion of hydrolytic enzymes). Making the genome sequence of C. obsidiansis OB47^T available will allow comprehensive comparisons with other members of the genus and enable further investigation into the mechanisms employed by microorganisms to solubilize lignocellulosic materials at elevated temperatures.     

Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp,Ctenopharyngodon idellus

Background  

Hypoxia-inducible factors (HIFs) are involved in adaptive and survival responses to hypoxic stress in mammals. In fish, very little is known about the functions of HIFs.     

Structural integrity of collagen and elastin in SynerGraft® decellularized-cryopreserved human heart valves

SynerGraft® (SG) decellularized–cryopreserved cardiac valve allografts have been developed to provide a valve replacement option that has reduced antigenicity, retained structural integrity, and the ability to be stored long-term until needed for implantation. However, it is critical to ensure that both the SG processing and cryopreservation of these allografts do not detrimentally affect the extracellular matrix architecture within the tissue. This study evaluates the effects of SG decellularization and subsequent cryopreservation on the extracellular matrix integrity of allograft heart valves. Human aortic and pulmonary valves were trisected, with one-third of each either left fresh (no further processing after dissection), decellularized, or decellularized and cryopreserved. Two-photon laser scanning confocal microscopy was used to visualize collagen and elastin in leaflets and conduits. The optimized percent laser transmission (OPLT) required for full dynamic range imaging of each site was determined, and changes in OPLT were used to infer changes in collagen and elastin signal intensity. Collagen fiber crimp period and collagen and elastin fiber diameter were measured in leaflet tissue. Statistically significant differences in OPLT and the dimensional characteristics of collagen and elastin in study groups were determined through single factor ANOVA. The majority of donor-aggregated average OPLT observations showed no statistically significant differences among all groups, indicating no difference in collagen or elastin signal strength. Morphometric analysis of collagen and elastin fibers revealed no significant alterations in treated leaflet tissues relative to fresh tissues. Collagen and elastin structural integrity within allograft heart valves are maintained through SynerGraft® decellularization and subsequent cryopreservation.