Systemic Sclerosis-Associated Pulmonary Arterial Hypertension: From Bedside to Bench and Back Again.

Systemic sclerosis (SSc) is a heterogeneous disease characterized by autoimmunity, vasculopathy, and fibrosis which affects the skin and internal organs. One key aspect of SSc vasculopathy is pulmonary arterial hypertension (SSc-PAH) which represents a leading cause of morbidity and mortality in patients with SSc. The pathogenesis of pulmonary hypertension is complex, with multiple vascular cell types, inflammation, and intracellular signaling pathways contributing to vascular pathology and remodeling. In this review, we focus on shared molecular features of pulmonary hypertension and those which make SSc-PAH a unique entity. We highlight advances in the understanding of the clinical and translational science pertinent to this disease. We first review clinical presentations and phenotypes, pathology, and novel biomarkers, and then highlight relevant animal models, key cellular and molecular pathways in pathogenesis, and explore emerging treatment strategies in SSc-PAH.

S6K1 blockade overcomes acquired resistance to EGFR-TKIs in non-small cell lung cancer.

The development of resistance to EGFR Tyrosine kinase inhibitors (TKIs) in NSCLC with activating EGFR mutations is a critical limitation of this therapy. In addition to genetic alterations such as EGFR secondary mutation causing EGFR-TKI resistance, compensatory activation of signaling pathways without interruption of genome integrity remains to be defined. In this study, we identified S6K1/MDM2 signaling axis as a novel bypass mechanism for the development of EGFR-TKI resistance. The observation of S6K1 as a candidate mechanism for resistance to EGFR TKI therapy was investigated by interrogation of public databases and a clinical cohort to establish S6K1 expression as a prognostic/predictive biomarker. The role of S6K1 in TKI resistance was determined in in vitro gain-and-loss of function studies and confirmed in subcutaneous and orthotopic mouse lung cancer models. Blockade of S6K1 by a specific inhibitor PF-4708671 synergistically enhanced the efficacy of TKI without showing toxicity. The mechanistic study showed the inhibition of EGFR caused nuclear translocation of S6K1 for binding with MDM2 in resistant cells. MDM2 is a downstream effector of S6K1-mediated TKI resistance. Taken together, we present evidence for the reversal of resistance to EGFR TKI by the addition of small molecule S6K1/MDM2 antagonists that could have clinical benefit.

Arsenic-induced metabolic shift triggered by the loss of miR-199a-5p through Sp1-dependent DNA methylation.

Inorganic arsenic is an environmental carcinogen that poses a major global public health risk. A high percentage of drinking water from wells in the U.S. contains higher-than-normal levels of arsenic, suggesting an increased risk of arsenic-induced deleterious effects. In addition to primary preventive measures, therapeutic strategies need to effectively address and integrate multiple molecular mechanisms underlying arsenic-induced carcinogenesis. We previously showed that the loss of miR-199a-5p in arsenic-transformed cells is pivotal to promote arsenic-induced angiogenesis and tumor growth in lung epithelial cells. In this study, we further showed that subacute or chronic exposure to arsenic diminished miR-199a-5p levels largely due to DNA methylation, which was achieved by increased DNA methyltransferase-1 (DNMT1) activity, mediated by the formation of specific protein 1 (Sp1)/DNMT1 complex. In addition to the DNA hypermethylation, arsenic exposure also repressed miR-199a transcription through a transcriptional repressor Sp1. We further identified an association between miR-199a-5p repression and the arsenic-mediated energy metabolic shift, as reflected by mitochondria defects and a switch to glycolysis, in which a glycolytic enzyme pyruvate kinase 2 (PKM2) was a functional target of miR-199a-5p. Taken together, the repression of miR-199a-5p through both Sp1-dependent DNA methylation and Sp1 transcriptional repression promotes an arsenic-mediated metabolic shift from mitochondria respiration to aerobic glycolysis via PKM2.

Influence of Intestinal Microbiota Transplantation and NleH Expression on Citrobacter rodentium Colonization of Mice.

The intestinal microbiota plays an important role in regulating host resistance to enteric pathogens. The relative abundance of the microbiota is dependent upon both genetic and environmental factors. The attaching and effacing pathogens enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium cause diarrheal disease and translocate type III secretion system effector proteins into host cells to inhibit pro-inflammatory host responses. Here we determined the influence of both the intestinal microbiota and the expression of the C. rodentium NleH effector on C. rodentium colonization in different mouse models. We performed fecal transplantation experiments between C57BL/6J and C57BL/10ScNJ mice and found that such microbiota transfers altered both the host resistance to C. rodentium infection as well as the benefit or detriment of expressing NleH to C. rodentium intestinal colonization.

Enterotoxigenic Escherichia coli Flagellin Inhibits TNF-Induced NF-κB Activation in Intestinal Epithelial Cells.

Enterotoxigenic Escherichia coli (ETEC) causes childhood diarrhea in developing countries. ETEC strains produce the heat-labile enterotoxin (LT) and/or heat-stable enterotoxins (ST) and encode a diverse set of colonization factors used for adherence to intestinal epithelial cells. We previously found that ETEC secretes a heat-stable protein we designated as ETEC Secreted Factor (ESF) that inhibits the extent of NF-κB activation normally induced by tumor necrosis factor alpha (TNF). Here we fractionated ETEC supernatants using fast protein liquid chromatography (FPLC) and determined that ETEC flagellin was necessary and sufficient to protect IκBα from degradation in response to TNF stimulation. These data suggest a potentially novel mechanism by which ETEC may evade the host innate immune response by down-regulating NF-κB-dependent host responses.

Protein extraction from the earthworm Eisenia fetida for 2-DE.

We identified an efficient protocol for extracting proteins from whole earthworm, Eisenia fetida, for 2-DE. Sample preparation is a critical step in a 2-DE proteome approach and is absolutely essential for obtaining good results. Six protein extraction protocols based on different protein precipitation agents were tested and evaluated using 2-DE. The methods generated remarkably different 2-DE protein spot patterns. We conclude that trichloroacetic acid (TCA)-A eliminates interfering compounds, thus allowing for the efficient resolubilization of proteins. TCA-A gives good distinction, more bands in 1-DE gels, and the most number of protein spots in 2-DE gels. It is also rapid, provides the higher protein yield, and has the less number of steps. To demonstrate the quality of the extracted proteins, we cut several protein spots that were common to four methods from 2-DE gels, analyzed them using MALDI-TOF/TOF MS, and tentatively identified them. The classic TCA-A method proved to be most useful as a standard method of extracting proteins from E. fetida.