Structural Insights into the Regulatory Mechanism of the Response Regulator RocR from Pseudomonas aeruginosa in Cyclic Di-GMP Signaling

The nucleotide messenger cyclic di-GMP (c-di-GMP) plays a central role in the regulation of motility, virulence, and biofilm formation in many pathogenic bacteria. EAL domain-containing phosphodiesterases are the major signaling proteins responsible for the degradation of c-di-GMP and maintenance of its cellular level. We determined the crystal structure of a single mutant (R286W) of the response regulator RocR from Pseudomonas aeruginosa to show that RocR exhibits a highly unusual tetrameric structure arranged around a single dyad, with the four subunits adopting two distinctly different conformations. Subunits A and B adopt a conformation with the REC domain located above the c-di-GMP binding pocket, whereas subunits C and D adopt an open conformation with the REC domain swung to the side of the EAL domain. Remarkably, the access to the substrate-binding pockets of the EAL domains of the open subunits C and D are blocked in trans by the REC domains of subunits A and B, indicating that only two of the four active sites are engaged in the degradation of c-di-GMP. In conjunction with biochemical and biophysical data, we propose that the structural changes within the REC domains triggered by the phosphorylation are transmitted to the EAL domain active sites through a pathway that traverses the dimerization interfaces composed of a conserved regulatory loop and the neighboring motifs. This exquisite mechanism reinforces the crucial role of the regulatory loop and suggests that similar regulatory mechanisms may be operational in many EAL domain proteins, considering the preservation of the dimerization interface and the spatial arrangement of the regulatory domains.     

Cardioprotective effects of nitroparacetamol and paracetamol in acute phase of myocardial infarction in experimental rats

We aimed to determine whether nitroparacetamol (NO-paracetamol) and paracetamol exhibit cardioprotective effects. Myocardial infarction (MI) was induced in rats, and drug treatment was started 1 wk before surgery. Mortality rate and infarct size at 2 days after MI were compared. Treatment groups included vehicle (saline), paracetamol (5 mg x kg(-1) x day(-1)) and NO-paracetamol (15 mg x kg(-1) x day(-1)). Mortality rates for vehicle (n = 80), paracetamol (n = 79), and NO-paracetamol (n = 76) groups were 37.5%, 21.5%, and 26.3%, respectively. Infarct size for the vehicle group was 44.8% (+/-6.1%) of the left ventricle (LV). For the paracetamol and NO-paracetamol groups, infarct size was 31.3% (+/-5.6%) and 30.7% (+/-8.1%) of the LV, respectively. Both paracetamol- and NO-paracetamol-treated groups showed increased activities of catalase and SOD compared with the vehicle group. They could attenuate endothelial, inducible, and neuronal nitric oxide synthase and cyclooxygenase-1 and -2 gene expression after MI. The observation indicates the potential clinical significance of the cardioprotective effects of these drugs.     

Inter- and intra-researcher variation in measurement of morphometrics on Steller sea lions (<Emphasis Type="Italic">Eumetopias jubatus</Emphasis>)

Substantial effort has been made to increase the accuracy of photogrammetric measurement techniques, especially on marine mammals. However, accuracy of the physical measurements is rarely considered when calculating the photogrammetric accuracy. We examined inter- and intra-researcher variation in the measurement of physical morphometrics of Steller sea lions (Eumetopias jubatus) to assess their potential effects on photogrammetric accuracy and body mass predictions based on photogrammetrically derived morphometrics. Significant differences were found inter-researcher in median standard length and axillary girth and intra-researcher when measuring axillary girth of the same animals. Photogrammetrically derived estimates of these parameters are comparable to physical measurements when taking into account the inherent inter- and intra-researcher variation.     

Evolution of an adenocarcinoma in response to selection by targeted kinase inhibitors

Background

Adenocarcinomas of the tongue are rare and represent the minority (20 to 25%) of salivary gland tumors affecting the tongue. We investigated the utility of massively parallel sequencing to characterize an adenocarcinoma of the tongue, before and after treatment.

Results

In the pre-treatment tumor we identified 7,629 genes within regions of copy number gain. There were 1,078 genes that exhibited increased expression relative to the blood and unrelated tumors and four genes contained somatic protein-coding mutations. Our analysis suggested the tumor cells were driven by the RET oncogene. Genes whose protein products are targeted by the RET inhibitors sunitinib and sorafenib correlated with being amplified and or highly expressed. Consistent with our observations, administration of sunitinib was associated with stable disease lasting 4 months, after which the lung lesions began to grow. Administration of sorafenib and sulindac provided disease stabilization for an additional 3 months after which the cancer progressed and new lesions appeared. A recurring metastasis possessed 7,288 genes within copy number amplicons, 385 genes exhibiting increased expression relative to other tumors and 9 new somatic protein coding mutations. The observed mutations and amplifications were consistent with therapeutic resistance arising through activation of the MAPK and AKT pathways.

Conclusions

We conclude that complete genomic characterization of a rare tumor has the potential to aid in clinical decision making and identifying therapeutic approaches where no established treatment protocols exist. These results also provide direct in vivo genomic evidence for mutational evolution within a tumor under drug selection and potential mechanisms of drug resistance accrual.     

Complete Genome Sequence of Staphylococcus aureus Strain JKD6008, an ST239 Clone of Methicillin-Resistant Staphylococcus aureus with Intermediate-Level Vancomycin Resistance

We report here the complete 2.92-Mb genome sequence of a clinical isolate of methicillin-resistant Staphylococcus aureus subsp. aureus that demonstrates intermediate-level vancomycin resistance. The strain, named JKD6008, belongs to multilocus sequence type 239 and was isolated from the bloodstream of a patient in New Zealand in 2003.We have previously described the in vivo evolution of low-level vancomycin resistance in Staphylococcus aureus through comparative and functional genomic assessment of a pair of isogenic methicillin-resistant Staphylococcus aureus (MRSA) strains. The vancomycin-susceptible S. aureus (VSSA) strain JKD6009 was a patient wound isolate, whereas vancomycin-intermediate S. aureus (VISA) strain JKD6008 was recovered from the bloodstream of the same patient after 42 days of vancomycin treatment (5). Comparison of the partially assembled genomes of the two isolates revealed a single-point mutation in the sensor region of the two-component regulatory gene graS, which caused a significant reduction in the vancomycin susceptibility of JKD6008 (6). Here we report the fully assembled and annotated genome of S. aureus JKD6008.The genome sequence of S. aureus strain JKD6008 was determined by whole-genome shotgun sequencing using single-read 454 GS20 (Roche Diagnostics, Basel, Switzerland), Sanger (Applied Biosystems), and SOLiD (Applied Biosystems) sequencing technologies, producing approximately 20 times, 4 times, and 225 times coverage of the genome, respectively. GS20 reads were assembled using gsAssembler v2.0 software, resulting in 131 contigs (≥500 bp) totaling 2.83 Mbp (6, 10). Sanger paired-end reads (clone insert size, 3 to 5 kb) were combined with the GS20 contigs using Gap4 v4.11 software (3). Mate-pair SOLiD reads (3 to 5 kb) were aligned to the contigs using SHRiMP 1.3.2 software to identify and correct sequencing errors (11). Optical mapping produced a high-resolution XbaI chromosome restriction map, to which the contigs were aligned using MapSolver 2.1.1 (Opgen) to determine misassemblies. Gap closures were performed by PCR, followed by Sanger sequencing and primer walking of amplification products (3730S DNA Analyzer sequencer; Applied Biosystems). The assembly of the completed genome was confirmed to be correct by reference to the XbaI optical map.Protein-coding regions were predicted using GeneMarkS 4.6b software, tRNA genes using tRNAscan-SE 1.23, and rRNA genes using RNAmmer 1.2 (2, 8, 9). Gene products were assigned using HMMER 3.0 against the Pfam database (release 23) and BLAST 2.2.23 against RefSeq proteins (April 2010) and the Conserved Domain Database (v2.22) (1, 4). These automated analyses were followed by manual curation and comparisons with other completed S. aureus genomes.The genome of S. aureus strain JKD6008 consists of a circular 2,924,344-bp chromosome with a 34% G+C content and no extrachromosomal elements. A total of 2,766 coding DNA sequences, 82 tRNA genes, and 5 rRNA loci were detected. Over 70% of genes were assigned to specific Clusters of Orthologous Groups (COG) functional groups, and 42% were assigned an enzyme classification number (12).Initial analysis of the whole-genome sequence of JKD6008 confirmed it as a member of the ST239 complex, sharing 2,504 orthologous coding sequences (CDSs) with the recently described ST239 member TW20 (EMBL accession no. {"type":"entrez-nucleotide","attrs":{"text":"FN433596.1","term_id":"269939526","term_text":"FN433596.1"}}FN433596.1). There are 17 copies of IS256 and a type III staphylococcal cassette chromosome mec element (SCCmec). Comparisons with 19 published S. aureus genomes revealed 20 CDS not present in any other S. aureus genome, although some of these 20 CDS have orthologs in other Staphylococcus species. JKD6008 also harbors a 28-kb integrated pSK1-like plasmid that is predicted to confer resistance to aminoglycosides and trimethoprim, as well as efflux-mediated antiseptic and disinfectant resistance (7).Nucleotide sequence accession number. The complete genome sequence has been deposited in NCBI GenBank under accession number {"type":"entrez-nucleotide","attrs":{"text":"CP002120","term_id":"302749911","term_text":"CP002120"}}CP002120.     

Reduced fat mass in mice lacking orphan nuclear receptor estrogen-related receptor alpha

The estrogen-related receptor alpha (ERRalpha) is an orphan member of the superfamily of nuclear hormone receptors expressed in tissues that preferentially metabolize fatty acids. Despite the molecular characterization of ERRalpha and identification of target genes, determination of its physiological function has been hampered by the lack of a natural ligand. To further understand the in vivo function of ERRalpha, we generated and analyzed Estrra-null (ERRalpha-/-) mutant mice. Here we show that ERRalpha-/- mice are viable, fertile and display no gross anatomical alterations, with the exception of reduced body weight and peripheral fat deposits. No significant changes in food consumption and energy expenditure or serum biochemistry parameters were observed in the mutant animals. However, the mutant animals are resistant to a high-fat diet-induced obesity. Importantly, DNA microarray analysis of gene expression in adipose tissue demonstrates altered regulation of several enzymes involved in lipid, eicosanoid, and steroid synthesis, suggesting that the loss of ERRalpha might interfere with other nuclear receptor signaling pathways. In addition, the microarray study shows alteration in the expression of genes regulating adipogenesis as well as energy metabolism. In agreement with these findings, metabolic studies showed reduced lipogenesis in adipose tissues. This study suggests that ERRalpha functions as a metabolic regulator and that the ERRalpha-/- mice provide a novel model for the investigation of metabolic regulation by nuclear receptors.