Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and often fatal disorder. Two U.S. Food and Drug Administration-approved antifibrotic drugs, nintedanib and pirfenidone, slow the rate ...of decline in lung function, but responses are variable and side effects are common.
Using an
data-driven approach, we identified a robust connection between the transcriptomic perturbations in IPF disease and those induced by saracatinib, a selective Src kinase inhibitor originally developed for oncological indications. Based on these observations, we hypothesized that saracatinib would be effective at attenuating pulmonary fibrosis.
We investigated the antifibrotic efficacy of saracatinib relative to nintedanib and pirfenidone in three preclinical models:
)
in normal human lung fibroblasts;
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in bleomycin and recombinant Ad-TGF-β (adenovirus transforming growth factor-β) murine models of pulmonary fibrosis; and
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in mice and human precision-cut lung slices from these two murine models as well as patients with IPF and healthy donors.
In each model, the effectiveness of saracatinib in blocking fibrogenic responses was equal or superior to nintedanib and pirfenidone. Transcriptomic analyses of TGF-β-stimulated normal human lung fibroblasts identified specific gene sets associated with fibrosis, including epithelial-mesenchymal transition, TGF-β, and WNT signaling that was uniquely altered by saracatinib. Transcriptomic analysis of whole-lung extracts from the two animal models of pulmonary fibrosis revealed that saracatinib reverted many fibrogenic pathways, including epithelial-mesenchymal transition, immune responses, and extracellular matrix organization. Amelioration of fibrosis and inflammatory cascades in human precision-cut lung slices confirmed the potential therapeutic efficacy of saracatinib in human lung fibrosis.
These studies identify novel Src-dependent fibrogenic pathways and support the study of the therapeutic effectiveness of saracatinib in IPF treatment.
IL-1 family members are central mediators of host defense. In this article, we show that the novel IL-1 family member IL-36γ was expressed during experimental colitis and human inflammatory bowel ...disease. Germ-free mice failed to induce IL-36γ in response to dextran sodium sulfate (DSS)-induced damage, suggesting that gut microbiota are involved in its induction. Surprisingly, IL-36R-deficient (Il1rl2(-/-)) mice exhibited defective recovery following DSS-induced damage and impaired closure of colonic mucosal biopsy wounds, which coincided with impaired neutrophil accumulation in the wound bed. Failure of Il1rl2(-/-) mice to recover from DSS-induced damage was associated with a profound reduction in IL-22 expression, particularly by colonic neutrophils. Defective recovery of Il1rl2(-/-) mice could be rescued by an aryl hydrocarbon receptor agonist, which was sufficient to restore IL-22 expression and promote full recovery from DSS-induced damage. These findings implicate the IL-36/IL-36R axis in the resolution of intestinal mucosal wounds.
Glucocorticoids are the most frequently used anti-inflammatory drugs in dermatology. However, the molecular signature of glucocorticoids and their receptor in human skin is largely unknown. Our ...validated bioinformatics analysis of human skin transcriptome induced by topical glucocorticoid clobetasol propionate (CBP) in healthy volunteers identified numerous unreported glucocorticoid-responsive genes, including over a thousand noncoding RNAs. We observed sexual and racial dimorphism in the CBP response including a shift toward IFN-α/IFN-γ and IL-6/Jak/Signal transducer and activator of transcription (STAT) 3 signaling in female skin; and a larger response to CBP in African-American skin. Weighted gene coexpression network analysis unveiled a dense skin network of 41 transcription factors including circadian Kruppel-like factor 9 (KLF9), and ∼260 of their target genes enriched for functional pathways representative of the entire CBP transcriptome. Using keratinocytes with Kruppel-like factor 9 knockdown, we revealed a feedforward loop in glucocorticoid receptor signaling, previously unreported. Interestingly, many of the CBP-regulated transcription factors were involved in the control of development, metabolism, circadian clock; and 80% of them were associated with skin aging showing similarities between glucocorticoid-treated and aged skin. Overall, these findings indicate that glucocorticoid receptor acts as an important regulator of gene expression in skin—both at the transcriptional and posttranscriptional level—via multiple mechanisms including regulation of noncoding RNAs and multiple core transcription factors.
The hematologic and metabolic benefits of high altitude exposure have been extensively studied in athletes due to their promising performance enhancing effects. However, despite the increased ...research and development of various high altitude protocols for achieving peak performance, the reproducibility of the results at the individual level remains sparse. To systematically address this limitation and establish a more effective method to achieve consistent results at the individual level, we conducted a multi-dimensional study of one elite endurance athlete in two Phases. In Phase 1, we applied the standard protocol of LHTH (Live-High-Train-High) using a commercially available, at-home, normobaric, high altitude simulation tent under the SHTL (Sleep-High-Train-Low) model. Then, we developed the athlete's personalized protocol for peak hematologic parameters during their off-season. This protocol determined the exact total high altitude exposure time required to achieve peak hematologic parameters, which in the case of this athlete, amounted to 45 nights with approximately 8hrs per night. In Phase 2, we replicated the Phase 1 protocol during the athlete's in-season and observed the same or even higher hematologic and metabolic benefits compared to Phase 1. During both phases, we collected thousands of multi-dimensional data points to ensure that the athlete's lifestyle and environmental factors remained stable, and to increase the likelihood that physiological changes resulted primarily from the high altitude exposure. The data trends in both Phases validated that, for this athlete, hematologic measures such as red blood cell count, hematocrit, and hemoglobin, as well as electrolyte content, body weight and gut microbiome composition improved to their personal best values after a total of approximately 15 days of high altitude exposure (45 nights with roughly 8hrs per night totaling 360hrs or 15days). These improvements did not occur after the 21 days recommended by the LHTH protocol highlighting the significance of personalization in high altitude protocols that are designed for peak performance parameters. Therefore, to maximize the benefits in hematologic and other metabolic values and thus increase muscle oxygen supply and peak aerobic capacity through high altitude exposure, each athlete may require a unique total duration of high altitude exposure tailored to their individual physiology. This duration must be determined by their specific response in hematologic peaking. Therefore, initially establishing a personalized protocol for an athlete by determining their required total duration of high altitude exposure for peak hematologic values during their off-season and applying this protocol during their in-season phase may lead to more successful and reproducible benefits compared to following a generalized protocol alone.
While a range of methods for stool collection exist, many require complicated, self-directed protocols and stool transfer. In this study, we introduce and validate a novel, wipe-based approach to ...fecal sample collection and stabilization for metagenomics analysis. A total of 72 samples were collected across four different preservation types: freezing at -20°C, room temperature storage, a commercial DNA preservation kit, and a dissolvable wipe used with DESS (dimethyl sulfoxide, ethylenediaminetetraacetic acid, sodium chloride) solution. These samples were sequenced and analyzed for taxonomic abundance metrics, bacterial metabolic pathway classification, and diversity analysis. Overall, the DESS wipe results validated the use of a wipe-based capture method to collect stool samples for microbiome analysis, showing an R
2
of 0.96 for species across all kingdoms, as well as exhibiting a maintenance of Shannon diversity (3.1-3.3) and species richness (151-159) compared to frozen samples. Moreover, DESS showed comparable performance to the commercially available preservation kit (R
2
of 0.98), and samples consistently clustered by subject across each method. These data support that the DESS wipe method can be used for stable, room temperature collection and transport of human stool specimens.
Manganese (Mn) is an essential trace element, which also causes neurotoxicity in exposed occupational workers. Mn causes mitochondrial toxicity; however, little is known about transcriptional ...responses discriminated by physiological and toxicological levels of Mn. Identification of such mechanisms could provide means to evaluate risk of Mn toxicity and also potential avenues to protect against adverse effects. To study the Mn dose-response effects on transcription, analyzed by RNA-Seq, we used human SH-SY5Y neuroblastoma cells exposed for 5 h to Mn (0 to 100 μM), a time point where no immediate cell death occurred at any of the doses. Results showed widespread effects on abundance of protein-coding genes for metabolism of reactive oxygen species, energy sensing, glycolysis, and protein homeostasis including the unfolded protein response and transcriptional regulation. Exposure to a concentration (10 μM Mn for 5 h) that did not result in cell death after 24-h increased abundance of differentially expressed genes (DEGs) in the protein secretion pathway that function in protein trafficking and cellular homeostasis. These include
(Golgi vesicular membrane-trafficking protein),
(ADAM metallopeptidase domain 10), and
(ADP-ribosylation factor GTPase-activating protein 3). In contrast, 5-h exposure to 100 μM Mn, a concentration that caused cell death after 24 h, increased abundance of DEGs for components of the mitochondrial oxidative phosphorylation pathway. Integrated pathway analysis results showed that protein secretion gene set was associated with amino acid metabolites in response to 10 μM Mn, while oxidative phosphorylation gene set was associated with energy, lipid, and neurotransmitter metabolites at 100 μM Mn. These results show that differential effects of Mn occur at a concentration which does not cause subsequent cell death compared to a concentration that causes subsequent cell death. If these responses translate to effects on the secretory pathway and mitochondrial functions
, differential activities of these systems could provide a sensitive basis to discriminate sub-toxic and toxic environmental and occupational Mn exposures.
While metastasis ranks among the most lethal of all cancer-associated processes, on the molecular level, it remains one of the least well understood. One model that has gained credibility in recent ...years is that metastasizing cells at least partially recapitulate the developmental process of epithelial-to-mesenchymal transition (EMT) in their transit from primary to metastatic sites. While experimentally supported by cell culture and animal model studies, the lack of unambiguous confirmatory evidence in cancer patients has led to persistent challenges to the model's relevance in humans.
Gene expression profiling (Affymetrix, U133) was carried out on 14 matched sets of primary (ovary) and metastatic (omentum) ovarian cancer (serous adenocarcinoma) patient samples. Hierarchical clustering and functional pathway algorithms were used in the data analysis.
While histological examination reveled no morphological distinction between the matched sets of primary and metastatic samples, gene expression profiling clearly distinguished two classes of metastatic samples. One class displayed expression patterns statistically indistinguishable from primary samples isolated from the same patients while a second class displayed expression patterns significantly different from primary samples. Further analyses focusing on genes previously associated with EMT clearly distinguished the primary from metastatic samples in all but one patient.
Our results are consistent with a role of EMT in most if not all ovarian cancer metastases and demonstrate that identical morphologies between primary and metastatic cancer samples is insufficient evidence to negate a role of EMT in the metastatic process.
Expression levels of the miR-200 family of miRNAs are significantly reduced during the epithelial-to-mesenchymal transition (EMT) and consequent metastasis of ovarian and other cancers. Consistently, ...ectopic over-expression of miR-200 family miRNAs in mesenchymal-like cells reverses the process by converting treated cells to an epithelial phenotype, thereby reducing invasiveness and increasing sensitivity to chemotherapeutic drugs. To better understand the dynamics and molecular processes underlying miRNA-induced mesenchymal-to mesenchymal transition (MET), a time-course study was conducted where miRNA-induced morphological and molecular changes associated with MET were monitored over a period of 144 h. Morphological transition from an elongated mesenchymal-like to a cuboidal epithelial-like phenotype is maximized at 48 h with cells returning to the elongated phenotype by 144 h. Changes in the expression of >3000 genes, including many previously associated with epithelial-to-mesenchymal transition (EMT), are most pronounced at 48 h, and approach starting levels of expression by 144 h. The majority of these genes are not direct targets of miR-429. Targeted (siRNA) inhibition of key miR-429 regulated genes previously implicated as drivers of EMT/MET, do not recapitulate miR-429 induced MET indicating that the underlying molecular processes are complex.
•miR-429 induces mesenchymal-to-epithelial transition (MET) in ovarian cancer cells.•Most of the >3000 differentially expressed genes are indirect targets of miR-429.•miR-429 induced MET involves multiple pathways previously associated with MET.•Knockdown of genes associated with MET do not recapitulate miR-429 induced MET.
The conditions under which plasmids are predicted to persist remain controversial. Here, we reevaluate the ordinary differential equations used previously to model plasmid persistence and conclude ...that the parameter space required for maintenance is far less stringent than has been supposed. Strikingly, our model demonstrates that purely parasitic plasmids may persist, even in the absence of heterogeneity in the host population, and that this persistence is expressed by oscillations or damped oscillations between the plasmid-bearing and the plasmid-free class.
There is a growing body of evidence that targeted gene therapy holds great promise for the future treatment of cancer. A crucial step in this therapy is the accurate identification of appropriate ...candidate genes/pathways for targeted treatment. One approach is to identify variant genes/pathways that are significantly enriched in groups of afflicted individuals relative to control subjects. However, if there are multiple molecular pathways to the same cancer, the molecular determinants of the disease may be heterogeneous among individuals and possibly go undetected by group analyses.
In an effort to explore this question in pancreatic cancer, we compared the most significantly differentially expressed genes/pathways between cancer and control patient samples as determined by group versus personalized analyses.
We found little to no overlap between genes/pathways identified by gene expression profiling using group analyses relative to those identified by personalized analyses.
Our results indicate that personalized and not group molecular profiling is the most appropriate approach for the identification of putative candidates for targeted gene therapy of pancreatic and perhaps other cancers with heterogeneous molecular etiology.
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