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Drug-induced kidney injury, a major cause of acute kidney injury, results in progressive kidney disease and is linked to increased mortality in hospitalized patients. Primary injury sites of drug-induced kidney injury are proximal tubules. Clinically, kidney injury molecule-1, an established tubule-specific biomarker, is monitored to assess the presence and progression of injury. The ability to accurately predict drug-related nephrotoxicity preclinically would reduce patient burden and drug attrition rates, yet state-of-the-art in vitro and animal models fail to do so. In this study, we demonstrate the use of kidney injury molecule-1 measurement in the kidney microphysiological system as a preclinical model for drug toxicity assessment. To show clinical relevance, we use quantitative systems pharmacology computational models for in vitro-in vivo translation of the experimental results and to identify favorable dosing regimens for one of the tested drugs.

Mısır ıslahı ve yetiştiriciliğine yönelik araştırmalarda koçan ve tane özelliklerinin belirlenmesine yönelik öçümlere ihtiyaç duyulmaktadır. Geleneksel ölçümler oldukça fazla zaman alan ve fazla işgücü gerektiren yöntemelere dayanmaktadır. Son yıllarda bilgisayar bilimlerinin tarımsal araştırmalarda kullanımı oldukça yaygınlaşmış ve koçan/tane özelliklerinin görüntü analizlerine dayalı olarak belirleyebilen çeşitli alternatifler yazılımlar geliştirilmiştir. Bu çalışmada açıkta ve kontorllü tozlama yöntemleri ile 20 farklı genotipten elde edilen koçan örnekleri kullanılarak iki farklı yazılımın karşılaştırılması amaçlanmıştır. Çalışmada görüntü işleme yazılımı olarak SmartGrain ve Fiji seçenekleri denenmiştir. Koçan uzunluğu (cm), koçan çapı (cm), koçanda tane sayısı (adet), tane çevresi (mm), tane boyu (mm) ile ilgili görüntü işleme yazılımları ile alınan sonuçlar kıyaslanmıştır. Araştırma bulgularına göre mısırda koçan ve tane gelişimini önemli şekilde etkileyen tozlama yöntemlerinin ölçülen özelliklerden bazıları üzerine önemli etkilere sahip olduğu görülmüştür. Kullanılan genotip gruplarına göre tozlama yöntemlerinin etkileri değişkenlik göstermiştir. İncelenen özelliklerden koçanda tane sayısının her iki yazılım ile de mükemmel bir şekilde tespit edilebileceği (r=1), buna karşın tane uzunluğu ve tane çevresi gibi ölçümlerde yazılımlardan elde edilen sonuçlar arasındaki benzerliğin zayıf olduğu dikkat çekmiştir., In studies on maize breeding and cultivation, ear and kernel measurements are needed. Traditional measurements are based on methods that are very time consuming and labor intensive. In recent years, the use of computer science in agricultural research has become widespread and various alternative software has been developed that can determine the ear/kernel characteristics based on image analysis. In this study, it is aimed to compare two different softwares by using ear samples obtained from 20 different genotypes by open and controlled pollination methods. In the study, SmartGrain and Fiji options were tried as image processing software. The results obtained with the image processing software were compared regarding the ear length (cm), the ear diameter (cm), the number of kernels per ear (number), the kernel circumference (mm), the kernel lenght (mm). According to the findings of the research, it has been observed that pollination methods that significantly affect the ear and kernel development in maize have significant effects on some of the measured traits. The effects of pollination methods varied according to the genotype used. It was noted that the number of kernels per cob could be determined perfectly by both software (r=1), but the similarity between the results obtained from the software was weak in measurements such as grain lenght and grain circumference.

The development and application of quantitative systems pharmacology models in neuroscience have been modest relative to other fields, such as oncology and immunology, which may reflect the complexity of the brain. Technological and methodological advancements have enhanced the quantitative understanding of brain physiology and pathophysiology and the effects of pharmacological interventions. To maximize the knowledge gained from these novel data types, pharmacometrics modelers may need to expand their toolbox to include additional mathematical and statistical frameworks. A session was held at the 10th annual American Conference on Pharmacometrics (ACoP10) to highlight several recent advancements in quantitative and systems neuroscience. In this mini‐review, we provide a brief overview of technological and methodological advancements in the neuroscience therapeutic area that were discussed during the session and how these can be leveraged with quantitative systems pharmacology modeling to enhance our understanding of neurological diseases. Microphysiological systems using human induced pluripotent stem cells (IPSCs), digital biomarkers, and large‐scale imaging offer more clinically relevant experimental datasets, enhanced granularity, and a plethora of data to potentially improve the preclinical‐to‐clinical translation of therapeutics. Network neuroscience methodologies combined with quantitative systems models of neurodegenerative disease could help bridge the gap between cellular and molecular alterations and clinical end points through the integration of information on neural connectomics. Additional topics, such as the neuroimmune system, microbiome, single‐cell transcriptomic technologies, and digital device biomarkers, are discussed in brief.

Microphysiological systems (MPS) hold promise for improving therapeutic drug approval rates by providing more physiological, human-based, in vitro assays for preclinical drug development activities compared to traditional in vitro and animal models. Here, we first summarize why MPSs are needed in pharmaceutical development, and examine how MPS technologies can be utilized to improve preclinical efforts. We then provide the perspective that the full impact of MPS technologies will be realized only when robust approaches for in vitro-in vivo (MPS-to-human) translation are developed and utilized, and explain how the burgeoning field of quantitative systems pharmacology (QSP) can fill that need.

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.

Early assessment of adverse drug effects in humans is critical to avoid long-lasting harm. However, current approaches for early detection of adverse effects still lack predictive and organ-specific biomarkers to evaluate undesired responses in humans. Microphysiological systems (MPSs) are in vitro representations of human tissues and provide organ-specific translational insights for physiological processes. In this study, a brain MPS was utilized to assess molecular signatures of neurotoxic and non-neurotoxic compounds using targeted and untargeted molecular approaches. The brain MPS comprising of human embryonic stem (ES) cell-derived neural progenitor cells seeded on three-dimensional (3D), chemically defined, polyethylene glycol hydrogels was treated with the neurotoxic drug, bortezomib and the non-neurotoxic drug, tamoxifen over 14-days. Possible toxic effects were monitored with human N-acetylaspartic acid (NAA) kinetics, which correlates the neuronal function/health and DJ-1/PARK7, an oxidative stress biomarker. Changes in NAA levels were observed as early as 2-days post-bortezomib treatment, while onset detection of oxidative stress (DJ-1) was delayed until 4-days post-treatment. Separately, the untargeted extracellular metabolomics approach revealed distinct fingerprints 2-days post-bortezomib treatment as perturbations in cysteine and glycerophospholipid metabolic pathways. These results suggest accumulation of reactive oxygen species associated with oxidative stress, and disruption of membrane structure and integrity. The NAA response was strongly correlated with changes in a subset of the detected metabolites at the same time point 2-days post-treatment. Moreover, these metabolite changes correlated strongly with DJ-1 levels measured at the later time point (4-days post-treatment). This suggests that early cellular metabolic dysfunction leads to later DJ-1 leakage and cell death, and that early measurement of this subset of metabolites could predict the later occurrence of cell death. While the approach demonstrated here provides an individual case study for proof of concept, we suggest that this approach can be extended for preclinical toxicity screening and biomarker discovery studies.

Microphysiological systems (MPS) provide relevant physiological environments in vitro for studies of pharmacokinetics, pharmacodynamics and biological mechanisms for translational research. Designing multi-MPS platforms is essential to study multi-organ systems. Typical design approaches, including direct and allometric scaling, scale each MPS individually and are based on relative sizes not function. This study's aim was to develop a new multi-functional scaling approach for integrated multi-MPS platform design for specific applications. We developed an optimization approach using mechanistic modeling and specification of an objective that considered multiple MPS functions, e.g., drug absorption and metabolism, simultaneously to identify system design parameters. This approach informed the design of two hypothetical multi-MPS platforms consisting of gut and liver (multi-MPS platform I) and gut, liver and kidney (multi-MPS platform II) to recapitulate in vivo drug exposures in vitro. This allows establishment of clinically relevant drug exposure-response relationships, a prerequisite for efficacy and toxicology assessment. Design parameters resulting from multi-functional scaling were compared to designs based on direct and allometric scaling. Human plasma time-concentration profiles of eight drugs were used to inform the designs, and profiles of an additional five drugs were calculated to test the designed platforms on an independent set. Multi-functional scaling yielded exposure times in good agreement with in vivo data, while direct and allometric scaling approaches resulted in short exposure durations. Multi-functional scaling allows appropriate scaling from in vivo to in vitro of multi-MPS platforms, and in the cases studied provides designs that better mimic in vivo exposures than standard MPS scaling methods., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), National Institutes of Health (U.S.). Microphysiological Systems Program (4-UH3-TR000496-03)

In this work, we first describe the population variability in hepatic drug metabolism using cryopreserved hepatocytes from five different donors cultured in a perfused three-dimensional human liver microphysiological system, and then show how the resulting data can be integrated with a modeling and simulation framework to accomplish in vitro-in vivo translation. For each donor, metabolic depletion profiles of six compounds (phenacetin, diclofenac, lidocaine, ibuprofen, propranolol, and prednisolone) were measured, along with metabolite formation, mRNA levels of 90 metabolism-related genes, and markers of functional viability [lactate dehydrogenase (LDH) release, albumin, and urea production]. Drug depletion data were analyzed with mixed-effects modeling. Substantial interdonor variability was observed with respect to gene expression levels, drug metabolism, and other measured hepatocyte functions. Specifically, interdonor variability in intrinsic metabolic clearance ranged from 24.1% for phenacetin to 66.8% for propranolol (expressed as coefficient of variation). Albumin, urea, LDH, and cytochrome P450 mRNA levels were identified as significant predictors of in vitro metabolic clearance. Predicted clearance values from the liver microphysiological system were correlated with the observed in vivo values. A population physiologically based pharmacokinetic model was developed for lidocaine to illustrate the translation of the in vitro output to the observed pharmacokinetic variability in vivo. Stochastic simulations with this model successfully predicted the observed clinical concentration-time profiles and the associated population variability. This is the first study of population variability in drug metabolism in the context of a microphysiological system and has important implications for the use of these systems during the drug development process., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (Grant W911NF-12-2-0039), National Institutes of Health (U.S.). Microphysiological Systems Program (Grant 4-UH3-TR000496-03)

Microphysiological systems (MPS), consisting of tissue constructs, biomaterials, and culture media, aim to recapitulate relevant organ functions in vitro. MPS components are housed in fluidic hardware with operational protocols, such as periodic complete media replacement. Such batch-like operations provide relevant nutrients and remove waste products but also reset cell-secreted mediators (e.g. cytokines, hormones) and potentially limit exposure to drugs (and metabolites). While each component plays an essential role for tissue functionality, MPS-specific nutrient needs are not yet well-characterized nor utilized to operate MPSs at more physiologically-relevant conditions. MPS-specific nutrient needs for gut (immortalized cancer cells), liver (human primary hepatocytes) and cardiac (iPSC-derived cardiomyocytes) MPSs were experimentally quantified. In a long-term study of the gut MPS (10 days), this knowledge was used to design operational protocols to maintain glucose and lactate at desired levels. This quasi-steady state operation was experimentally validated by monitoring glucose and lactate as well as MPS functionality. In a theoretical study, nutrient needs of an integrated multi-MPS platform (gut, liver, cardiac MPSs) were computationally simulated to identify long-term quasi-steady state operations. This integrative experimental and computational approach demonstrates the utilization of quantitative multi-scale characterization of MPSs and incorporating MPS-specific information to establish more physiologically-relevant experimental operations., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), National Institutes of Health (U.S.) (U24TR001951), National Institutes of Health (U.S.). Microphysiological Systems Program (4-UH3-TR000496-03)

Microphysiological systems (MPSs) are in vitro models that capture facets of in vivo organ function through use of specialized culture microenvironments, including 3D matrices and microperfusion. Here, we report an approach to co-culture multiple different MPSs linked together physiologically on re-useable, open-system microfluidic platforms that are compatible with the quantitative study of a range of compounds, including lipophilic drugs. We describe three different platform designs – “4-way”, “7-way”, and “10-way” – each accommodating a mixing chamber and up to 4, 7, or 10 MPSs. Platforms accommodate multiple different MPS flow configurations, each with internal re-circulation to enhance molecular exchange, and feature on-board pneumatically-driven pumps with independently programmable flow rates to provide precise control over both intra- and inter-MPS flow partitioning and drug distribution. We first developed a 4-MPS system, showing accurate prediction of secreted liver protein distribution and 2-week maintenance of phenotypic markers. We then developed 7-MPS and 10-MPS platforms, demonstrating reliable, robust operation and maintenance of MPS phenotypic function for 3 weeks (7-way) and 4 weeks (10-way) of continuous interaction, as well as PK analysis of diclofenac metabolism. This study illustrates several generalizable design and operational principles for implementing multi-MPS “physiome-on-a-chip” approaches in drug discovery.

Scaling of a microphysiological system (MPS) or physiome-on-a-chip is arguably two interrelated, modeling-based activities: on-platform scaling and in vitro-in vivo translation. This dual approach reduces the need to perfectly rescale and mimic in vivo physiology, an aspiration that is both extremely challenging and not substantively meaningful because of uncertain relevance of any specific physiological condition. Accordingly, this perspective offers a tractable approach for designing interacting MPSs and relating in vitro results to analogous context in vivo., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (Grant W911NF-12-2-0039), National Institutes of Health (U.S.) Microphysiological Systems Program (Grant 4-UH3-TR000496-03), United States. Army Research Office (Institute for Collaborative Biotechnologies. Grant W911NF-09- 0001)

Our goal in developing Microphysiological Systems (MPS) technology is to provide an improved approach for more predictive preclinical drug discovery via a highly integrated experimental/computational paradigm. Success will require quantitative characterization of MPSs and mechanistic analysis of experimental findings sufficient to translate resulting insights from in vitro to in vivo. We describe herein a systems pharmacology approach to MPS development and utilization that incorporates more mechanistic detail than traditional pharmacokinetic/pharmacodynamic (PK/PD) models. A series of studies illustrates diverse facets of our approach. First, we demonstrate two case studies: a PK data analysis and an inflammation response––focused on a single MPS, the liver/immune MPS. Building on the single MPS modeling, a theoretical investigation of a four-MPS interactome then provides a quantitative way to consider several pharmacological concepts such as absorption, distribution, metabolism, and excretion in the design of multi-MPS interactome operation and experiments., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), National Institutes of Health (U.S.) Microphysiological Systems Program (4-UH3-TR000496-03), Massachusetts Institute of Technology. Center for Environmental Health Sciences (NIEHS Grant P30-ES002109)

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut-liver tissue interactions under normal and inflammatory contexts, via an integrative multi-organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long-term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut-liver crosstalk. Moreover, significant non-linear modulation of cytokine responses was observed under inflammatory gut-liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA-seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut-liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut-liver interaction also negatively affected tissue-specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi-tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk., National Institutes of Health (U.S.) (grant UH3TR00069), United States. Defense Advanced Research Projects Agency (grant Microphysiological Systems Program (W911NF-12-2-00))

Investigation of the pharmacokinetics (PK) of a compound is of significant importance during the early stages of drug development, and therefore several in vitro systems are routinely employed for this purpose. However, the need for more physiologically realistic in vitro models has recently fueled the emerging field of tissue-engineered 3D cultures, also referred to as organs-on-chips, or microphysiological systems (MPSs). We have developed a novel fluidic platform that interconnects multiple MPSs, allowing PK studies in multi-organ in vitro systems along with the collection of high-content quantitative data. This platform was employed here to integrate a gut and a liver MPS together in continuous communication, and investigate simultaneously different PK processes taking place after oral drug administration in humans (e.g., intestinal permeability, hepatic metabolism). Measurement of tissue-specific phenotypic metrics indicated that gut and liver MPSs can be fluidically coupled with circulating common medium without compromising their functionality. The PK of diclofenac and hydrocortisone was investigated under different experimental perturbations, and results illustrate the robustness of this integrated system for quantitative PK studies. Mechanistic model-based analysis of the obtained data allowed the derivation of the intrinsic parameters (e.g., permeability, metabolic clearance) associated with the PK processes taking place in each MPS. Although these processes were not substantially affected by the gut-liver interaction, our results indicate that inter-MPS communication can have a modulating effect (hepatic metabolism upregulation). We envision that our integrative approach, which combines multi-cellular tissue models, multi-MPS platforms, and quantitative mechanistic modeling, will have broad applicability in pre-clinical drug development., United States. Defense Advanced Research Projects Agency (Grant W911NF-12-2-0039), National Institutes of Health (U.S.) (Grant 4-UH3-TR000496-03)

“Mice are not little people” – a refrain becoming louder as the gaps between animal models and human disease become more apparent. At the same time, three emerging approaches are headed toward integration: powerful systems biology analysis of cell-cell and intracellular signaling networks in patient-derived samples; 3D tissue engineered models of human organ systems, often made from stem cells; and micro-fluidic and meso-fluidic devices that enable living systems to be sustained, perturbed and analyzed for weeks in culture. Integration of these rapidly moving fields has the potential to revolutionize development of therapeutics for complex, chronic diseases, including those that have weak genetic bases and substantial contributions from gene-environment interactions. Technical challenges in modeling complex diseases with “organs on chips” approaches include the need for relatively large tissue masses and organ-organ cross talk to capture systemic effects, such that current microfluidic formats often fail to capture the required scale and complexity for interconnected systems. These constraints drive development of new strategies for designing in vitro models, including perfusing organ models, as well as “mesofluidic” pumping and circulation in platforms connecting several organ systems, to achieve the appropriate physiological relevance.

Signal transduction networks in mammalian cells, comprising a limited set of interacting biochemical pathways, are accessed by various growth factor and cytokine receptors to elicit distinct cell responses. This raises the question as to how specificity of the stimulus–response relationship is encoded at the molecular level. It has been proposed that specificity arises not only from the activation of unique signalling pathways, but also from quantitative differences in the activation and regulation of shared receptor-proximal signalling proteins. To address such hypotheses, data sets with greater precision and coverage of experimental conditions will need to be acquired, and rigorous frameworks that codify and parameterize the inherently non-linear relationships among signalling activities will need to be developed. In the present study we apply a systematic approach combining quantitative measurements and mathematical modelling to compare the signalling networks accessed by FGF (fibroblast growth factor) and PDGF (platelet-derived growth factor) receptors in mouse fibroblasts, in which the ERK (extracellular-signal-regulated kinase) cascade is activated by Ras- and PI3K (phosphoinositide 3-kinase)-dependent pathways. We show that, whereas the FGF stimulation of PI3K signalling is relatively weak, this deficiency is compensated for by a more potent Ras-dependent activation of ERK. Thus, as the modelling would predict, the ERK pathway is activated to a greater extent in cells co-stimulated with FGF and PDGF, relative to the saturated levels achieved with either ligand alone. It is envisaged that similar approaches will prove valuable in the elucidation of quantitative differences among other closely related receptor signalling networks.

The extracellular signal‐regulated kinase (ERK) signaling pathway controls cell proliferation and differentiation in metazoans. Two hallmarks of its dynamics are adaptation of ERK phosphorylation, which has been linked to negative feedback, and nucleocytoplasmic shuttling, which allows active ERK to phosphorylate protein substrates in the nucleus and cytosol. To integrate these complex features, we acquired quantitative biochemical and live‐cell microscopy data to reconcile phosphorylation, localization, and activity states of ERK. While maximal growth factor stimulation elicits transient ERK phosphorylation and nuclear translocation responses, ERK activities available to phosphorylate substrates in the cytosol and nuclei show relatively little or no adaptation. Free ERK activity in the nucleus temporally lags the peak in nuclear translocation, indicating a slow process. Additional experiments, guided by kinetic modeling, show that this process is consistent with ERK's modification of and release from nuclear substrate anchors. Thus, adaptation of whole‐cell ERK phosphorylation is a by‐product of transient protection from phosphatases. Consistent with this interpretation, predictions concerning the dose‐dependence of the pathway response and its interruption by inhibition of MEK were experimentally confirmed.

Objective: This study aimed to compare the efficaciesof 3-day and 10-day courses of methylprednisolone (MP) treatment insevere COPD exacerbations necessitating hospitalization for respiratoryfailure. Design: Prospective, randomized, single-blindstudy. Settings: Tertiary-care center. Patients and methods: Thirty-six patients were included in the study and randomized into two groups: group 1 received MP, 0.5mg/kg q6h for 3 days, and group 2 was administered the same dosage of MP for the first 3 days, after which it was tapered and terminated onthe tenth day. There was no difference between the groups for age, baseline FEV 1 , Pa o 2 ,Pa co 2 , and pH levels. One patient in group 1who developed pneumothorax and one patient in group 2 who hadsteroid-related psychosis could not complete the study. Results: Both groups showed significant improvements inPa o 2 and FEV 1 levels, but thesewere more marked in group 2 (p = 0.012 and p = 0.019,respectively). There was a significant increase in FVC levels in group2 only (p = 0.003). Group 2 also had a more marked improvement indyspnea on exertion. There was no difference between the two groupswith regards to other parameters, including pH, Pa co 2 levels, and other symptom scores. Sixpatients in group 1 and five patients in group 2 developed newexacerbations with in the following 6 months. Hyperglycemia occurred intwo patients in each group. Conclusion: In severe COPDexacerbations, a 10-day course of steroid treatment is more effectivethan a 3-day course in improving the outcome, but has no benefit inreducing exacerbation rates.

Inhibition of the ubiquitin-proteasome protein degradation pathway has been identified as a viable strategy for anti-tumor therapy based on its broad effects on cell proliferation. By the same token, the variety of elicited effects confounds the interpretation of cell-based experiments using proteasome inhibitors such as MG132. It has been proposed that MG132 treatment reduces growth factor-stimulated phosphorylation of extracellular signal-regulated kinases (ERKs), at least in part through upregulation of dual specificity phosphatases (DUSPs). Here, we show that the effects of MG132 treatment on ERK signaling are more widespread, leading to a reduction in activation of the upstream kinase MEK. This suggests that MG132 systemically perturbs the intracellular phosphoproteome, impacting ERK signaling by reducing phosphorylation status at multiple levels of the kinase cascade.

Ras is a key signal transduction protein in the cell. Mutants of Gly(12) and Gln(61) impair GTPase activity and are found prominently in cancers. In wild type Ras-GTP, an allosteric switch promotes disorder to order transition in switch II, placing Gln(61) in the active site. We show that the "on" and "off" conformations of the allosteric switch can also be attained in RasG12V and RasQ61L. Although both mutants have similarly impaired active sites in the on state, RasQ61L stabilizes an anti-catalytic conformation of switch II in the off state of the allosteric switch when bound to Raf. This translates into more potent activation of the MAPK pathway involving Ras, Raf kinase, MEK, and ERK (Ras/Raf/MEK/ERK) in cells transfected with RasQ61L relative to RasG12V. This differential is not observed in the Raf-independent pathway involving Ras, phosphoinositide 3-kinase (PI3K), and Akt (Ras/PI3K/Akt). Using a combination of structural analysis, hydrolysis rates, and experiments in NIH-3T3 cells, we link the allosteric switch to the control of signaling in the Ras/Raf/MEK/ERK pathway, supporting a GTPase-activating protein-independent model for duration of the Ras-Raf complex.

Cell responses are actuated by tightly controlled signal transduction pathways. Although the concept of an integrated signaling network replete with interpathway cross-talk and feedback regulation is broadly appreciated, kinetic data of the type needed to characterize such interactions in conjunction with mathematical models are lacking. In mammalian cells, the Ras/ERK pathway controls cell proliferation and other responses stimulated by growth factors, and several cross-talk and feedback mechanisms affecting its activation have been identified. In this work, we take a systematic approach to parse the magnitudes of multiple regulatory mechanisms that attenuate ERK activation through canonical (Ras-dependent) and non-canonical (PI3K-dependent) pathways. In addition to regulation of receptor and ligand levels, we consider three layers of ERK-dependent feedback: desensitization of Ras activation, negative regulation of MEK kinase (e.g. Raf) activities, and up-regulation of dual-specificity ERK phosphatases. Our results establish the second of these as the dominant mode of ERK self-regulation in mouse fibroblasts. We further demonstrate that kinetic models of signaling networks, trained on a sufficient diversity of quantitative data, can be reasonably comprehensive, accurate, and predictive in the dynamical sense.

Productive cell migration requires the spatiotemporal coordination of cell adhesion, membrane protrusion, and actomyosin-mediated contraction. Integrins, engaged by the extracellular matrix (ECM), nucleate the formation of adhesive contacts at the cell's leading edge(s), and maturation of nascent adhesions to form stable focal adhesions constitutes a functional switch between protrusive and contractile activities. To shed additional light on the coupling between integrin-mediated adhesion and membrane protrusion, we have formulated a quantitative model of leading edge dynamics combining mechanistic and phenomenological elements and studied its features through classical bifurcation analysis and stochastic simulation. The model describes in mathematical terms the feedback loops driving, on the one hand, Rac-mediated membrane protrusion and rapid turnover of nascent adhesions, and on the other, myosin-dependent maturation of adhesions that inhibit protrusion at high ECM density. Our results show that the qualitative behavior of the model is most sensitive to parameters characterizing the influence of stable adhesions and myosin. The major predictions of the model, which we subsequently confirmed, are that persistent leading edge protrusion is optimal at an intermediate ECM density, whereas depletion of myosin IIA relieves the repression of protrusion at higher ECM density., Author Summary Cell migration is fundamental to human physiology and a phenomenon of long-standing interest in cell biology. It requires the concerted regulation of several dynamic processes that mediate physical anchorage of the cell and productive generation of protrusion and traction forces that propel the cell forward. In this work, we have developed a mathematical model that describes this interplay, cast at the level of biochemical signaling pathways activated at the front of a moving cell. Based on our analysis of the model and experimental confirmation of its basic predictions, we assert that coupled, counteracting feedback loops constitute a functional switch between maintenance and stalling of the cell protrusion speed. Our model successfully explains the dependence of this switch on the abundance of adhesive molecules in the cell's immediate surroundings and sheds light on how non-muscle myosin shapes that dependence.

Although it is appreciated that canonical signal-transduction pathways represent dominant modes of regulation embedded in larger interaction networks, relatively little has been done to quantify pathway cross-talk in such networks. Through quantitative measurements that systematically canvas an array of stimulation and molecular perturbation conditions, together with computational modeling and analysis, we have elucidated cross-talk mechanisms in the platelet-derived growth factor (PDGF) receptor signaling network, in which phosphoinositide 3-kinase (PI3K) and Ras/extracellular signal-regulated kinase (Erk) pathways are prominently activated. We show that, while PI3K signaling is insulated from cross-talk, PI3K enhances Erk activation at points both upstream and downstream of Ras. The magnitudes of these effects depend strongly on the stimulation conditions, subject to saturation effects in the respective pathways and negative feedback loops. Motivated by those dynamics, a kinetic model of the network was formulated and used to precisely quantify the relative contributions of PI3K-dependent and -independent modes of Ras/Erk activation.

This study was planned in order to determine the fungal spores in the air of inside the homes of asthmatic patients living in Isparta (from southwest region of Turkey). The seasonal properties of mold spores in the air of homes of 24 asthmatic and 14 control subjects living in the city of Isparta over a period of one year were investigated. Viable molds were recovered from all 38 houses. Twenty different molds were isolated and identified from the indoor air of the houses in which asthmatic patients and controls lived. The most common isolated genera were Penicillium spp. (27.9%), followed by Cladosporium spp. (26.3%), Aspergillus spp. (14.7%) and Alternaria spp. (13.1%) in the indoor air of the houses of asthmatic patients. No differences in colony numbers were observed between asthmatics and control groups. The percentage of molds was higher in kitchens than other parts of the houses such as living rooms and bedrooms (p0.05). A seasonal variety of the fungal flora in Isparta city region was observed. It is concluded that viable molds are common in houses in Isparta. Reducing indoor molds may improve the health of individuals with fungal-induced diseases like asthma.

INTRODUCTION The present study was designed to evaluate inhaler techniques and patient satisfaction with fixed-combination budesonide/formoterol dry-powder inhaler chronic obstructive pulmonary disease (COPD) in Turkey in real-life clinical practice. PATIENTS AND METHODS A total of 442 patients with COPD [mean (SD) age: 63.2 (10.6) years, 76.5% were males] were included in this cross-sectional study conducted at 25 outpatient clinics across Turkey. Data on socio-demographic characteristics, characteristics of COPD, inhaler technique and satisfaction with dry-powder inhaler were recorded at a single crosssectional visit performed at the study enrolment. RESULTS Patients were characterized by prominence of moderate to severe (78.1%) COPD, high rate of regular use of overall COPD medications (89.4%) and Turbuhaler® for an average of 33.7 months, predominance of males (76.5%), primary education (85.7%), urban location (68.3), ex-smokers (61.1%) and spending time outdoors for ≥ 4 hour/day (62.0%). Use of correct techniques was evident in majority of patients (≥ 94%), whereas inhalation maneuvers including breathing out gently away from mouthpiece without blowing into it (71.9%) and holding the breath for 5-10 seconds (78.3%) were performed correctly by lesser percent of patients especially in the older group (≥ 65 years, p< 0.05). Overall percent of patients with the feeling that she/he used the inhaler very/fairly correctly was 73.3%, while 86% of patients identified that they were very/fairly satisfied with the inhaler, irrespective of age and educational status. CONCLUSION In conclusion, our findings revealed the majority of patients are able to use Turbuhaler® correctly regardless of the educational status, while older age was associated with higher rate of errors in inhalation maneuvers in the real clinical practice in Turkey. Majority of our patients identified Turbuhaler® to be very/fairly convenient regarding ease of use, portability, and usability with an overall self-confidence in using the inhaler correctly among 73% and the satisfaction rate of 86%; irrespective of age and educational level.