Data integration methodology that couples novel bioreactor monitoring tools, automated sampling, and applied mathematics to redefine bioproduction processes

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Botulinum toxin A-induced muscle paralysis stimulates Hdac4 and differential miRNA expression.

At sufficient dose, intramuscular injection of Botulinum toxin A causes muscle wasting that is physiologically consistent with surgical denervation and other types of neuromuscular dysfunction. The aim of this study was to clarify early molecular and micro-RNA alterations in skeletal muscle following Botulinum toxin A-induced muscle paralysis. Quadriceps were analyzed for changes in expression of micro- and messenger RNA and protein levels after a single injection of 0.4, 2 or 4U Botulinum toxin A (/100g body weight). After injection with 2.0U Botulinum toxin A, quadriceps exhibited significant reduction in muscle weight and increased levels of ubiquitin ligase proteins at 7, 14 and 28 days. Muscle miR-1 and miR-133a/b levels were decreased at these time points, whereas a dose-responsive increase in miR-206 expression at day 14 was observed. Expression of the miR-133a/b target genes RhoA, Tgfb1 and Ctfg, and the miR-1/206 target genes Igf-1 and Hdac4, were upregulated at 28 days after Botulinum toxin A injection. Increased levels of Hdac4 protein were observed after injection, consistent with anticipated expression changes in direct and indirect Hdac4 target genes, such as Myog. Our results suggest Botulinum toxin A-induced denervation of muscle shares molecular characteristics with surgical denervation and other types of neuromuscular dysfunction, and implicates miR-133/Tgf-β1/Ctfg and miR-1/Hdac4/Myog signaling during the resultant muscle atrophy

Systems-Based Identification of Temporal Processing Pathways during Bone Cell Mechanotransduction

<div><p>Bone has long been established to be a highly mechanosensitive tissue. When subjected to mechanical loading, bone exhibits profoundly different anabolic responses depending on the temporal pattern in which the stimulus is applied. This phenomenon has been termed temporal processing, and involves complex signal amplification mechanisms that are largely unidentified. In this study, our goal was to characterize transcriptomic perturbations arising from the insertion of intermittent rest periods (a temporal variation with profound effects on bone anabolism) in osteoblastic cells subjected to fluid flow, and assess the utility of these perturbations to identify signaling pathways that are differentially activated by this temporal variation. At the level of the genome, we found that the common and differential alterations in gene expression arising from the two flow conditions were distributionally distinct, with the differential alterations characterized by many small changes in a large number of genes. Using bioinformatics analysis, we identified distinct up- and down-regulation transcriptomic signatures associated with the insertion of rest intervals, and found that the up-regulation signature was significantly associated with MAPK signaling. Confirming the involvement of the MAPK pathway, we found that the insertion of rest intervals significantly elevated flow-induced p-ERK1/2 levels by enabling a second spike in activity that was not observed in response to continuous flow. Collectively, these studies are the first to characterize distinct transcriptomic perturbations in bone cells subjected to continuous and intermittent stimulation, and directly demonstrate the utility of systems-based transcriptomic analysis to identify novel acute signaling pathways underlying temporal processing in bone cells.</p></div

Pathways analysis of transcriptomic perturbations implicates MAPK signaling.

<p>(A–B): Results from KEGG analysis for Groups A and B. Group A was significantly associated with MAPK signaling (an intracellular signaling pathway) as well as two pathways associated with extracellular signal transduction: focal adhesion and cytokine-cytokine receptor interactions. Group B was significantly associated with cytokine-cytokine receptor interactions only. *Bayes Factor >6 (C–D): Results from SPEED analysis for Groups A and B. SPEED analysis revealed a significant overlap in Group A genes with the MAPK gene expression signature, and a significant overlap of Group B genes with the TNF-alpha pathway. *FDR <0.05.</p

Continuous and rest-inserted fluid flow give rise to acute differences in gene expression that are highly transient.

<p>(A) Panel of 20 genes screened for differential expression immediately following cessation of continuous or rest-inserted flow. Though most of the genes assessed did not exhibit significant alterations by the insertion of rest intervals, we observed two sub-clusters (highlighted in blue and yellow) that exhibited detectable differences in expression following rest-inserted and continuous flow. Of these six genes, four were found to be significantly different under the two flow conditions: OPG, RUNX2, DSCR1, and OPN. (B–D) Time course of gene expression for these four genes following exposure to one hour of continuous or rest-inserted flow. Differences in expression observed immediately following continuous or rest-inserted flow were highly transient and tended to be focused within the 0–1 h period follow cessation of the flow. *p<0.05, **p<0.01, ***p<0.001 for rest-inserted flow vs. continuous flow.</p

Rest intervals enhance flow-induced ERK1/2 activation by enabling recurring spikes in activity.

<p>(A) Western blot for p-ERK1/2 and ERK1/2 for cells subjected to no flow (NF), rest-inserted flow (RF), and continuous flow (CF) following cessation of each of the four flow bouts. (B) Quantification of Western blots from three separate experiments. Cells exposed to continuous flow exhibited a single spike in p-ERK1/2. In contrast, cells exposed to rest-inserted flow exhibited multiple spikes in p-ERK1/2 following each flow bout. We observed only modest increases in ERK1/2 activity after the second and fourth bouts but a significant increase after the third bout, suggesting that ERK1/2 may possess a refractory period of longer than 15 min but shorter than 45 min. *,**p<0.05, p<0.01 when compared to no flow at same time point, respectively. #p<0.05 when compared to continuous flow at same time point.</p

Establishment of Group A and B gene sets.

<p>(A) Visualization of Group A (black circles) and Group B (white circles), which consisted of genes with the top 1% of positive and negative values of y’ respectively. Physically, gene members of Group A and B were those that were the most differentially expressed and which were up-regulated and down-regulated in response to rest, respectively. (B–C) GO analysis results for Groups A and B. The most significant GO terms for Group A were associated with morphogenesis and developmental processes, while Group B terms were primarily associated with inflammatory and defense responses.</p

Temsirolimus with or without megestrol acetate and tamoxifen for endometrial cancer: a gynecologic oncology group study

OBJECTIVES: To determine the response, toxicities, and progression free survival of a regimen of temsirolimus with or without hormonal therapy in the treatment of advanced, or recurrent endometrial carcinoma. BACKGROUND: Preclinical evidence suggested that blockade of the PI3K/AKT/mTOR pathway might overcome resistance to hormonal therapy. METHODS: We performed a randomized phase II trial of intravenous temsirolimus 25mg weekly versus the combination of weekly temsirolimus with a regimen of megestrol acetate 80 mg bid for three weeks alternating with tamoxifen 20mg bid for three weeks in women with recurrent or metastatic endometrial carcinoma. RESULTS: There were 71 eligible patients who received at least one dose of therapy with 21 of these treated on the combination arm which was closed early because of an excess of venous thrombosis, with 5 episodes of deep venous thrombosis (DVT) and 2 pulmonary emboli. There were three responses observed in that arm (14%). A total of 50 eligible patients were treated on the single agent arm with 3 episodes of DVT and 11 responses (22%). Response rates were similar in patients with prior chemotherapy (7 of 29; 24%) and those with no prior chemotherapy (4 of 21; 19%). Two of four patients with clear cell carcinoma responded. CONCLUSIONS: Adding the combination of megestrol acetate and tamoxifen to temsirolimus therapy did not enhance activity and the combination was associated with an excess of venous thrombosis. Temsirolimus activity was preserved in patients with prior adjuvant chemotherapy

Genome-wide gene expression profiling reveals transcriptomic variations arising from rest-inserted flow.

<p>(A) Microarray gene expression data plotted in (x,y) space. Genes were distributed within an approximately elliptical-shaped region with major axis aligned parallel to y = x. The quantities x’ and y’ (inset) provide measures of common and differential gene expression alterations under the two flow conditions, respectively. (B–C) Density distributions for |x| and |y| (black and red lines respectively in B) and |x’| and |y’| (black and red lines respectively in C). (D) Relative distributions for |y|/|x| (black) |y’|/|x’| (red). For |y|/|x|, the distribution was essentially uniform and equal to one. This is in contrast to the relative distribution for |y’|/|x’|, which exhibited an inverse relation between relative density and quantile.</p