In serum veritas—in serum sanitas? Cell non-autonomous aging compromises differentiation and survival of mesenchymal stromal cells via the oxidative stress pathway

Even tissues capable of complete regeneration, such as bone, show an age-related reduction in their healing capacity. Here, we hypothesized that this decline is primarily due to cell non-autonomous (extrinsic) aging mediated by the systemic environment. We demonstrate that culture of mesenchymal stromal cells (MSCs) in serum from aged Sprague–Dawley rats negatively affects their survival and differentiation ability. Proteome analysis and further cellular investigations strongly suggest that serum from aged animals not only changes expression of proteins related to mitochondria, unfolded protein binding or involved in stress responses, it also significantly enhances intracellular reactive oxygen species production and leads to the accumulation of oxidatively damaged proteins. Conversely, reduction of oxidative stress levels in vitro markedly improved MSC function. These results were validated in an in vivo model of compromised bone healing, which demonstrated significant increase regeneration in aged animals following oral antioxidant administration. These observations indicate the high impact of extrinsic aging on cellular functions and the process of endogenous (bone) regeneration. Thus, addressing the cell environment by, for example, systemic antioxidant treatment is a promising approach to enhance tissue regeneration and to regain cellular function especially in elderly patients.     

In or out? Regulating nuclear transport

The compartmentalization of proteins within the nucleus or cytoplasm of a eukaryotic cell offers opportunity for regulation of cell cycle progression and signalling pathways. Nuclear localization of proteins is determined by their ability to interact with specific nuclear import and export factors. In the past year, substrate phosphorylation has emerged as a common mechanism for controlling this interaction.     

Adrenomedullin in Ovarian Cancer: Foe In Vitro and Friend In Vivo?

Stromal elements within a tumor interact with cancer cells to create a microenvironment that supports tumor growth and survival. Adrenomedullin (ADM) is an autocrine/paracrine factor produced by both stromal cells and cancer cells to create such a microenvironment. During differentiation of macrophages, ADM is produced in response to pro-inflammatory stimuli and hypoxia. In this study we investigated the role of ADM as a growth factor for ovarian cancer cells and as a modulator of macrophages. We also analyzed ADM expression levels in a retrospective clinical study using nanofluidic technology and assessment of ADM at the gene level in 220 ovarian cancer patients. To study the effects of ADM, ovarian cancer cell lines A2780, OVCAR-3, and HEY and their drug-resistant counterparts were used for proliferation assays, while monocytes from healthy donors were differentiated in vitro. ADM was a weak growth factor, as revealed by proliferation assays and cell cycle analysis. After culturing cancer cells under stressing conditions, such as serum starvation and/or hypoxia, ADM was found to be a survival factor in HEY but not in other cell lines. In macrophages, ADM showed activity on proliferation/differentiation, primarily in type 2 macrophages (M2). Unexpectedly, the clinical study revealed that high expression of ADM was linked to positive outcome and to cancer with low Ca125. In conclusion, although in vitro ADM was a potential factor in biological aggressiveness, this possibility was not confirmed in patients. Therefore, use of an ADM antagonist would be inappropriate in managing ovarian cancer patients.     

In Vitro and In Vivo Neuronal Electrotaxis: A Potential Mechanism for Restoration?

Electrical brain stimulation used to treat a variety of neurological and psychiatric diseases is entering a new period. The technique is well established and the potential complications are well known and generally manageable. Recent studies demonstrated that electrical fields (EFs) can enhance neuroplasticity-related processes. EFs applied in the physiological range induce migration of different neural cell types from different species in vitro. There are some evidences that also the speed and directedness of cell migration are enhanced by EFs. However, it is still unclear how electrical signals from the extracellular space are translated into intracellular actions resulting in the so-called electrotaxis phenomenon. Here, we aim to provide a comprehensive review of the data on responses of cells to electrical stimulation and the relation to functional recovery.     

In what sense is contemporary medicine dualistic?

Medicine's inability to humanely respond to the concerns of its patients has often been attributed to its Cartesian dualism of mind and body. More recently, this inability has been ascribed to medicine's penchant for isolating biological disease as its sole proper concern to the exclusion of experienced illness. This dualism of disease and illness is claimed to be an outgrowth of the Cartesian dualism but the differences and similiarities between these two forms of dualism is not clear. This paper seeks to clarify their relationship through an examination of their historical origins.Disease is currently identified and characterized by a process of clinico-pathological correlation. By this means clinical impressions are corrected in light of autopsy findings. Our current mode of clinico-pathological correlation originated in Paris in the early nineteenth century with the work of Xavier Bichat and others. The theory of knowledge implicit within this clinico-pathological approach to the body is described and compared to that of Descartes. Though medicine does pursue certain Cartesian goals for knowledge, such as knowledge of the patient that does not rely upon his candor, it ultimately espouses neither a Cartesian theory of knowledge nor a Cartesian theory of the body. With pathological inquiry modeled after the autopsy as the final word in the identification and characterization of disease, the patient's capacity for self-knowledge and interpretation — not an entity called mind — is isolated away from his body. This approach to the body means that objective evidence of disease is valued to the exclusion of subjective evidence.If the shortcomings of modern biomedicine are to be effectively addressed, not only must the interdependence of disease and illness be acknowledged. The very canons of medical evidence must be revised. Subjective evidence must be rehabilitated and rejuvenated with better methods of subjective clinical investigation. Host factors relevant to the healing and knowing of sickness must be elucidated so that medicine may rediscover the sentience of its patients. Department of Psychiatry and Behavioral Sciences University of Washington Seattle, WA 98195, U.S.A.     

A Deconvolution-Based Method with High Sensitivity and Temporal Resolution for Detection of Spontaneous Synaptic Currents In?Vitro and?In?Vivo

Spontaneous postsynaptic currents (PSCs) provide key information about the mechanisms of synaptic transmission and the activity modes of neuronal networks. However, detecting spontaneous PSCs in vitro and in vivo has been challenging, because of the small amplitude, the variable kinetics, and the undefined time of generation of these events. Here, we describe a, to our knowledge, new method for detecting spontaneous synaptic events by deconvolution, using a template that approximates the average time course of spontaneous PSCs. A recorded PSC trace is deconvolved from the template, resulting in a series of delta-like functions. The maxima of these delta-like events are reliably detected, revealing the precise onset times of the spontaneous PSCs. Among all detection methods, the deconvolution-based method has a unique temporal resolution, allowing the detection of individual events in high-frequency bursts. Furthermore, the deconvolution-based method has a high amplitude resolution, because deconvolution can substantially increase the signal/noise ratio. When tested against previously published methods using experimental data, the deconvolution-based method was superior for spontaneous PSCs recorded in vivo. Using the high-resolution deconvolution-based detection algorithm, we show that the frequency of spontaneous excitatory postsynaptic currents in dentate gyrus granule cells is 4.5 times higher in vivo than in vitro.     

Is Redox-Cycling Ubiquinone Involved In Mitochondrial Oxygen Activation?

In most tissues mitochondria consume more than 90% of cellular oxygen. Although the greatest part of it undergoes tetravalent reduction thereby conserving free energy changes in the form of ATP. a great deal of evidence exists in the literature that also univalently reduced dioxygen is released during respiration. Redox-cycling ubiquinone was considered most frequently to be involved in this univalent e^? transfer to oxygen out of sequence however, other components of the respiratory chain could not be excluded. Our investigations on this problem questioned the role of redox-cycling ubiquinone in mitochondrial O^?₂ formation while H₂O₂ is supposed to accept e^? from this source. The paper provides experimental evidence that H₂O₂ in fact may operate as an oxidant of ubisemiquinone while dioxygen requires protons for such a reaction which are not available in the phospholipid bilayer where ubiquinone undergoes one e^?redox-cycling     

Why Are Lipid Rafts Not Observed In Vivo?

The existence of lipid rafts in live cells remains a topic of lively debate. Although large, micrometer-sized rafts are readily observed in artificial membranes, attempts to observe analogous domains in live cells place an upper limit of approximately 5 nm on their size. We suggest that integral membrane proteins attached to the cytoskeleton act as obstacles that limit the size of lipid domains. Computer simulations of a binary lipid mixture show that the presence of protein obstacles at only 5-10% by area dramatically reduces the tendency of the lipids to phase separate. These calculations emphasize the importance of spatial heterogeneity in cell membranes, which limits the transferability of conclusions drawn from artificial membranes to live cells.     

Is Redox-Cycling Ubiquinone Involved In Mitochondrial Oxygen Activation?

In most tissues mitochondria consume more than 90% of cellular oxygen. Although the greatest part of it undergoes tetravalent reduction thereby conserving free energy changes in the form of ATP. a great deal of evidence exists in the literature that also univalently reduced dioxygen is released during respiration. Redox-cycling ubiquinone was considered most frequently to be involved in this univalent e^- transfer to oxygen out of sequence however, other components of the respiratory chain could not be excluded. Our investigations on this problem questioned the role of redox-cycling ubiquinone in mitochondrial O^-₂ formation while H₂O₂ is supposed to accept e^- from this source. The paper provides experimental evidence that H₂O₂ in fact may operate as an oxidant of ubisemiquinone while dioxygen requires protons for such a reaction which are not available in the phospholipid bilayer where ubiquinone undergoes one e^-redox-cycling     

Simulation of In Vitro Dissolution Behavior Using DDDPlus?

Dissolution testing is a performance test for many dosage forms including tablets and capsules. The objective of this study was to evaluate if computer simulations can predict the in vitro dissolution of two model drugs for which different dissolution data were available. Published montelukast sodium and glyburide dissolution data was used for the simulations. Different pharmacopeial and biorelevant buffers, volumes, and rotations speeds were evaluated. Additionally, a pH change protocol was evaluated using these buffers. DDDPlus™ 3, Beta version (Simulation Plus, Inc.), was used to simulate the in vitro dissolution data. The simulated data were compared with the in vitro data. A regression coefficient between predicted and observed data was used to assess the simulations. The statistical analysis of Montelukast sodium showed that there was a significant correlation between the in vitro release data and the predicted data for all cases except for one buffer. For glyburide, there was also a significant correlation between the experimental data and the predicted data using single pH conditions. Using the dynamic pH protocol, a correlation was significant for one biorelevant media. The simulations showed that both in vitro drug releases were sensitive to solubility effects which confirmed their BCS class II category. Computer simulations of the in vitro release using DDDPlus™ have the potential to estimate the in vivo dissolution at an early stage in the drug development process. This might be used to choose the most appropriate dissolution condition to establish IVIVC and to develop biorelevant in vitro performance tests to capture critical product attributes for quality control procedures in quality by design environments.KEY WORDS: biorelevant media, DDDPlus, dissolution testing, QbD, QC     

Personalized In Vitro Cancer Modeling — Fantasy or Reality?

With greater technological advancements and understanding of pathophysiology, “personalized medicine” has become a more realistic goal. In the field of cancer, personalized medicine is the ultimate objective, as each cancer is unique and each tumor is heterogeneous. For many decades, researchers have relied upon studying the histopathology of tumors in the hope that it would provide clues to understanding the pathophysiology of cancer. Current preclinical research relies heavily upon two-dimensional culture models. However, these models have had limited success in recreating the complex interactions between cancer cells and the stroma environment in vivo. Thus, there is increasing impetus to shift to three-dimensional models, which more accurately reflect this phenomenon. With a more accurate in vitro tumor model, drug sensitivity can be tested to determine the best treatment option based on the tumor characteristics. Many methods have been developed to create tumor models or “tumoroids,” each with its advantages and limitations. One significant problem faced is the replication of angiogenesis that is characteristic of tumors in vivo. Nonetheless, if three-dimensional models could be standardized and implemented as a preclinical research tool for therapeutic testing, we would be taking a step towards making personalized cancer medicine a reality.