An electrostatic mechanism for Ca(2+)-mediated regulation of gap junction channels.

Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca(2+) blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca(2+). The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca(2+) coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca(2+)chelation. Computational analysis revealed that Ca(2+)-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K(+) into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore

Cx43-hemichannel function and regulation in physiology and pathophysiology: insights from the bovine corneal endothelial cell system and beyond

Intercellular communication in primary bovine corneal endothelial cells (BCECs) is mainly driven by the release of extracellular ATP through Cx43 hemichannels. Studying the characteristics of Ca2+-wave propagation in BCECs, an important form of intercellular communication, in response to physiological signaling events has led to the discovery of important insights in the functional properties and regulation of native Cx43 hemichannels. Together with ectopic expression models for Cx43 hemichannels and truncated/mutated Cx43 versions, it became very clear that loop/tail interactions play a key role in controlling the activity of Cx43 hemichannels. Interestingly, the negative regulation of Cx43 hemichannels by enhanced actin/myosin contractility seems to impinge upon loss of these loop/tail interactions essential for opening Cx43 hemichannels. Finally, these molecular insights have spurred the development of novel peptide tools that can selectively inhibit Cx43 hemichannels, but neither Cx43 gap junctions nor hemichannels formed by other Cx isoforms. These tools now set the stage to hunt for novel physiological functions for Cx43 hemichannels in primary cells and tissues and to tackle disease conditions associated with excessive, pathological Cx43-hemichannel openings

Isolation of equine endothelial cells and life cell angiogenesis assay

Arterial or venous thromboses are frequent clinical complications with the risk of fatal progression. Recent studies suggest the disruption of angiogenesis in the course of thrombus resolution as the underlying pathomechanism. Very similar to the situation in human patients, equine vessels have been described to be particularly susceptible to thrombosis. In contrast to humans, equine donors are readily available to obtain organs and tissues for isolation of endothelial cells. Objective of this study was to isolate equine endothelial cells and develop an angiogenesis assay from primary cultures. Macrovascular endothelial cells were obtained from jugular veins and carotid arteries of nine horses, one of which suffered from inflammatory processes. After enzymatic isolation, the cells were incubated in different selective primary media. Phenotypic identification of endothelial cells was accomplished by morphology and positive staining to von Willebrand factor. The reliable, inexpensive, and standardized combination of methods presented here resulted in pure endothelial cultures for angiogenesis assays that can be used in any cell culture laboratory. Inverted phase microscopy and life cell imaging was used to characterize the stages of the angiogenic cascade of the endothelial cells. Life cell imaging gave new insights into the in vitro formation of capillary like structures including exocytosis of microparticles from endothelial cells before integration into the three-dimensional structure. We hypothesize that a specific population of endothelial cells showing a highly active migration pattern in life cell imaging might play a role in the resolution of thrombosis

2015 Space Radiation Standing Review Panel

The 2015 Space Radiation Standing Review Panel (from here on referred to as the SRP) met for a site visit in Houston, TX on December 8 - 9, 2015. The SRP met with representatives from the Space Radiation Element and members of the Human Research Program (HRP) to review the updated research plan for the Risk of Radiation Carcinogenesis Cancer Risk. The SRP also reviewed the newly revised Evidence Reports for the Risk of Acute Radiation Syndromes Due to Solar Particle Events (SPEs) (Acute Risk), the Risk of Acute (In-flight) and Late Central Nervous System Effects from Radiation Exposure (CNS Risk), and the Risk of Cardiovascular Disease and Other Degenerative Tissue Effects from Radiation (Degen Risk), as well as a status update on these Risks. The SRP would like to commend Dr. Simonsen, Dr. Huff, Dr. Nelson, and Dr. Patel for their detailed presentations. The Space Radiation Element did a great job presenting a very large volume of material. The SRP considers it to be a strong program that is well-organized, well-coordinated and generates valuable data. The SRP commended the tissue sharing protocols, working groups, systems biology analysis, and standardization of models. In several of the discussed areas the SRP suggested improvements of the research plans in the future. These include the following: It is important that the team has expanded efforts examining immunology and inflammation as important components of the space radiation biological response. This is an overarching and important focus that is likely to apply to all aspects of the program including acute, CVD, CNS, cancer and others. Given that the area of immunology/inflammation is highly complex (and especially so as it relates to radiation), it warrants the expansion of investigators expertise in immunology and inflammation to work with the individual research projects and also the NASA Specialized Center of Research (NSCORs). Historical data on radiation injury to be entered into the Watson big data study must be used with caution. The general scientific issues of reproducibility, details of experimental methods and data analysis from preclinical and basic research laboratories have been raised broadly over the last few years (not specific to this work) and indicate that caution must be applied in the ways these data are used. This pertains to preclinical data and also to phase 3 clinical trials in radiation oncology and medical oncology. Of course, appropriate use and analysis of these big-data sets also offer the potential of pinpointing limitations and extracting remaining useful information. Emphasis should be placed on the latter possibility. A key target is risk reduction from radiation exposure. Progress of the entire space program, now moving towards the Mars mission, requires timely answers to key components of human risk, which are known to be complex. Periodic review of progress should be conducted with additional resources directed into achieving critical milestones. Turning the long red bars to yellow and green (or for some risks such as CNS possibly to grey) must be high priority. That such progress will require new science and not engineering means that it should be viewed in a knowledge-based light. The technology-based aspects of engineering issues are certainly as important, however, science and knowledge-based problems are solved in a different way than engineering. Timelines for engineering are more predictable, while for science, progress can be methodical with occasional major incremental findings that can rapidly change the rate of progress. As opportunities for rapid incremental changes arise, periodic enhancement of investment is strongly recommended to enable such new knowledge to be quickly and efficiently exploited. Collaborations and linkages with National Institute of Allergy and Infectious Diseases (NIAID), the Biomedical Advanced Research and Development Authority (BARDA) and the Department of Defense (DoD) are in place and more are encouraged, where possible, with the radiation injury and medical countermeasure studies. This could include utilizing some of their animal model testing contracts to facilitate obtaining results using common platforms. Such approach will facilitate the comparison of results among laboratories, and will facilitate and accelerate the development of medical countermeasures. It is particularly noteworthy that the NASA Space Radiation Element is reaching out to the Multidisciplinary European Low Dose Initiative (MELODI) platform coordinating low dose radiation risk research, and to other international agencies that are studying low dose radiation effects in an effort to fill the void generated by the cancelation of the Department of Energy (DOE) low dose radiation program. While NASA is working actively with NIAID and BARDA to integrate their relevant findings of radiation mitigator investigations to NASA programs, the committee notes its disappointment that the United States currently lacks a dedicated low dose radiation program with clear mechanistic orientation and aimed at the quantification and mitigation of human radiation risk on Earth. This void gives to the NASA Space Radiation Program Element special societal value, but also makes its overall design more challenging

The prognostic value of the hypoxia markers CA IX and GLUT 1 and the cytokines VEGF and IL 6 in head and neck squamous cell carcinoma treated by radiotherapy ± chemotherapy

BACKGROUND: Several parameters of the tumor microenvironment, such as hypoxia, inflammation and angiogenesis, play a critical role in tumor aggressiveness and treatment response. A major question remains if these markers can be used to stratify patients to certain treatment protocols. The purpose of this study was to investigate the inter-relationship and the prognostic significance of several biological and clinicopathological parameters in patients with head and neck squamous cell carcinoma (HNSCC) treated by radiotherapy ± chemotherapy. METHODS: We used two subgroups of a retrospective series for which CT-determined tumoral perfusion correlated with local control. In the first subgroup (n = 67), immunohistochemistry for carbonic anhydrase IX (CA IX) and glucose transporter-1 (GLUT-1) was performed on the pretreatment tumor biopsy. In the second subgroup (n = 34), enzyme linked immunosorbent assay (ELISA) was used to determine pretreatment levels of the cytokines vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) in serum. Correlation was investigated between tumoral perfusion and each of these biological markers, as well as between the markers mutually. The prognostic value of these microenvironmental parameters was also evaluated. RESULTS: For CA IX and GLUT-1, the combined assessment of patients with both markers expressed above the median showed an independent correlation with local control (p = 0.02) and disease-free survival (p = 0.04) with a trend for regional control (p = 0.06). In the second subgroup, IL-6 pretreatment serum level above the median was the only independent predictor of local control (p = 0.009), disease-free survival (p = 0.02) and overall survival (p = 0.005). CONCLUSION: To our knowledge, we are the first to report a link in HNSCC between IL-6 pretreatment serum levels and radioresistance in vivo. This link is supported by the strong prognostic association of pretreatment IL-6 with local control, known to be the most important parameter to judge radiotherapy responses. Furthermore, the combined assessment of CA IX and GLUT-1 correlated independently with prognosis. This is a valuable indication that a combined approach is important in the investigation of prognostic markers

High-contrast three-dimensional imaging of the Arabidopsis leaf enables the analysis of cell dimensions in the epidermis and mesophyll

UMR DAP, équipe PHIV; UMR LEPSEInternational audienceABSTRACT: BACKGROUND: Despite the wide spread application of confocal and multiphoton laser scanning microscopy in plant biology, leaf phenotype assessment still relies on two-dimensional imaging with a limited appreciation of the cells' structural context and an inherent inaccuracy of cell measurements. Here, a successful procedure for the three-dimensional imaging and analysis of plant leaves is presented. RESULTS: The procedure was developed based on a range of developmental stages, from leaf initiation to senescence, of soil-grown Arabidopsis thaliana (L.) Heynh. Rigorous clearing of tissues, made possible by enhanced leaf permeability to clearing agents, allowed the optical sectioning of the entire leaf thickness by both confocal and multiphoton microscopy. The superior image quality, in resolution and contrast, obtained by the latter technique enabled the three-dimensional visualisation of leaf morphology at the individual cell level, cell segmentation and the construction of structural models. Image analysis macros were developed to measure leaf thickness and tissue proportions, as well as to determine for the epidermis and all layers of mesophyll tissue, cell density, volume, length and width. For mesophyll tissue, the proportion of intercellular spaces and the surface areas of cells were also estimated. The performance of the procedure was demonstrated for the expanding 6th leaf of the Arabidopsis rosette. Furthermore, it was proven to be effective for leaves of another dicotyledon, apple (Malus domestica Borkh.), which has a very different cellular organisation. CONCLUSIONS: The pipeline for the three-dimensional imaging and analysis of plant leaves provides the means to include variables on internal tissues in leaf growth studies and the assessment of leaf phenotypes. It also allows the visualisation and quantification of alterations in leaf structure alongside changes in leaf functioning observed under environmental constraints. Data obtained using this procedure can further be integrated in leaf development and functioning models

Continuous culture of a marine heterotrophic microorganism in multiple substrate solution

Thesis (Ph.D.) University of Alaska Fairbanks, 197

Structure-Dependent Effects of Phthalates on Intercellular and Intracellular Communication in Liver Oval Cells

Humans are exposed to phthalates released from plastics, cosmetics, or food on a daily basis. Phthalates have low acute liver toxicity, but their chronic exposures could induce molecular and cellular effects linked to adverse health outcomes, such as liver tumor promotion or chronic liver diseases. The alternation of gap junctional intercellular communication (GJIC) and MAPK-Erk1/2 pathways in liver progenitor or oval cells can disrupt liver tissue homeostatic mechanisms and affect the development and severity of these adverse outcomes. Our study with 20 different phthalates revealed their structurally dependent effects on liver GJIC and MAPK-Erk1/2 signaling in rat liver WB-F344 cell line with characteristics of liver oval cells. The phthalates with a medium-length side chain (3-6 C) were the most potent dysregulators of GJIC and activators of MAPK-Erk1/2. The effects occurred rapidly, suggesting the activation of non-genomic (non-transcriptional) mechanisms directly by the parental compounds. Short-chain phthalates (1-2 C) did not dysregulate GJIC even after longer exposures and did not activate MAPK-Erk1/2. Longer chain (>= 7 C) phthalates, such as DEHP or DINP, moderately activated MAPK-Erk1/2, but inhibited GJIC only after prolonged exposures (>12 h), suggesting that GJIC dysregulation occurs via genomic mechanisms, or (bio)transformation. Overall, medium-chain phthalates rapidly affected the key tissue homeostatic mechanisms in the liver oval cell population via non-genomic pathways, which might contribute to the development of chronic liver toxicity and diseases

Elastic and ultradeformable liposomes for transdermal delivery of active pharmaceutical ingredients (APIs)

Administration of active pharmaceutical ingredients (APIs) through the skin, by means of topical drug delivery systems, is an advanced therapeutic approach. As the skin is the largest organ of the human body, primarily acting as a natural protective barrier against permeation of xenobiotics, specific strategies to overcome this barrier are needed. Liposomes are nanometric-sized delivery systems composed of phospholipids, which are key components of cell membranes, making liposomes well tolerated and devoid of toxicity. As their lipid compositions are similar to those of the skin, liposomes are used as topical, dermal, and transdermal delivery systems. However, permeation of the first generation of liposomes through the skin posed some limitations; thus, a second generation of liposomes has emerged, overcoming permeability problems. Various mechanisms of permeation/penetration of elastic/ultra-deformable liposomes into the skin have been proposed; however, debate continues on their extent/mechanisms of permeation/penetration. In vivo bioavailability of an API administered in the form of ultra-deformable liposomes is similar to the bioavailability achieved when the same API is administered in the form of a solution by subcutaneous or epi-cutaneous injection, which demonstrates their applicability in transdermal drug delivery.This research was funded by the Portuguese Science and Technology Foundation (FCT/ MCT) and European Funds (PRODER/COMPETE), under the project reference UIDB/04469/2020 (strategic fund), co-financed by FEDER, under the Partnership Agreement PT2020. The work is also supported by the National Science Centre within the MINIATURA 4 for a single research activity (grant No: 2020/04/X/ST5/00789) and by the START 2021 Program of the Foundation for Polish Science (FNP) granted to Aleksandra Zielinska.info:eu-repo/semantics/publishedVersio