Pathogenic Mechanisms Underlying Idiopathic Pulmonary Fibrosis.

The pathogenesis of idiopathic pulmonary fibrosis (IPF) involves a complex interplay of cell types and signaling pathways. Recurrent alveolar epithelial cell (AEC) injury may occur in the context of predisposing factors (e.g., genetic, environmental, epigenetic, immunologic, and gerontologic), leading to metabolic dysfunction, senescence, aberrant epithelial cell activation, and dysregulated epithelial repair. The dysregulated epithelial cell interacts with mesenchymal, immune, and endothelial cells via multiple signaling mechanisms to trigger fibroblast and myofibroblast activation. Recent single-cell RNA sequencing studies of IPF lungs support the epithelial injury model. These studies have uncovered a novel type of AEC with characteristics of an aberrant basal cell, which may disrupt normal epithelial repair and propagate a profibrotic phenotype. Here, we review the pathogenesis of IPF in the context of novel bioinformatics tools as strategies to discover pathways of disease, cell-specific mechanisms, and cell-cell interactions that propagate the profibrotic niche.

Candida species in community-acquired pneumonia in patients with chronic aspiration.

BACKGROUND: When Candida species is found in a sputum culture, clinicians generally dismiss it as a contaminant. We sought to identify cases of community-acquired pneumonia (CAP) in which Candida might play a contributory etiologic role. METHODS: In a convenience sample of patients hospitalized for CAP, we screened for "high-quality sputum" by Gram stain (> 20 WBC/epithelial cell) and performed quantitative sputum cultures. Criteria for a potential etiologic role for Candida included the observation of large numbers of yeast forms on Gram stain, intracellular organisms and > 106 CFU/ml Candida in sputum. We gathered clinical information on cases that met these criteria for possible Candida infection. RESULTS: Sputum from 6 of 154 consecutive CAP patients had large numbers of extra- and intracellular yeast forms on Gram stain, with > 106 CFU/ml Candida albicans, glabrata, or tropicalis on quantitative culture. In all 6 patients, the clinical diagnoses at admission included chronic aspiration. Greater than 105 CFU/ml of a recognized bacterial pathogen (Streptococcus pneumoniae, Staphylococcus aureus, or Pseudomonas) or > 106 CFU/ml of other 'normal respiratory flora' (Lactobacillus species) were present together with Candida spp. in every case. Blood cultures yielded Candida in 2 cases, and 1,3-beta-D glucan was > 500 ng/mL in 3 of 3 cases in which it was assayed. Since all patients were treated with anti-bacterial and anti-fungal drugs, no inference about etiology can be derived from therapeutic response. CONCLUSIONS: Candida spp. together with a recognized bacterial pathogen or normal respiratory flora may contribute to the cause of CAP in patients who chronically aspirate.

Corrigendum to: Normal Respiratory Flora as a Cause of Community-Acquired Pneumonia.

[This corrects the article DOI: 10.1093/ofid/ofaa307.].

Normal Respiratory Flora as a Cause of Community-Acquired Pneumonia.

BACKGROUND: Intensive studies have failed to identify an etiologic agent in >50% cases of community-acquired pneumonia (CAP). Bacterial pneumonia follows aspiration of recognized bacterial pathogens (RBPs) such as Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus after they have colonize the nasopharynx. We hypothesized that aspiration of normal respiratory flora (NRF) might also cause CAP. METHODS: We studied 120 patients hospitalized for CAP who provided a high-quality sputum specimen at, or soon after admission, using Gram stain, quantitative sputum culture, bacterial speciation by matrix-assisted laser desorption ionization time-of-flight, and viral polymerase chain reaction. Thresholds for diagnosis of bacterial infection were ≥105 colony-forming units (cfu)/mL sputum for RBPs and ≥106 cfu for NRF. RESULTS: Recognized bacterial pathogens were found in 68 of 120 (56.7%) patients; 14 (20.1%) of these had a coinfecting respiratory virus. Normal respiratory flora were found in 31 (25.8%) patients; 10 (32.2%) had a coinfecting respiratory virus. Infection by ≥2 RBPs occurred in 10 cases and by NRF together with RBPs in 13 cases. Among NRF, organisms identified as Streptococcus mitis, which share many genetic features of S pneumoniae, predominated. A respiratory virus alone was found in 16 of 120 (13.3%) patients. Overall, an etiologic diagnosis was established in 95.8% of cases. CONCLUSIONS: Normal respiratory flora, with or without viral coinfection, appear to have caused one quarter of cases of CAP and may have played a contributory role in an additional 10.8% of cases caused by RBPs. An etiology for CAP was identified in >95% of patients who provided a high-quality sputum at, or soon after, the time of admission.

The CaM Kinase CMK-1 Mediates a Negative Feedback Mechanism Coupling the C. elegans Glutamate Receptor GLR-1 with Its Own Transcription.

Regulation of synaptic AMPA receptor levels is a major mechanism underlying homeostatic synaptic scaling. While in vitro studies have implicated several molecules in synaptic scaling, the in vivo mechanisms linking chronic changes in synaptic activity to alterations in AMPA receptor expression are not well understood. Here we use a genetic approach in C. elegans to dissect a negative feedback pathway coupling levels of the AMPA receptor GLR-1 with its own transcription. GLR-1 trafficking mutants with decreased synaptic receptors in the ventral nerve cord (VNC) exhibit compensatory increases in glr-1 mRNA, which can be attributed to increased glr-1 transcription. Glutamatergic transmission mutants lacking presynaptic eat-4/VGLUT or postsynaptic glr-1, exhibit compensatory increases in glr-1 transcription, suggesting that loss of GLR-1 activity is sufficient to trigger the feedback pathway. Direct and specific inhibition of GLR-1-expressing neurons using a chemical genetic silencing approach also results in increased glr-1 transcription. Conversely, expression of a constitutively active version of GLR-1 results in decreased glr-1 transcription, suggesting that bidirectional changes in GLR-1 signaling results in reciprocal alterations in glr-1 transcription. We identify the CMK-1/CaMK signaling axis as a mediator of the glr-1 transcriptional feedback mechanism. Loss-of-function mutations in the upstream kinase ckk-1/CaMKK, the CaM kinase cmk-1/CaMK, or a downstream transcription factor crh-1/CREB, result in increased glr-1 transcription, suggesting that the CMK-1 signaling pathway functions to repress glr-1 transcription. Genetic double mutant analyses suggest that CMK-1 signaling is required for the glr-1 transcriptional feedback pathway. Furthermore, alterations in GLR-1 signaling that trigger the feedback mechanism also regulate the nucleocytoplasmic distribution of CMK-1, and activated, nuclear-localized CMK-1 blocks the feedback pathway. We propose a model in which synaptic activity regulates the nuclear localization of CMK-1 to mediate a negative feedback mechanism coupling GLR-1 activity with its own transcription.

The DAF-7/TGF-ß signaling pathway regulates abundance of the Caenorhabditis elegans glutamate receptor GLR-1.

Transforming growth factor-ß (TGF-ß) family signaling pathways have roles in both neuronal development and the regulation of synaptic function. Here we identify a novel role for the Caenorhabditis elegans DAF-7/TGF-ß signaling pathway in the regulation of the AMPA-type glutamate receptor GLR-1. We found that the abundance of GLR-1 increases at synapses in the ventral nerve cord (VNC) of animals with loss-of-function mutations in multiple DAF-7/TGF-ß pathway components including the TGF-ß ligand DAF-7, the type I receptor DAF-1, and the Smads DAF-8 and DAF-14. The GLR-1 defect can be rescued by expression of daf-8 specifically in glr-1-expressing interneurons. The effect on GLR-1 was specific for the DAF-7 pathway because mutations in the DBL-1/TGF-ß family pathway did not increase GLR-1 levels in the VNC. Immunoblot analysis indicates that total levels of GLR-1 protein are increased in neurons of DAF-7/TGF-ß pathway mutants. The increased abundance of GLR-1 in the VNC of daf-7 pathway mutants is dependent on the transcriptional regulator DAF-3/Smad suggesting that DAF-3-dependent transcription controls GLR-1 levels. Furthermore, we found that glr-1 transcription is increased in daf-7 mutants based on a glr-1 transcriptional reporter. Together these results suggest that the DAF-7/TGF-ß signaling pathway functions in neurons and negatively regulates the abundance of GLR-1, in part, by controlling transcription of the receptor itself. Finally, DAF-7/TGF-ß pathway mutants exhibit changes in spontaneous locomotion that are dependent on endogenous GLR-1 and consistent with increased glutamatergic signaling. These results reveal a novel mechanism by which TGF-ß signaling functions in the nervous system to regulate behavior.

The AP2 clathrin adaptor protein complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans.

Regulation of glutamate receptor (GluR) abundance at synapses by clathrin-mediated endocytosis can control synaptic strength and plasticity. We take advantage of viable, null mutations in subunits of the clathrin adaptor protein 2 (AP2) complex in Caenorhabditis elegans to characterize the in vivo role of AP2 in GluR trafficking. In contrast to our predictions for an endocytic adaptor, we found that levels of the GluR GLR-1 are decreased at synapses in the ventral nerve cord (VNC) of animals with mutations in the AP2 subunits APM-2/µ2, APA-2/α, or APS-2/σ2. Rescue experiments indicate that APM-2/µ2 functions in glr-1-expressing interneurons and the mature nervous system to promote GLR-1 levels in the VNC. Genetic analyses suggest that APM-2/µ2 acts upstream of GLR-1 endocytosis in the VNC. Consistent with this, GLR-1 accumulates in cell bodies of apm-2 mutants. However, GLR-1 does not appear to accumulate at the plasma membrane of the cell body as expected, but instead accumulates in intracellular compartments including Syntaxin-13- and RAB-14-labeled endosomes. This study reveals a novel role for the AP2 clathrin adaptor in promoting the abundance of GluRs at synapses in vivo, and implicates AP2 in the regulation of GluR trafficking at an early step in the secretory pathway.

Discovery of non-ETS gene fusions in human prostate cancer using next-generation RNA sequencing.

Half of prostate cancers harbor gene fusions between TMPRSS2 and members of the ETS transcription factor family. To date, little is known about the presence of non-ETS fusion events in prostate cancer. We used next-generation transcriptome sequencing (RNA-seq) in order to explore the whole transcriptome of 25 human prostate cancer samples for the presence of chimeric fusion transcripts. We generated more than 1 billion sequence reads and used a novel computational approach (FusionSeq) in order to identify novel gene fusion candidates with high confidence. In total, we discovered and characterized seven new cancer-specific gene fusions, two involving the ETS genes ETV1 and ERG, and four involving non-ETS genes such as CDKN1A (p21), CD9, and IKBKB (IKK-beta), genes known to exhibit key biological roles in cellular homeostasis or assumed to be critical in tumorigenesis of other tumor entities, as well as the oncogene PIGU and the tumor suppressor gene RSRC2. The novel gene fusions are found to be of low frequency, but, interestingly, the non-ETS fusions were all present in prostate cancer harboring the TMPRSS2-ERG gene fusion. Future work will focus on determining if the ETS rearrangements in prostate cancer are associated or directly predispose to a rearrangement-prone phenotype.

FusionSeq: a modular framework for finding gene fusions by analyzing paired-end RNA-sequencing data.

We have developed FusionSeq to identify fusion transcripts from paired-end RNA-sequencing. FusionSeq includes filters to remove spurious candidate fusions with artifacts, such as misalignment or random pairing of transcript fragments, and it ranks candidates according to several statistics. It also has a module to identify exact sequences at breakpoint junctions. FusionSeq detected known and novel fusions in a specially sequenced calibration data set, including eight cancers with and without known rearrangements.

N-myc downstream regulated gene 1 (NDRG1) is fused to ERG in prostate cancer.

A step toward the molecular classification of prostate cancer was the discovery of recurrent erythroblast transformation-specific rearrangements, most commonly fusing the androgen-regulated TMPRSS2 promoter to ERG. The TMPRSS2-ERG fusion is observed in around 90% of tumors that overexpress the oncogene ERG. The goal of the current study was to complete the characterization of these ERG-overexpressing prostate cancers. Using fluorescence in situ hybridization and reverse transcription-polymerase chain reaction assays, we screened 101 prostate cancers, identifying 34 cases (34%) with the TMPRSS2-ERG fusion. Seven cases demonstrated ERG rearrangement by fluorescence in situ hybridization without the presence of TMPRSS2-ERG fusion messenger RNA transcripts. Screening for known 5' partners, we determined that three cases harbored the SLC45A3-ERG fusion. To discover novel 5' partners in these ERG-overexpressing and ERG-rearranged cases, we used paired-end RNA sequencing. We first confirmed the utility of this approach by identifying the TMPRSS2-ERG fusion in a known positive prostate cancer case and then discovered a novel fusion involving the androgen-inducible tumor suppressor, NDRG1 (N-myc downstream regulated gene 1), and ERG in two cases. Unlike TMPRSS2-ERG and SCL45A3-ERG fusions, the NDRG1-ERG fusion is predicted to encode a chimeric protein. Like TMPRSS2, SCL45A3 and NDRG1 are inducible not only by androgen but also by estrogen. This study demonstrates that most ERG-overexpressing prostate cancers harbor hormonally regulated TMPRSS2-ERG, SLC45A3-ERG, or NDRG1-ERG fusions. Broader implications of this study support the use of RNA sequencing to discover novel cancer translocations.

EML4-ALK fusion lung cancer: a rare acquired event.

A recurrent gene fusion between EML4 and ALK in 6.7% of non-small cell lung cancers (NSCLCs) and NKX2-1 (TTF1, TITF1) high-level amplifications in 12% of adenocarcinomas of the lung were independently reported recently. Because the EML4-ALK fusion was only shown by a reverse transcription-polymerase chain reaction approach, we developed fluorescent in situ hybridization assays to interrogate more than 600 NSCLCs using break-apart probes for EML4 and ALK. We found that EML4-ALK fusions occur in less than 3% of NSCLC samples and that EML4 and/or ALK amplifications also occur. We also observed that, in most cases in which an EML4/ALK alteration is detected, not all of the tumor cells harbor the lesion. By using a detailed multi-fluorescent in situ hybridization probe assay and reverse transcription-polymerase chain reaction, we have evidence that other, more common mechanisms besides gene inversion exist including the possibility of other fusion partners for ALK and EML4. Furthermore, we confirmed the NKX2-1 high-level amplification in a significant subset of NSCLC and found this amplification to be mutually exclusive to ALK and EML4 rearrangements.