Multiplex transcriptional analysis of paraffin-embedded liver needle biopsy from patients with liver fibrosis

BACKGROUND: The possibility of extracting RNA and measuring RNA expression from paraffin sections can allow extensive investigations on stored paraffin samples obtained from diseased livers and could help with studies of the natural history of liver fibrosis and inflammation, and in particular, correlate basic mechanisms to clinical outcomes. RESULTS: To address this issue, a pilot study of multiplex gene expression using branched-chain DNA technology was conducted to directly measure mRNA expression in formalin-fixed paraffin-embedded needle biopsy samples of human liver. Twenty-five genes were selected for evaluation based on evidence obtained from human fibrotic liver, a rat BDL model and in vitro cultures of immortalized human hepatic stellate cells. The expression levels of these 25 genes were then correlated with liver fibrosis and inflammation activity scores. Statistical analysis revealed that three genes (COL3A1, KRT18, and TUBB) could separate fibrotic from non-fibrotic samples and that the expression of ten genes (ANXA2, TIMP1, CTGF, COL4A1, KRT18, COL1A1, COL3A1, ACTA2, TGFB1, LOXL2) were positively correlated with the level of liver inflammation activity. CONCLUSION: This is the first report describing this multiplex technique for liver fibrosis and has provided the proof of concept of the suitability of RNA extracted from paraffin sections for investigating the modulation of a panel of proinflammatory and profibrogenic genes. This pilot study suggests that this technique will allow extensive investigations on paraffin samples from diseased livers and possibly from any other tissue. Using identical or other genes, this multiplex expression technique could be applied to samples obtained from extensive patient cohorts with stored paraffin samples in order to correlate gene expression with valuable clinically relevant information. This method could be used to provide a better understanding of the mechanisms of liver fibrosis and inflammation, its progression, and help development of new therapeutic approaches for this indication

Insulin Sensitizing Pharmacology of Thiazolidinediones Correlates with Mitochondrial Gene Expression rather than Activation of PPARγ

Insulin sensitizing thiazolidinediones (TZDs) are generally considered to work as agonists for the nuclear receptor peroxisome proliferative activated receptor-gamma (PPARγ). However, TZDs also have acute, non-genomic metabolic effects and it is unclear which actions are responsible for the beneficial pharmacology of these compounds. We have taken advantage of an analog, based on the metabolism of pioglitazone, which has much reduced ability to activate PPARγ. This analog (PNU-91325) was compared to rosiglitazone, the most potent PPARγ activator approved for human use, in a variety of studies both in vitro and in vivo. The data demonstrate that PNU-91325 is indeed much less effective than rosiglitazone at activating PPARγ both in vitro and in vivo. In contrast, both compounds bound similarly to a mitochondrial binding site and acutely activated PI-3 kinase-directed phosphorylation of AKT, an action that was not affected by elimination of PPARγ activation. The two compounds were then compared in vivo in both normal C57 mice and diabetic KKAy mice to determine whether their pharmacology correlated with biomarkers of PPARγ activation or with the expression of other gene transcripts. As expected from previous studies, both compounds improved insulin sensitivity in the diabetic mice, and this occurred in spite of the fact that there was little increase in expression of the classic PPARγ target biomarker adipocyte binding protein-2 (aP2) with PNU-91325 under these conditions. An examination of transcriptional profiling of key target tissues from mice treated for one week with both compounds demonstrated that the relative pharmacology of the two thiazolidinediones correlated best with an increased expression of an array of mitochondrial proteins and with expression of PPARγ coactivator 1-alpha (PGC1α), the master regulator of mitochondrial biogenesis. Thus, important pharmacology of the insulin sensitizing TZDs may involve acute actions, perhaps on the mitochondria, that are independent of direct activation of the nuclear receptor PPARγ. These findings suggest a potential alternative route to the discovery of novel insulin sensitizing drugs

The TropD software package (v1): standardized methods for calculating tropical-width diagnostics

Observational and modeling studies suggest that Earth's tropical belt has widened over the late 20th century and will continue to widen throughout the 21st century. Yet, estimates of tropical-width variations differ significantly across studies. This uncertainty, to an unknown degree, is partly due to the large variety of methods used in studies of the tropical width. Here, methods for eight commonly used metrics of the tropical width are implemented in the Tropical-width Diagnostics (TropD) code package in the MATLAB programming language. To consolidate the various methods, the operations used in each of the implemented methods are reduced to two basic calculations: finding the latitude of a zero crossing and finding the latitude of a maximum. A detailed description of the methods implemented in the code and of the code syntax is provided, followed by a method sensitivity analysis for each of the metrics. The analysis provides information on how to reduce the methodological component of the uncertainty associated with fundamental aspects of the calculations, such as monthly vs. seasonal averaging biases, grid dependence, sensitivity to noise, and sensitivity to threshold criteria

Selective modification of recombinant bovine placental lactogen by site-directed mutagenesis at its C terminus

International audienc

Tropical Widening: From Global Variations to Regional Impacts

AbstractOver the past 15 years, numerous studies have suggested that the sinking branches of Earth’s Hadley circulation and the associated subtropical dry zones have shifted poleward over the late twentieth century and early twenty-first century. Early estimates of this tropical widening from satellite observations and reanalyses varied from 0.25° to 3° latitude per decade, while estimates from global climate models show widening at the lower end of the observed range. In 2016, two working groups, the U.S. Climate Variability and Predictability (CLIVAR) working group on the Changing Width of the Tropical Belt and the International Space Science Institute (ISSI) Tropical Width Diagnostics Intercomparison Project, were formed to synthesize current understanding of the magnitude, causes, and impacts of the recent tropical widening evident in observations. These working groups concluded that the large rates of observed tropical widening noted by earlier studies resulted from their use of metrics that poorly capture changes in the Hadley circulation, or from the use of reanalyses that contained spurious trends. Accounting for these issues reduces the range of observed expansion rates to 0.25°–0.5° latitude decade‒1—within the range from model simulations. Models indicate that most of the recent Northern Hemisphere tropical widening is consistent with natural variability, whereas increasing greenhouse gases and decreasing stratospheric ozone likely played an important role in Southern Hemisphere widening. Whatever the cause or rate of expansion, understanding the regional impacts of tropical widening requires additional work, as different forcings can produce different regional patterns of widening.</jats:p