Classification of tolerable/intolerable mucosal toxicity of head-and-neck radiotherapy schedules with a biomathematical model of cell dynamics

Purpose: The purpose of this study is to present a biomathematical model based on the dynamics of cell populations to predict the tolerability/intolerability of mucosal toxicity in head-and-neck radiotherapy. Methods and Materials: Our model is based on the dynamics of proliferative and functional cell populations in irradiated mucosa, and incorporates the three As: Accelerated proliferation, loss of Asymmetric proliferation, and Abortive divisions. The model consists of a set of delay differential equations, and tolerability is based on the depletion of functional cells during treatment. We calculate the sensitivity (sen) and specificity (spe) of the model in a dataset of 108 radiotherapy schedules, and compare the results with those obtained with three phenomenological classification models, two based on a biologically effective dose (BED) function describing the tolerability boundary (Fowler and Fenwick) and one based on an equivalent dose in 2 Gy fractions (EQD2) boundary (Strigari). We also perform a machine learning-like cross-validation of all the models, splitting the database in two, one for training and one for validation. Results: When fitting our model to the whole dataset, we obtain predictive values (sen + spe) up to 1.824. The predictive value of our model is very similar to that of the phenomenological models of Fowler (1.785), Fenwick (1.806), and Strigari (1.774). When performing a k = 2 cross-validation, the specificity and sensitivity in the validation dataset decrease for all models, from ˜1.82 to ˜1.55–1.63. For Fowler, the worsening is higher, down to 1.49. Conclusions: Our model has proved useful to predict the tolerability/intolerability of a dataset of 108 schedules. As the model is more mechanistic than other available models, it could prove helpful when designing unconventional dose fractionations, schedules not covered by datasets to which phenomenological models of toxicity have been fitted

A mathematical model of dynamics of cell populations in squamous epithelium after irradiation

Purpose: To develop multi-compartment mechanistic models of dynamics of stem and functional cell populations in epithelium after irradiation. Methods and materials: We present two models, with three (3C) and four (4C) compartments respectively. We use delay differential equations, and include accelerated proliferation, loss of division asymmetry, progressive death of abortive stem cells, and turnover of functional cells. The models are used to fit experimental data on the variations of the number of cells in mice mucosa after irradiation with 13 Gy and 20 Gy. Akaike information criteria (AIC) was used to evaluate the performance of each model. Results: Both 3C and 4C models provide good fits to experimental data for 13 Gy. Fits for 20 Gy are slightly poorer and may be affected by larger uncertainties and fluctuations of experimental data. Best fits are obtained by imposing constraints on the fitting parameters, so to have values that are within experimental ranges. There is some degeneration in the fits, as different sets of parameters provide similarly good fits. Conclusions: The models provide good fits to experimental data. Mechanistic approaches like this can facilitate the development of mucositis response models to nonstandard schedules/treatment combinations not covered by datasets to which phenomenological models have been fitted. Studying the dynamics of cell populations in multifraction treatments, and finding links with induced toxicity, is the next step of this work

A Biomathematical Model of Tumor Response to Radioimmunotherapy with PDL1 and CTLA4

There is evidence of synergy between radiotherapy and immunotherapy. Radiotherapy can increase liberation of tumor antigens, causing activation of antitumor T-cells. This effect can be boosted with immunotherapy. Radioimmunotherapy has potential to increase tumor control rates. Biomathematical models of response to radioimmunotherapy may help on understanding of the mechanisms affecting response, and assist clinicians on the design of optimal treatment strategies. In this work we present a biomathematical model of tumor response to radioimmunotherapy. The model uses the linear-quadratic response of tumor cells to radiation (or variation of it), and builds on previous developments to include the radiation-induced immune effect. We have focused this study on the combined effect of radiotherapy and PDL1/CTLA4 therapies. The model can fit preclinical data of volume dynamics and control obtained with different dose fractionations and PDL1/CTLA4. A biomathematical study of optimal combination strategies suggests that a good understanding of the involved biological delays, the biokinetics of the immunotherapy drug, and the interplay between them, may be of paramount importance to design optimal radioimmunotherapy schedules. Biomathematical models like the one we present can help to interpret experimental data on the synergy between radiotherapy and immunotherapy, and to assist in the design of more effective treatments

Upstream kinases of plant SnRKs are involved in salt stress tolerance

Sucrose-Non-Fermenting1-related protein kinases (SnRKs) are important for plant growth and stress responses. This family has three clades: SnRK1, SnRK2, and SnRK3. Although plant SnRKs are thought to be activated by upstream kinases, the overall mechanism remains obscure. Geminivirus Rep-Interacting Kinase (GRIK)1 and GRIK2 phosphorylate SnRK1s, which are involved in sugar/energy sensing, and the grik1-1 grik2-1 double mutant shows growth retardation under regular growth conditions. In this study, we established another Arabidopsis mutant line harbouring a different allele of gene GRIK1 (grik1-2 grik2-1) that grows similarly to the wild type, enabling us to evaluate the function of GRIKs under stress conditions. In the grik1-2 grik2-1 double mutant, phosphorylation of SnRK1.1 was reduced, but not eliminated, suggesting that the grik1-2 mutation is a weak allele. In addition to high sensitivity to glucose, the grik1-2 grik2-1 mutant was sensitive to high salt, indicating that GRIKs are also involved in salinity signalling pathways. Salt Overly Sensitive (SOS)2, a member of the SnRK3 subfamily, is a critical mediator of the response to salinity. GRIK1 phosphorylated SOS2 in vitro, resulting in elevated kinase activity of SOS2. The salt tolerance of sos2 was restored to normal levels by wild-type SOS2, but not by a mutated form of SOS2 lacking the T168 residue phosphorylated by GRIK1. Activation of SOS2 by GRIK1 was also demonstrated in a reconstituted system in yeast. Our results indicate that GRIKs phosphorylate and activate SnRK1 and other members of the SnRK3 family and that they play important roles in multiple signalling pathways in vivo. This article is protected by copyright. All rights reserved

A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

Embryonic stem (ES) cell self-renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self-renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP-Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple-repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal

High-throughput in vivo vertebrate screening

We demonstrate a high-throughput platform for cellular-resolution in vivo chemical and genetic screens on zebrafish larvae. The system automatically loads zebrafish from reservoirs or multiwell plates, and positions and rotates them for high-speed confocal imaging and laser manipulation of both superficial and deep organs within 19 s without damage. We performed small-scale test screening of retinal axon guidance mutants and neuronal regeneration assays in combination with femtosecond laser microsurgery.National Institutes of Health (U.S.) (Director’s Innovator Award 1-DP2-OD002989–01)David & Lucile Packard Foundation (Award in Science and Engineering)Alfred P. Sloan Foundation (Award)Broad Institute of MIT and Harvard (Sparc Grant)National Science Foundation (U.S.) (Fellowship)Foxconn (Sponsorship

Dual isotope analyses indicate efficient processing of atmospheric nitrate by forested watersheds in the northeastern U.S.

Author Posting. © Springer, 2008. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Biogeochemistry 90 (2008): 15-27, doi:10.1007/s10533-008-9227-2.Nitrogen from atmospheric deposition serves as the dominant source of new nitrogen to forested ecosystems in the northeastern U.S.. By combining isotopic data obtained using the denitrifier method, with chemistry and hydrology measurements we determined the relative importance of sources and control mechanisms on nitrate (NO3-) export from five forested watersheds in the Connecticut River watershed. Microbially produced NO3- was the dominant source (82-100%) of NO3- to the sampled streams as indicated by the δ15N and δ18O of NO3-. Seasonal variations in the δ18O-NO3- in streamwater are controlled by shifting hydrology and temperature affects on biotic processing, resulting in a relative increase in unprocessed NO3- export during winter months. Mass balance estimates find that the unprocessed atmospherically derived NO3- stream flux represents less than 3% of the atmospherically delivered wet NO3- flux to the region. This suggests that despite chronically elevated nitrogen deposition these forests are not nitrogen saturated and are retaining, removing, and reprocessing the vast majority of NO3- delivered to them throughout the year. These results confirm previous work within Northeastern U.S. forests and extend observations to watersheds not dominated by a snow-melt driven hydrology. In contrast to previous work, unprocessed atmospherically derived NO3- export is associated with the period of high recharge and low biotic activity as opposed to spring snowmelt and other large runoff events.This work was funded by an EPA STAR Fellowship (FP-91637501-1) and a grant from QLF/The Sound Conservancy to RTB

Effects of long-term low-dose oxygen supplementation on the epithelial function, collagen metabolism and interstitial fibrogenesis in the guinea pig lung

<p>Abstract</p> <p>Background</p> <p>The patient population receiving long-term oxygen therapy has increased with the rising morbidity of COPD. Although high-dose oxygen induces pulmonary edema and interstitial fibrosis, potential lung injury caused by long-term exposure to low-dose oxygen has not been fully analyzed. This study was designed to clarify the effects of long-term low-dose oxygen inhalation on pulmonary epithelial function, edema formation, collagen metabolism, and alveolar fibrosis.</p> <p>Methods</p> <p>Guinea pigs (n = 159) were exposed to either 21% or 40% oxygen for a maximum of 16 weeks, and to 90% oxygen for a maximum of 120 hours. Clearance of inhaled technetium-labeled diethylene triamine pentaacetate (Tc-DTPA) and bronchoalveolar lavage fluid-to-serum ratio (BAL/Serum) of albumin (ALB) were used as markers of epithelial permeability. Lung wet-to-dry weight ratio (W/D) was measured to evaluate pulmonary edema, and types I and III collagenolytic activities and hydroxyproline content in the lung were analyzed as indices of collagen metabolism. Pulmonary fibrotic state was evaluated by histological quantification of fibrous tissue area stained with aniline blue.</p> <p>Results</p> <p>The clearance of Tc-DTPA was higher with 2 week exposure to 40% oxygen, while BAL/Serum Alb and W/D did not differ between the 40% and 21% groups. In the 40% oxygen group, type I collagenolytic activities at 2 and 4 weeks and type III collagenolytic activity at 2 weeks were increased. Hydroxyproline and fibrous tissue area were also increased at 2 weeks. No discernible injury was histologically observed in the 40% group, while progressive alveolar damage was observed in the 90% group.</p> <p>Conclusion</p> <p>These results indicate that epithelial function is damaged, collagen metabolism is affected, and both breakdown of collagen fibrils and fibrogenesis are transiently induced even with low-dose 40% oxygen exposure. However, these changes are successfully compensated even with continuous exposure to low-dose oxygen. We conclude that long-term low-dose oxygen exposure does not significantly induce permanent lung injury in guinea pigs.</p

Perforin Rapidly Induces Plasma Membrane Phospholipid Flip-Flop

The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes) from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN) and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm) when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB) treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells