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Objective: To examine the association between anxiety symptoms, depression symptoms, and life satisfaction 1 year after SCI., Design: Cross-sectional analysis of data from the SCIRehab Project. A linear regression model estimated the association between anxiety symptoms and life satisfaction and tested the moderating effect of depression symptoms on the association between anxiety symptoms and depression symptoms with an interaction term., Setting: Six rehabilitation facilities across the United States., Participants: A total to 940 persons older than 12 years who received inpatient spinal cord injury (SCI) rehabilitation between 2007 and 2009 were followed up 1 year post injury (n=940)., Interventions: None., Main Outcome Measures: Life satisfaction 1 year after SCI measured via the Satisfaction With Life Scale., Results: Unadjusted analyses showed anxiety symptoms were associated with decreased life satisfaction for individuals with SCI. In adjusted analyses, anxiety symptoms were not associated with life satisfaction. In adding an interaction term, anxiety symptoms were associated with 2 points lower life satisfaction holding the other variables constant ( P =.02). There was a moderating effect of depression symptoms on the association between anxiety symptoms and life satisfaction. Persons with anxiety symptoms had lower life satisfaction scores at lower levels of depression symptoms but higher life satisfaction scores at higher levels of depression symptoms than persons with no anxiety., Conclusions: In clinical settings, both anxiety and depression symptoms should be monitored, measured, and treated together to optimally improve life satisfaction for persons with SCI. Prioritizing interventions known to have transdiagnostic effects may achieve the best results., (© 2022 The Authors.)

High-throughput RNA sequencing enables quantification of transcripts (both known and novel), exon/exon junctions and fusions of exons from different genes. Discovery of gene fusions-particularly those expressed with low abundance- is a challenge with short- and medium-length sequencing reads. To address this challenge, we implemented an RNA-Seq mapping pipeline within the LifeScope software. We introduced new features including filter and junction mapping, annotation-aided pairing rescue and accurate mapping quality values. We combined this pipeline with a Suffix Array Spliced Read (SASR) aligner to detect chimeric transcripts. Performing paired-end RNA-Seq of the breast cancer cell line MCF-7 using the SOLiD system, we called 40 gene fusions among over 120,000 splicing junctions. We validated 36 of these 40 fusions with TaqMan assays, of which 25 were expressed in MCF-7 but not the Human Brain Reference. An intra-chromosomal gene fusion involving the estrogen receptor alpha gene ESR1, and another involving the RPS6KB1 (Ribosomal protein S6 kinase beta-1) were recurrently expressed in a number of breast tumor cell lines and a clinical tumor sample.

We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding approximately 18x haploid coverage of aligned sequence and close to 300x clone coverage. Over 98% of the reference genome is covered with at least one uniquely placed read, and 99.65% is spanned by at least one uniquely placed mate-paired clone. We identify over 3.8 million SNPs, 19% of which are novel. Mate-paired data are used to physically resolve haplotype phases of nearly two-thirds of the genotypes obtained and produce phased segments of up to 215 kb. We detect 226,529 intra-read indels, 5590 indels between mate-paired reads, 91 inversions, and four gene fusions. We use a novel approach for detecting indels between mate-paired reads that are smaller than the standard deviation of the insert size of the library and discover deletions in common with those detected with our intra-read approach. Dozens of mutations previously described in OMIM and hundreds of nonsynonymous single-nucleotide and structural variants in genes previously implicated in disease are identified in this individual. There is more genetic variation in the human genome still to be uncovered, and we provide guidance for future surveys in populations and cancer biopsies.

The type III secretion system (TTSS) is a dedicated bacterial pathogen protein targeting system that directly affects host cell signalling and response pathways. Our goal was to identify host responses to the Pseudomonas aeruginosa effectors, introduced into target cells utilizing the TTSS. We carried out expression profiling of a human lung pneumocyte cell line A549 exposed to isogenic mutants of P. aeruginosa PAK lacking individual or a combination of TTSS components. We then devised a data analysis method to isolate the key responses to specific secreted bacterial effector proteins as well as components of the TTSS machinery. Individually, the effector proteins elicited host responses consistent with their known functions, many of which were cell cycle-related. However, our analysis has shown that the effector proteins elicit a distinct host transcriptional response when present in combination, suggesting a synergistic effect. Furthermore, the pattern of host transcriptional responses is consistent with the pore forming ability of the TTSS needle complex. This study shows that the individual components of the TTSS define an integrated system and that a systems biology approach is required to fully understand the complex interplay between pathogen and host.

Chronic lung infection by Pseudomonas aeruginosa causes significant morbidity in cystic fibrosis patients initiated by the failure of innate immune responses. We used microarray analysis and real-time PCR to detect transcriptional changes associated with cytokine production in isogenic bronchial epithelial cell lines with either wild-type (WT) or mutant cystic fibrosis transmembrane conductance regulator (CFTR) in response to P. aeruginosa infection. The transcription of four NF-kappaB-regulated cytokine genes was maximal in the presence of WT CFTR: the interleukin-8 (IL-8), IL-6, CXCL1, and intracellular adhesion molecule 1 (ICAM-1) genes. Analysis of protein expression in two cell lines paired for wild-type and mutant CFTR with three P. aeruginosa strains showed IL-6 and IL-8 expressions were consistently enhanced by the presence of WT CFTR in both cell lines with all three strains of P. aeruginosa, although some strains gave small IL-8 increases in cells with mutant CFTR. CXCL1 production showed consistent enhancement in cells with WT CFTR using all three bacterial strains in one cell line, whereas in the other cell line, CXCL1 showed a significant increase in cells with either WT or mutant CFTR. ICAM-1 was unchanged at the protein level in one of the cell lines but did show mild enhancement with WT CFTR in the other cell pair. Inhibitions of NF-kappaB prior to infection indicated differing degrees of dependence on NF-kappaB for production of the cytokines, contingent on the cell line. Cytokine effectors of innate immunity to P. aeruginosa were found to be positively influenced by the presence of WT CFTR, indicating a role in resistance to P. aeruginosa infection.

The role of epithelial polarity and bacterial factors in the control of the innate immune response of airway epithelial cells to Pseudomonas aeruginosa PAK was investigated using a human, nasal cystic fibrosis (DeltaF508/DeltaF508) epithelial cell line CF15 grown as confluent layers on permeable supports. Addition of PAK to the basal surface of CF15 layers caused significant expression changes in 1525 different genes (out of 12 625 examined), including the cytokines IL-6, IL-8, IL-1beta and TNF-alpha, as well as genes associated with leucocyte adhesion, antibacterial factors, and NF-kappaB signalling. Confocal microscopy showed that nuclear migration of NF-kappaB in all CF15 cells was preceded by PAK binding to the basal and lateral surfaces of some cells. Addition of PAK to the apical surface of CF15 monolayers elicited changes in expression of only 602 genes, including 256 not affected during basolateral PAK exposure. Over time, cytokine expression during apical PAK was similar to that exhibited by basal PAK, but the magnitudes during apical treatment were much smaller with little/no nuclear migration of NF-kappaB in CF15 cells. Furthermore, these responses depended on the presence of flagellin, but not pili on the bacteria. Thus, P. aeruginosa triggered a strong innate immune response that depended on the apical versus basolateral polarity of CF15 cells and the presence of flagellin on the bacteria.

Pseudomonas aeruginosa strains from the chronic lung infections of cystic fibrosis (CF) patients are phenotypically and genotypically diverse. Using strain PAO1 whole genome DNA microarrays, we assessed the genomic variation in P. aeruginosa strains isolated from young children with CF (6 months to 8 years of age) as well as from the environment. Eighty-nine to 97% of the PAO1 open reading frames were detected in 20 strains by microarray analysis, while subsets of 38 gene islands were absent or divergent. No specific pattern of genome mosaicism defined strains associated with CF. Many mosaic regions were distinguished by their low G + C content; their inclusion of phage related or pyocin genes; or by their linkage to a vgr gene or a tRNA gene. Microarray and phenotypic analysis of sequential isolates from individual patients revealed two deletions of greater than 100 kbp formed during evolution in the lung. The gene loss in these sequential isolates raises the possibility that acquisition of pyomelanin production and loss of pyoverdin uptake each may be of adaptive significance. Further characterization of P. aeruginosa diversity within the airways of individual CF patients may reveal common adaptations, perhaps mediated by gene loss, that suggest new opportunities for therapy.

Pseudomonas aeruginosa is an opportunistic pathogen that plays a major role in lung function deterioration in cystic fibrosis patients. To identify critical host responses during infection, we have used high-density DNA microarrays, consisting of 1,506 human cDNA clones, to monitor gene expression in the A549 lung pneumocyte cell line during exposure to P. aeruginosa. We have identified host genes that are differentially expressed upon infection, several of which require interaction with P. aeruginosa and the expression of the major subunit of type IV pili, PilA. Differential expression of genes involved in various cellular functions was identified, and we selected the gene encoding the transcription factor interferon regulatory factor 1 (IRF-1) for further analysis. The levels of the IRF-1 transcript increased 3- to 4-fold in A549 cells after adherence by P. aeruginosa. A similar increase of IRF-1 mRNA was observed in A549 cells exposed to wild-type P. aeruginosa when compared to an isogenic, nonpiliated strain. However, this difference was abolished when serum was present during the incubation of bacteria. Exposure of A549 cells to purified P. aeruginosa lipopolysaccharide did not result in a significant increase in IRF-1 mRNA. Although the P. aeruginosa-induced increased IRF-1 expression depends on the presence of bacterial adhesin, our findings do not preclude the possibility that other bacterial products are responsible for IRF-1 activation, which is enhanced by bacterial adherence to cells. These data show that microarray technology can be an important tool for studying the complex interplay between bacterial pathogens and host.

We show that the open reading frame in the Thermotoga maritima genome tentatively identified as the pcm gene (R. V. Swanson et al., J. Bacteriol. 178, 484-489, 1996) does indeed encode a protein L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) and that this protein repair enzyme displays several novel features. We expressed the 317 amino acid pcm gene product of this thermophilic bacterium in Escherichia coli as a fusion protein with an N-terminal 20 residue hexa-histidine-containing sequence. This protein contains a C-terminal domain of approximately 100 residues not previously seen in this enzyme from various prokaryotic or eukaryotic species and which does not have sequence similarity to any other entry in the GenBank databases. The C-terminal region appears to be required for enzymatic function as no activity is detected in two recombinant constructs lacking this domain. Sedimentation equilibrium analysis indicated that the enzyme is monomeric in solution. The Km values for measured for peptide and protein substrates were found to be intermediate between those of the high-affinity human enzyme and those of the lower-affinity wheat, nematode, and E. coli enzymes. The enzyme was extremely heat stable, with no loss of activity after 60 min at 100 degreesC. Enzyme activity was observed at temperatures as high as 93 degreesC with an optimal activity of 164 nmol/min/mg protein at 85 degreesC. This activity is approximately 18-fold higher than the maximal activities of mesophilic homologs at 37 degreesC. These data suggest that the Thermotoga enzyme has unique features for initiating repair in damaged proteins containing L-isoaspartyl residues at elevated temperatures., (Copyright 1998 Academic Press.)

The Escherichia coli surE gene is co-transcribed with pcm, encoding the L-isoaspartyl protein repair methyltransferase, and is highly conserved among both the Eubacteria and the Archaea; however, no biochemical function has yet been identified for this gene. Isoaspartyl accumulation during stationary phase was much higher in a pcm surE double mutant than in either single mutant, suggesting that the two genes may represent two parallel pathways by which E. coli can respond to protein damage. A null mutation in surE also suppressed stress-survival defects previously observed in a pcm mutant strain, providing further evidence for an interaction between the two gene products.

We determined the DNA sequence of a 2,232-bp region immediately upstream of the pcm gene at 59 min on the Escherichia coli chromosome that encodes an L-isoaspartyl protein methyltransferase with an important role in stationary-phase survival. Two open reading frames of 477 and 1,524 bp were found oriented in the same direction as that of the pcm gene. The latter open reading frame overlapped the 5' end of the pcm gene by 4 bp. Coupled in vitro transcription-translation analysis of DNA containing the 1,524-bp open reading frame directly demonstrated the production of a 37,000-Da polypeptide corresponding to a RNA species generated from a promoter within the open reading frame. The deduced amino acid sequence showed no similarity to known protein sequences. To test the function of this gene product, we constructed a mutant strain in which a kanamycin resistance element was inserted at a BstEII site in the middle of its coding region in an orientation that does not result in reduction of Pcm methyltransferase activity. These cells were found to survive poorly in stationary phase, at elevated temperatures, and in high-salt media compared with parent cells containing the intact gene, and we thus designate this gene surE (survival). surE appears to be the first gene of a bicistronic operon also containing the pcm gene. The phenotypes of mutations in either gene are very similar and indicate that both gene products are important for the viability of E. coli cells under stressful conditions.

We report a 1.432-kb DNA sequence at 59 min on the Escherichia coli chromosome that connects the published sequences of the pcm gene for the isoaspartyl protein methyltransferase and that of the katF or rpoS (katF/rpoS) gene for a sigma factor involved in stationary-phase gene expression. Analysis of the DNA sequence reveals an open reading frame potentially encoding a polypeptide of 379 amino acids. The polypeptide sequence includes a consensus bacterial lipidation sequence present at residues 23 to 26 (Leu-Ala-Gly-Cys), four octapeptide proline- and glutamine-rich repeats of consensus sequence QQPQIQPV, and four heptapeptide threonine- and serine-rich repeats of consensus sequence PTA(S,T)TTE. The deduced amino acid sequence, especially in the C-terminal region, is similar to that of the Haemophilus somnus LppB lipoprotein outer membrane antigen (40% overall sequence identity; 77% identity in last 95 residues). The LppB lipoprotein binds Congo red dye and has been proposed to be a virulence determinant in H. somnus. Utilizing a plasmid construct with the E. coli gene under the control of a phage T7 promoter, we demonstrate the lipidation of this gene product by the incorporation of [3H]palmitic acid into a 42-kDa polypeptide. We also show that treatment of E. coli cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 46-kDa precursor. We thus designate the protein NlpD (new lipoprotein D). E. coli cells overexpressing NlpD bind Congo red dye, suggesting a common function with the H. somnus LppB protein. Disruption of the chromosomal E. coli nlpD gene by insertional mutagenesis results in decreased stationary-phase survival after 7 days.