Grain boundary engineering to control the discontinuous precipitation in multicomponent U10Mo alloy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Devaraj, Arun; Kovarik, Libor; Kautz, Elizabeth

Grain boundaries in metallic alloys often play a crucial role, not only in determining the mechanical properties or thermal stability of alloys, but also in dictating the phase transformation kinetics during thermomechanical processing. We demonstrate that locally stabilized structure and compositional segregation at grain boundaries—“grain boundary complexions”—in a complex multicomponent alloy can be modified to influence the kinetics of cellular transformation during subsequent thermomechanical processing. Using aberration-corrected scanning transmission electron microscopy and atom probe tomography analysis of a metallic nuclear fuel highly relevant to worldwide nuclear non-proliferation efforts —uranium-10 wt% molybdenum (U-10Mo) alloy, new evidence for the existence of grainmore » boundary complexion is provided. We then modified the concentration of impurities dissolved in Υ-UMo grain interiors and/or segregated to Υ-UMo grain boundaries by changing the homogenization treatment, and these effects were used used to retard the kinetics of cellular transformation during subsequent sub-eutectoid annealing in this U-10-Mo alloy during sub-eutectoid annealing. Thus, this work provided insights on tailoring the final microstructure of the U-10Mo alloy, which can potentially improve the irradiation performance of this important class of alloy fuels.« less

An evolving paradigm for the secretory pathway?

PubMed Central

Lippincott-Schwartz, Jennifer

2011-01-01

The paradigm that the secretory pathway consists of a stable endoplasmic reticulum and Golgi apparatus, using discrete transport vesicles to exchange their contents, gained important support from groundbreaking biochemical and genetic studies during the 1980s. However, the subsequent development of new imaging technologies with green fluorescent protein introduced data on dynamic processes not fully accounted for by the paradigm. As a result, we may be seeing an example of how a paradigm is evolving to account for the results of new technologies and their new ways of describing cellular processes. PMID:22039065

An inquiry-based practical for a large, foundation-level undergraduate laboratory that enhances student understanding of basic cellular concepts and scientific experimental design.

PubMed

Bugarcic, A; Zimbardi, K; Macaranas, J; Thorn, P

2012-01-01

Student-centered education involving research experiences or inquiry have been shown to help undergraduate students understand, and become excited about, the process of scientific investigation. These benefits are particularly important for students in the early stages of their degree (Report and Kenny, http://naplesccsunysbedu/Pres/boyernsf/1998). However, embedding such experiences into the curriculum is particularly difficult when dealing with early stage students, who are in larger cohorts and often lack the background content knowledge necessary to engage with primary research literature and research level methods and equipment. We report here the design, delivery, assessment, and subsequent student learning outcomes of a 4-week practical module for 120 students at the beginning of their second year of university, which successfully engages students in designing cell culture experiments and in understanding the molecular processes and machinery involved in the basic cellular process of macropinocytosis. Copyright © 2011 Wiley Periodicals, Inc.

The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers.

PubMed

Newman, Joseph; Asfor, Amin S; Berryman, Stephen; Jackson, Terry; Curry, Stephen; Tuthill, Tobias J

2018-03-01

Productive picornavirus infection requires the hijacking of host cell pathways to aid with the different stages of virus entry, synthesis of the viral polyprotein, and viral genome replication. Many picornaviruses, including foot-and-mouth disease virus (FMDV), assemble capsids via the multimerization of several copies of a single capsid precursor protein into a pentameric subunit which further encapsidates the RNA. Pentamer formation is preceded by co- and posttranslational modification of the capsid precursor (P1-2A) by viral and cellular enzymes and the subsequent rearrangement of P1-2A into a structure amenable to pentamer formation. We have developed a cell-free system to study FMDV pentamer assembly using recombinantly expressed FMDV capsid precursor and 3C protease. Using this assay, we have shown that two structurally different inhibitors of the cellular chaperone heat shock protein 90 (hsp90) impeded FMDV capsid precursor processing and subsequent pentamer formation. Treatment of FMDV permissive cells with the hsp90 inhibitor prior to infection reduced the endpoint titer by more than 10-fold while not affecting the activity of a subgenomic replicon, indicating that translation and replication of viral RNA were unaffected by the drug. IMPORTANCE FMDV of the Picornaviridae family is a pathogen of huge economic importance to the livestock industry due to its effect on the restriction of livestock movement and necessary control measures required following an outbreak. The study of FMDV capsid assembly, and picornavirus capsid assembly more generally, has tended to be focused upon the formation of capsids from pentameric intermediates or the immediate cotranslational modification of the capsid precursor protein. Here, we describe a system to analyze the early stages of FMDV pentameric capsid intermediate assembly and demonstrate a novel requirement for the cellular chaperone hsp90 in the formation of these pentameric intermediates. We show the added complexity involved for this process to occur, which could be the basis for a novel antiviral control mechanism for FMDV. Copyright © 2018 Newman et al.

Protection against hydrogen peroxide cytotoxicity in rat-1 fibroblasts provided by the oncoprotein Bcl-2: maintenance of calcium homoeostasis is secondary to the effect of Bcl-2 on cellular glutathione.

PubMed Central

Rimpler, M M; Rauen, U; Schmidt, T; Möröy, T; de Groot, H

1999-01-01

The oncoprotein Bcl-2 protects cells against apoptosis, but the exact molecular mechanism that underlies this function has not yet been identified. Studying H2O2-induced cell injury in Rat-1 fibroblast cells, we observed that Bcl-2 had a protective effect against the increase in cytosolic calcium concentration and subsequent cell death. Furthermore, overexpression of Bcl-2 resulted in an alteration of cellular glutathione status: the total amount of cellular glutathione was increased by about 60% and the redox potential of the cellular glutathione pool was maintained in a more reduced state during H2O2 exposure compared with non-Bcl-2-expressing controls. In our cytotoxicity model, disruption of cellular glutathione homoeostasis closely correlated with the pathological elevation of cytosolic calcium concentration. Stabilization of the glutathione pool by Bcl-2, N-acetylcysteine or glucose delayed the cytosolic calcium increase and subsequent cell death, whereas depletion of glutathione by dl-buthionine-(S, R)-sulphoximine, sensitized Bcl-2-transfected cells towards cytosolic calcium increase and cell death. We therefore suggest that the protection exerted by Bcl-2 against H2O2-induced cytosolic calcium elevation and subsequent cell death is secondary to its effect on the cellular glutathione metabolism. PMID:10229685

Cellular Injuries in Cronobacter sakazakii CIP 103183T and Salmonella enterica Exposed to Drying and Subsequent Heat Treatment in Milk Powder

PubMed Central

Lang, Emilie; Guyot, Stéphane; Peltier, Caroline; Alvarez-Martin, Pablo; Perrier-Cornet, Jean-Marie; Gervais, Patrick

2018-01-01

Because of the ability of foodborne pathogens to survive in low-moisture foods, their decontamination is an important issue in food protection. This study aimed to clarify some of the cellular mechanisms involved in inactivation of foodborne pathogens after drying and subsequent heating. Individual strains of Salmonella Typhimurium, Salmonella Senftenberg, and Cronobacter sakazakii were mixed into whole milk powder and dried to different water activity levels (0.25 and 0.58); the number of surviving cells was determined after drying and subsequent thermal treatments in closed vessels at 90 and 100°C, for 30 and 120 s. For each condition, the percentage of unculturable cells was estimated and, in parallel, membrane permeability and respiratory activity were estimated by flow cytometry using fluorescent probes. After drying, it was clearly observable that the percentage of unculturable cells was correlated with the percentage of permeabilized cells (responsible for 20–40% of the total inactivated bacteria after drying), and to a lesser degree with the percentage of cells presenting with loss of respiratory activity. In contrast, the percentages of unculturable cells observed after heat treatment were strongly correlated with the loss of respiratory activity and weakly with membrane permeability (for 70–80% of the total inactivated bacteria after heat treatment). We conclude that cell inactivation during drying is closely linked to membrane permeabilization and that heat treatment of dried cells affects principally their respiratory activity. These results legitimize the use of time–temperature scales and allow better understanding of the cellular mechanisms of bacterial death during drying and subsequent heat treatment. These results may also allow better optimization of the decontamination process to ensure food safety by targeting the most deleterious conditions for bacterial cells without denaturing the food product. PMID:29593704

A New Cellular Architecture for Information Retrieval from Sensor Networks through Embedded Service and Security Protocols

PubMed Central

Shahzad, Aamir; Landry, René; Lee, Malrey; Xiong, Naixue; Lee, Jongho; Lee, Changhoon

2016-01-01

Substantial changes have occurred in the Information Technology (IT) sectors and with these changes, the demand for remote access to field sensor information has increased. This allows visualization, monitoring, and control through various electronic devices, such as laptops, tablets, i-Pads, PCs, and cellular phones. The smart phone is considered as a more reliable, faster and efficient device to access and monitor industrial systems and their corresponding information interfaces anywhere and anytime. This study describes the deployment of a protocol whereby industrial system information can be securely accessed by cellular phones via a Supervisory Control And Data Acquisition (SCADA) server. To achieve the study goals, proprietary protocol interconnectivity with non-proprietary protocols and the usage of interconnectivity services are considered in detail. They support the visualization of the SCADA system information, and the related operations through smart phones. The intelligent sensors are configured and designated to process real information via cellular phones by employing information exchange services between the proprietary protocol and non-proprietary protocols. SCADA cellular access raises the issue of security flaws. For these challenges, a cryptography-based security method is considered and deployed, and it could be considered as a part of a proprietary protocol. Subsequently, transmission flows from the smart phones through a cellular network. PMID:27314351

A New Cellular Architecture for Information Retrieval from Sensor Networks through Embedded Service and Security Protocols.

PubMed

Shahzad, Aamir; Landry, René; Lee, Malrey; Xiong, Naixue; Lee, Jongho; Lee, Changhoon

2016-06-14

Substantial changes have occurred in the Information Technology (IT) sectors and with these changes, the demand for remote access to field sensor information has increased. This allows visualization, monitoring, and control through various electronic devices, such as laptops, tablets, i-Pads, PCs, and cellular phones. The smart phone is considered as a more reliable, faster and efficient device to access and monitor industrial systems and their corresponding information interfaces anywhere and anytime. This study describes the deployment of a protocol whereby industrial system information can be securely accessed by cellular phones via a Supervisory Control And Data Acquisition (SCADA) server. To achieve the study goals, proprietary protocol interconnectivity with non-proprietary protocols and the usage of interconnectivity services are considered in detail. They support the visualization of the SCADA system information, and the related operations through smart phones. The intelligent sensors are configured and designated to process real information via cellular phones by employing information exchange services between the proprietary protocol and non-proprietary protocols. SCADA cellular access raises the issue of security flaws. For these challenges, a cryptography-based security method is considered and deployed, and it could be considered as a part of a proprietary protocol. Subsequently, transmission flows from the smart phones through a cellular network.

Characterization of carbon nanofibre-reinforced polypropylene foams.

PubMed

Antunes, M; Velasco, J I; Realinho, V; Arencón, D

2010-02-01

In this paper, carbon-nanofibre-reinforced polypropylene foams were prepared and characterized regarding their foaming behaviour, cellular structure and both thermo-mechanical as well as electrical properties. Polypropylene (PP) nanocomposites containing 5, 10 and 20 wt% of carbon nanofibres (CNF) and a chemical blowing agent were prepared by melt-mixing inside a twin-screw extruder and subsequently water-cooled and pelletized. The extruded nanocomposites were later foamed using a one-step compression-moulding process. The thermo-mechanical properties of the CNF-reinforced PP foams were studied, analyzing the influence of the carbon nanofibres on the cellular structure and subsequent thermo-mechanical behaviour of the foams. Carbon nanofibres not only seemed to act as nucleating agents, reducing the average cell size of the foams and increasing their cell density for similar expansion ratios, but also helped produce mechanically-improved foams, even reaching for the 20 wt% CNF-reinforced ones a specific modulus around 1.2 GPa x cm3/g for densities as low as 300 kg/m3. An increasingly higher electrical conductivity was assessed for both the solids as well as the foams with increasing the amount of carbon nanofibres.

Risky family processes prospectively forecast shorter telomere length mediated through negative emotions.

PubMed

Brody, Gene H; Yu, Tianyi; Shalev, Idan

2017-05-01

This study was designed to examine prospective associations of risky family environments with subsequent levels of negative emotions and peripheral blood mononuclear cell telomere length (TL), a marker of cellular aging. A second purpose was to determine whether negative emotions mediate the hypothesized link between risky family processes and diminished telomere length. Participants were 293 adolescents (age 17 years at the first assessment) and their primary caregivers. Caregivers provided data on risky family processes when the youths were age 17 years, youths reported their negative emotions at age 18 years, and youths' TL was assayed from a blood sample at age 22 years. The results revealed that (a) risky family processes forecast heightened negative emotions (β = .316, p < .001) and diminished TL (β = -.199, p = .003) among youths, (b) higher levels of negative emotions forecast shorter TL (β = -.187, p = .012), and (c) negative emotions served as a mediator connecting risky family processes with diminished TL (indirect effect = -0.012, 95% CI [-0.036, -0.002]). These findings are consistent with the hypothesis that risky family processes presage premature cellular aging through effects on negative emotions, with potential implications for lifelong health. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Risky Family Processes Prospectively Forecast Shorter Telomere Length Mediated through Negative Emotions

PubMed Central

Brody, Gene H.; Yu, Tianyi; Shalev, Idan

2016-01-01

Objective This study was designed to examine prospective associations of risky family environments with subsequent levels of negative emotions and peripheral blood mononuclear cell telomere length (TL), a marker of cellular aging. A second purpose was to determine whether negative emotions mediate the hypothesized link between risky family processes and diminished telomere length. Methods Participants were 293 adolescents (age 17 years at the first assessment) and their primary caregivers. Caregivers provided data on risky family processes when the youths were age 17 years, youths reported their negative emotions at age 18 years, and youths’ TL was assayed from a blood sample at age 22 years. Results The results revealed that (a) risky family processes forecast heightened negative emotions (β = .316, p < .001) and diminished TL (β = −.199, p = .003) among youths, (b) higher levels of negative emotions forecast shorter TL (β = −.187, p = .012), and (c) negative emotions served as a mediator connecting risky family processes with diminished TL (indirect effect = −0.012, 95% CI [−0.036, −0.002]). Conclusions These findings are consistent with the hypothesis that risky family processes presage premature cellular aging through effects on negative emotions, with potential implications for lifelong health. PMID:27831704

The distribution of DNA damage is defined by region-specific susceptibility to DNA damage formation rather than repair differences.

PubMed

Strand, Janne M; Scheffler, Katja; Bjørås, Magnar; Eide, Lars

2014-06-01

The cellular genomes are continuously damaged by reactive oxygen species (ROS) from aerobic processes. The impact of DNA damage depends on the specific site as well as the cellular state. The steady-state level of DNA damage is the net result of continuous formation and subsequent repair, but it is unknown to what extent heterogeneous damage distribution is caused by variations in formation or repair of DNA damage. Here, we used a restriction enzyme/qPCR based method to analyze DNA damage in promoter and coding regions of four nuclear genes: the two house-keeping genes Gadph and Tbp, and the Ndufa9 and Ndufs2 genes encoding mitochondrial complex I subunits, as well as mt-Rnr1 encoded by mitochondrial DNA (mtDNA). The distribution of steady-state levels of damage varied in a site-specific manner. Oxidative stress induced damage in nDNA to a similar extent in promoter and coding regions, and more so in mtDNA. The subsequent removal of damage from nDNA was efficient and comparable with recovery times depending on the initial damage load, while repair of mtDNA was delayed with subsequently slower repair rate. The repair was furthermore found to be independent of transcription or the transcription-coupled repair factor CSB, but dependent on cellular ATP. Our results demonstrate that the capacity to repair DNA is sufficient to remove exogenously induced damage. Thus, we conclude that the heterogeneous steady-state level of DNA damage in promoters and coding regions is caused by site-specific DNA damage/modifications that take place under normal metabolism. Copyright © 2014 Elsevier B.V. All rights reserved.

DNA replication components as regulators of epigenetic inheritance--lesson from fission yeast centromere.

PubMed

He, Haijin; Gonzalez, Marlyn; Zhang, Fan; Li, Fei

2014-06-01

Genetic information stored in DNA is accurately copied and transferred to subsequent generations through DNA replication. This process is accomplished through the concerted actions of highly conserved DNA replication components. Epigenetic information stored in the form of histone modifications and DNA methylation, constitutes a second layer of regulatory information important for many cellular processes, such as gene expression regulation, chromatin organization, and genome stability. During DNA replication, epigenetic information must also be faithfully transmitted to subsequent generations. How this monumental task is achieved remains poorly understood. In this review, we will discuss recent advances on the role of DNA replication components in the inheritance of epigenetic marks, with a particular focus on epigenetic regulation in fission yeast. Based on these findings, we propose that specific DNA replication components function as key regulators in the replication of epigenetic information across the genome.

Cystic Fibrosis, Cystic Fibrosis Transmembrane Conductance Regulator and Drugs: Insights from Cellular Trafficking.

PubMed

Bridges, Robert J; Bradbury, Neil A

2018-01-01

The eukaryotic cell is organized into membrane-delineated compartments that are characterized by specific cadres of proteins sustaining biochemically distinct cellular processes. The appropriate subcellular localization of proteins is key to proper organelle function and provides a physiological context for cellular processes. Disruption of normal trafficking pathways for proteins is seen in several genetic diseases, where a protein's absence for a specific subcellular compartment leads to organelle disruption, and in the context of an individual, a disruption of normal physiology. Importantly, several drug therapies can also alter protein trafficking, causing unwanted side effects. Thus, a deeper understanding of trafficking pathways needs to be appreciated as novel therapeutic modalities are proposed. Despite the promising efficacy of novel therapeutic agents, the intracellular bioavailability of these compounds has proved to be a potential barrier, leading to failures in treatments for various diseases and disorders. While endocytosis of drug moieties provides an efficient means of getting material into cells, the subsequent release and endosomal escape of materials into the cytosol where they need to act has been a barrier. An understanding of cellular protein/lipid trafficking pathways has opened up strategies for increasing drug bioavailability. Approaches to enhance endosomal exit have greatly increased the cytosolic bioavailability of drugs and will provide a means of investigating previous drugs that may have been shelved due to their low cytosolic concentration.

New cellular automaton designed to simulate geometration in gel electrophoresis

NASA Astrophysics Data System (ADS)

Krawczyk, M. J.; Kułakowski, K.; Maksymowicz, A. Z.

2002-08-01

We propose a new kind of cellular automaton to simulate transportation of molecules of DNA through agarose gel. Two processes are taken into account: reptation at strong electric field E, described in the particle model, and geometration, i.e. subsequent hookings and releases of long molecules at and from gel fibres. The automaton rules are deterministic and they are designed to describe both processes within one unified approach. Thermal fluctuations are not taken into account. The number of simultaneous hookings is limited by the molecule length. The features of the automaton are: (i) the size of the cell neighbourhood for the automaton rule varies dynamically, from nearest neighbors to the entire molecule; (ii) the length of the time step is determined at each step according to dynamic rules. Calculations are made up to N=244 reptons in a molecule. Two subsequent stages of the motion are found. Firstly, an initial set of random configurations of molecules is transformed into a more ordered phase, where most molecules are elongated along the applied field direction. After some transient time, the mobility μ reaches a constant value. Then, it varies with N as 1/ N for long molecules. The band dispersion varies with time t approximately as Nt1/2. Our results indicate that the well-known plateau of the mobility μ vs. N does not hold at large electric fields.

From single cells to tissue architecture-a bottom-up approach to modelling the spatio-temporal organisation of complex multi-cellular systems.

PubMed

Galle, J; Hoffmann, M; Aust, G

2009-01-01

Collective phenomena in multi-cellular assemblies can be approached on different levels of complexity. Here, we discuss a number of mathematical models which consider the dynamics of each individual cell, so-called agent-based or individual-based models (IBMs). As a special feature, these models allow to account for intracellular decision processes which are triggered by biomechanical cell-cell or cell-matrix interactions. We discuss their impact on the growth and homeostasis of multi-cellular systems as simulated by lattice-free models. Our results demonstrate that cell polarisation subsequent to cell-cell contact formation can be a source of stability in epithelial monolayers. Stroma contact-dependent regulation of tumour cell proliferation and migration is shown to result in invasion dynamics in accordance with the migrating cancer stem cell hypothesis. However, we demonstrate that different regulation mechanisms can equally well comply with present experimental results. Thus, we suggest a panel of experimental studies for the in-depth validation of the model assumptions.

Elastic force restricts growth of the murine utricle

PubMed Central

Gnedeva, Ksenia; Jacobo, Adrian; Salvi, Joshua D; Petelski, Aleksandra A; Hudspeth, A J

2017-01-01

Dysfunctions of hearing and balance are often irreversible in mammals owing to the inability of cells in the inner ear to proliferate and replace lost sensory receptors. To determine the molecular basis of this deficiency we have investigated the dynamics of growth and cellular proliferation in a murine vestibular organ, the utricle. Based on this analysis, we have created a theoretical model that captures the key features of the organ’s morphogenesis. Our experimental data and model demonstrate that an elastic force opposes growth of the utricular sensory epithelium during development, confines cellular proliferation to the organ’s periphery, and eventually arrests its growth. We find that an increase in cellular density and the subsequent degradation of the transcriptional cofactor Yap underlie this process. A reduction in mechanical constraints results in accumulation and nuclear translocation of Yap, which triggers proliferation and restores the utricle’s growth; interfering with Yap’s activity reverses this effect. DOI: http://dx.doi.org/10.7554/eLife.25681.001 PMID:28742024

Cellular Cations Control Conformational Switching of Inositol Pyrophosphate Analogues

PubMed Central

Hager, Anastasia; Wu, Mingxuan; Wang, Huanchen; Brown, Nathaniel W.; Shears, Stephen B.

2016-01-01

The inositol pyrophosphate messengers (PP-InsPs) are emerging as an important class of cellular regulators. These molecules have been linked to numerous biological processes, including insulin secretion and cancer cell migration, but how they trigger such a wide range of cellular responses has remained unanswered in many cases. Here, we show that the PP-InsPs exhibit complex speciation behaviour and propose that a unique conformational switching mechanism could contribute to their multifunctional effects. We synthesised non-hydrolysable bisphosphonate analogues and crystallised the analogues in complex with mammalian PPIP5K2 kinase. Subsequently, the bisphosphonate analogues were used to investigate the protonation sequence, metal-coordination properties, and conformation in solution. Remarkably, the presence of potassium and magnesium ions enabled the analogues to adopt two different conformations near physiological pH. Understanding how the intrinsic chemical properties of the PP-InsPs can contribute to their complex signalling outputs will be essential to elucidate their regulatory functions. PMID:27460418

Mammalian HspB1 (Hsp27) is a molecular sensor linked to the physiology and environment of the cell.

PubMed

Arrigo, André-Patrick

2017-07-01

Constitutively expressed small heat shock protein HspB1 regulates many fundamental cellular processes and plays major roles in many human pathological diseases. In that regard, this chaperone has a huge number of apparently unrelated functions that appear linked to its ability to recognize many client polypeptides that are subsequently modified in their activity and/or half-life. A major parameter to understand how HspB1 is dedicated to interact with particular clients in defined cellular conditions relates to its complex oligomerization and phosphorylation properties. Indeed, HspB1 structural organization displays dynamic and complex rearrangements in response to changes in the cellular environment or when the cell physiology is modified. These structural modifications probably reflect the formation of structural platforms aimed at recognizing specific client polypeptides. Here, I have reviewed data from the literature and re-analyzed my own studies to describe and discuss these fascinating changes in HspB1 structural organization.

M2-like macrophages are responsible for collagen degradation through a mannose receptor–mediated pathway

PubMed Central

Madsen, Daniel H.; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E.; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S.; Brenner, David A.; Burgdorf, Sven; Engelholm, Lars H.; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto

2013-01-01

Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase–dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor–associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process. PMID:24019537

The Cellular Pathology of Experimental Hypertension

PubMed Central

Wiener, Joseph; Giacomelli, Filiberto

1973-01-01

Acute hypertension was produced in rats by the infusion of angiotensin amide for 2 to 4 hours. These animals were injected intravenously prior to sacrifice with either colloidal carbon or iron dextran particles. The mesenteric vessels from hypertensive and control animals were processed for electron microscopy. Ultrastructural alterations are found in dilated segments of small arteries. Initially there is severe contraction of medial smooth muscle cells and the formation of processes of smooth muscle cytoplasm. This is followed by lysis of cell processes and bodies, and passage of plasma and colloidal iron into the media. Subsequently, carbon, platelets, fibrin and cellular debris are seen within these foci of medial necrosis. These changes appear as a sequence whose severity reflects the duration of the angiotensin infusion and degree of elevation of the systolic pressure. The morphologic alterations are discussed in relation to the generalized increase in vascular permeability that is associated with the hypertensive state. ImagesFig 5Fig 11Fig 12Fig 13Fig 14Fig 6Fig 7Fig 1Fig 2Fig 3Fig 4Fig 8Fig 9Fig 10 PMID:4124863

The Role of Mitophagy in Innate Immunity

PubMed Central

Gkikas, Ilias; Palikaras, Konstantinos; Tavernarakis, Nektarios

2018-01-01

Mitochondria are cellular organelles essential for multiple biological processes, including energy production, metabolites biosynthesis, cell death, and immunological responses among others. Recent advances in the field of immunology research reveal the pivotal role of energy metabolism in innate immune cells fate and function. Therefore, the maintenance of mitochondrial network integrity and activity is a prerequisite for immune system homeostasis. Mitochondrial selective autophagy, known as mitophagy, surveils mitochondrial population eliminating superfluous and/or impaired organelles and mediating cellular survival and viability in response to injury/trauma and infection. Defective removal of damaged mitochondria leads to hyperactivation of inflammatory signaling pathways and subsequently to chronic systemic inflammation and development of inflammatory diseases. Here, we review the molecular mechanisms of mitophagy and highlight its critical role in the innate immune system homeostasis.

Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

PubMed

Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K

2016-09-01

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

Synthesis of Lipidated Proteins.

PubMed

Mejuch, Tom; Waldmann, Herbert

2016-08-17

Protein lipidation is one of the major post-translational modifications (PTM) of proteins. The attachment of the lipid moiety frequently determines the localization and the function of the lipoproteins. Lipidated proteins participate in many essential biological processes in eukaryotic cells, including vesicular trafficking, signal transduction, and regulation of the immune response. Malfunction of these cellular processes usually leads to various diseases such as cancer. Understanding the mechanism of cellular signaling and identifying the protein-protein and protein-lipid interactions in which the lipoproteins are involved is a crucial task. To achieve these goals, fully functional lipidated proteins are required. However, access to lipoproteins by means of standard expression is often rather limited. Therefore, semisynthetic methods, involving the synthesis of lipidated peptides and their subsequent chemoselective ligation to yield full-length lipoproteins, were developed. In this Review we summarize the commonly used methods for lipoprotein synthesis and the development of the corresponding chemoselective ligation techniques. Several key studies involving full-length semisynthetic lipidated Ras, Rheb, and LC3 proteins are presented.

Conditional Depletion of Nuclear Proteins by the Anchor Away System (ms# CP-10-0125)

PubMed Central

Fan, Xiaochun; Geisberg, Joseph V.; Wong, Koon Ho; Jin, Yi

2011-01-01

Nuclear proteins play key roles in the regulation of many important cellular processes. In Saccharomyces cerevisiae, many genes encoding nuclear proteins are essential. Here we describe a method termed Anchor Away that can be used to conditionally and rapidly deplete nuclear proteins of interest. It involves conditional export of the protein of interest out of the nucleus and its subsequent sequestration in the cytoplasm. This method can be used to simultaneously deplete multiple proteins from nucleus. PMID:21225637

Recycling to discover something new: the role of autophagy in kidney disease.

PubMed

Leventhal, Jeremy S; Wyatt, Christina M; Ross, Michael J

2017-01-01

This year, the Nobel Prize in Physiology or Medicine was awarded to Yoshinori Ohsumi for his groundbreaking work in dissecting the mechanisms of autophagy, a cellular process resulting in the organized degradation of cytoplasmic components. Ohsumi's work paved the way for subsequent studies that demonstrated critical roles for autophagy in modulating both acute and chronic kidney injury. This work may lead to future therapeutic approaches that target the autophagy system to prevent or treat kidney diseases. Published by Elsevier Inc.

Cellular processing and destinies of artificial DNA nanostructures.

PubMed

Lee, Di Sheng; Qian, Hang; Tay, Chor Yong; Leong, David Tai

2016-08-07

Since many bionanotechnologies are targeted at cells, understanding how and where their interactions occur and the subsequent results of these interactions is important. Changing the intrinsic properties of DNA nanostructures and linking them with interactions presents a holistic and powerful strategy for understanding dual nanostructure-biological systems. With the recent advances in DNA nanotechnology, DNA nanostructures present a great opportunity to understand the often convoluted mass of information pertaining to nanoparticle-biological interactions due to the more precise control over their chemistry, sizes, and shapes. Coupling just some of these designs with an understanding of biological processes is both a challenge and a source of opportunities. Despite continuous advances in the field of DNA nanotechnology, the intracellular fate of DNA nanostructures has remained unclear and controversial. Because understanding its cellular processing and destiny is a necessary prelude to any rational design of exciting and innovative bionanotechnology, in this review, we will discuss and provide a comprehensive picture relevant to the intracellular processing and the fate of various DNA nanostructures which have been remained elusive for some time. We will also link the unique capabilities of DNA to some novel ideas for developing next-generation bionanotechnologies.

Grow-ING, Age-ING and Die-ING: ING proteins link cancer, senescence and apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Russell, Michael; Berardi, Philip; Gong Wei

The INhibitor of Growth (ING) family of plant homeodomain (PHD) proteins induce apoptosis and regulate gene expression through stress-inducible binding of phospholipids with subsequent nuclear and nucleolar localization. Relocalization occurs concomitantly with interaction with a subset of nuclear proteins, including PCNA, p53 and several regulators of acetylation such as the p300/CBP and PCAF histone acetyltransferases (HATs), as well as the histone deacetylases HDAC1 and hSir2. These interactions alter the localized state of chromatin compaction, subsequently affecting the expression of subsets of genes, including those associated with the stress response (Hsp70), apoptosis (Bax, MDM2) and cell cycle regulation (p21{sup WAF1}, cyclinmore » B) in a cell- and tissue-specific manner. The expression levels and subcellular localization of ING proteins are altered in a significant number of human cancer types, while the expression of ING isoforms changes during cellular aging, suggesting that ING proteins may play a role in linking cellular transformation and replicative senescence. The variety of functions attributed to ING proteins suggest that this tumor suppressor serves to link the disparate processes of cell cycle regulation, cell suicide and cellular aging through epigenetic regulation of gene expression. This review examines recent findings in the ING field with a focus on the functions of protein-protein interactions involving ING family members and the mechanisms by which these interactions facilitate the various roles that ING proteins play in tumorigenesis, apoptosis and senescence.« less

Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

PubMed Central

Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

2016-01-01

Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface. PMID:27353000

Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

NASA Astrophysics Data System (ADS)

Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

2016-06-01

Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface.

Molecular mechanisms regulating formation, trafficking and processing of annular gap junctions.

PubMed

Falk, Matthias M; Bell, Cheryl L; Kells Andrews, Rachael M; Murray, Sandra A

2016-05-24

Internalization of gap junction plaques results in the formation of annular gap junction vesicles. The factors that regulate the coordinated internalization of the gap junction plaques to form annular gap junction vesicles, and the subsequent events involved in annular gap junction processing have only relatively recently been investigated in detail. However it is becoming clear that while annular gap junction vesicles have been demonstrated to be degraded by autophagosomal and endo-lysosomal pathways, they undergo a number of additional processing events. Here, we characterize the morphology of the annular gap junction vesicle and review the current knowledge of the processes involved in their formation, fission, fusion, and degradation. In addition, we address the possibility for connexin protein recycling back to the plasma membrane to contribute to gap junction formation and intercellular communication. Information on gap junction plaque removal from the plasma membrane and the subsequent processing of annular gap junction vesicles is critical to our understanding of cell-cell communication as it relates to events regulating development, cell homeostasis, unstable proliferation of cancer cells, wound healing, changes in the ischemic heart, and many other physiological and pathological cellular phenomena.

Electromagnetic interference of bone-anchored hearing aids by cellular phones.

PubMed

Kompis, M; Negri, S; Häusler, R

2000-10-01

We report a case of electromagnetic interference between a bone-anchored hearing aid (BAHA) and a cellular phone. A 54-year-old women was successfully treated for severe mixed conductive and sensorineural hearing loss with a BAHA. Five years after implantation, the patient experienced a sudden feeling of dizziness, accompanied by a loud buzzing sound and by a sensation of head pressure while examining a digital mobile phone. During a subsequent experiment, the buzzing sound could be reproduced and was identified as electromagnetic interference between the BAHA and digital cellular phones. Seventeen adult BAHA users from our clinic participated in a subsequent survey. Of the 13 patients with some experience of digital cellular phones, 11 reported hearing annoying noises elicited by these devices. However, no other sensation, such as dizziness, was described. Owing to the increasing number of users of both hearing aids and cellular phones, the incidence of electromagnetic interference must be expected to increase as well. Although to date there is no evidence that such interference may be harmful or dangerous to users of conventional or bone-anchored hearing aids, unexpected interference can be a frightening experience.

dropEst: pipeline for accurate estimation of molecular counts in droplet-based single-cell RNA-seq experiments.

PubMed

Petukhov, Viktor; Guo, Jimin; Baryawno, Ninib; Severe, Nicolas; Scadden, David T; Samsonova, Maria G; Kharchenko, Peter V

2018-06-19

Recent single-cell RNA-seq protocols based on droplet microfluidics use massively multiplexed barcoding to enable simultaneous measurements of transcriptomes for thousands of individual cells. The increasing complexity of such data creates challenges for subsequent computational processing and troubleshooting of these experiments, with few software options currently available. Here, we describe a flexible pipeline for processing droplet-based transcriptome data that implements barcode corrections, classification of cell quality, and diagnostic information about the droplet libraries. We introduce advanced methods for correcting composition bias and sequencing errors affecting cellular and molecular barcodes to provide more accurate estimates of molecular counts in individual cells.

Movies of cellular and sub-cellular motion by digital holographic microscopy.

PubMed

Mann, Christopher J; Yu, Lingfeng; Kim, Myung K

2006-03-23

Many biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with sub-wavelength accuracy. Digital holography is an emergent phase contrast technique that offers an excellent approach in obtaining both qualitative and quantitative phase information from the hologram. A CCD camera is used to record a hologram onto a computer and numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. Another attractive feature of digital holography is the ability to focus on multiple focal planes from a single hologram, emulating the focusing control of a conventional microscope. A modified Mach-Zender off-axis setup in transmission is used to record and reconstruct a number of holographic amplitude and phase images of cellular and sub-cellular features. Both cellular and sub-cellular features are imaged with sub-micron, diffraction-limited resolution. Movies of holographic amplitude and phase images of living microbes and cells are created from a series of holograms and reconstructed with numerically adjustable focus, so that the moving object can be accurately tracked with a reconstruction rate of 300ms for each hologram. The holographic movies show paramecium swimming among other microbes as well as displaying some of their intracellular processes. A time lapse movie is also shown for fibroblast cells in the process of migration. Digital holography and movies of digital holography are seen to be useful new tools for visualization of dynamic processes in biological microscopy. Phase imaging digital holography is a promising technique in terms of the lack of coherent noise and the precision with which the optical thickness of a sample can be profiled, which can lead to images with an axial resolution of a few nanometres.

The role of focal adhesion kinase in the regulation of cellular mechanical properties

NASA Astrophysics Data System (ADS)

Mierke, Claudia Tanja

2013-12-01

The regulation of mechanical properties is necessary for cell invasion into connective tissue or intra- and extravasation through the endothelium of blood or lymph vessels. Cell invasion is important for the regulation of many healthy processes such as immune response reactions and wound healing. In addition, cell invasion plays a role in disease-related processes such as tumor metastasis and autoimmune responses. Until now the role of focal adhesion kinase (FAK) in regulating mechanical properties of cells and its impact on cell invasion efficiency is still not well known. Thus, this review focuses on mechanical properties regulated by FAK in comparison to the mechano-regulating protein vinculin. Moreover, it points out the connection between cancer cell invasion and metastasis and FAK by showing that FAK regulates cellular mechanical properties required for cellular motility. Furthermore, it sheds light on the indirect interaction of FAK with vinculin by binding to paxillin, which then impairs the binding of paxillin to vinculin. In addition, this review emphasizes whether FAK fulfills regulatory functions similar to vinculin. In particular, it discusses the differences and the similarities between FAK and vinculin in regulating the biomechanical properties of cells. Finally, this paper highlights that both focal adhesion proteins, vinculin and FAK, synergize their functions to regulate the mechanical properties of cells such as stiffness and contractile forces. Subsequently, these mechanical properties determine cellular invasiveness into tissues and provide a source sink for future drug developments to inhibit excessive cell invasion and hence, metastases formation.

In Vitro Experimental Model for the Long-Term Analysis of Cellular Dynamics During Bronchial Tree Development from Lung Epithelial Cells

PubMed Central

Maruta, Naomichi; Marumoto, Moegi

2017-01-01

Lung branching morphogenesis has been studied for decades, but the underlying developmental mechanisms are still not fully understood. Cellular movements dynamically change during the branching process, but it is difficult to observe long-term cellular dynamics by in vivo or tissue culture experiments. Therefore, developing an in vitro experimental model of bronchial tree would provide an essential tool for developmental biology, pathology, and systems biology. In this study, we succeeded in reconstructing a bronchial tree in vitro by using primary human bronchial epithelial cells. A high concentration gradient of bronchial epithelial cells was required for branching initiation, whereas homogeneously distributed endothelial cells induced the formation of successive branches. Subsequently, the branches grew in size to the order of millimeter. The developed model contains only two types of cells and it facilitates the analysis of lung branching morphogenesis. By taking advantage of our experimental model, we carried out long-term time-lapse observations, which revealed self-assembly, collective migration with leader cells, rotational motion, and spiral motion of epithelial cells in each developmental event. Mathematical simulation was also carried out to analyze the self-assembly process and it revealed simple rules that govern cellular dynamics. Our experimental model has provided many new insights into lung development and it has the potential to accelerate the study of developmental mechanisms, pattern formation, left–right asymmetry, and disease pathogenesis of the human lung. PMID:28471293

CPSF30 at the Interface of Alternative Polyadenylation and Cellular Signaling in Plants

PubMed Central

Chakrabarti, Manohar; Hunt, Arthur G.

2015-01-01

Post-transcriptional processing, involving cleavage of precursor messenger RNA (pre mRNA), and further incorporation of poly(A) tail to the 3' end is a key step in the expression of genetic information. Alternative polyadenylation (APA) serves as an important check point for the regulation of gene expression. Recent studies have shown widespread prevalence of APA in diverse systems. A considerable amount of research has been done in characterizing different subunits of so-called Cleavage and Polyadenylation Specificity Factor (CPSF). In plants, CPSF30, an ortholog of the 30 kD subunit of mammalian CPSF is a key polyadenylation factor. CPSF30 in the model plant Arabidopsis thaliana was reported to possess unique biochemical properties. It was also demonstrated that poly(A) site choice in a vast majority of genes in Arabidopsis are CPSF30 dependent, suggesting a pivotal role of this gene in APA and subsequent regulation of gene expression. There are also indications of this gene being involved in oxidative stress and defense responses and in cellular signaling, suggesting a role of CPSF30 in connecting physiological processes and APA. This review will summarize the biochemical features of CPSF30, its role in regulating APA, and possible links with cellular signaling and stress response modules. PMID:26061761

Contribution of vascular cell-derived cytokines to innate and inflammatory pathways in atherogenesis

PubMed Central

Loppnow, Harald; Buerke, Michael; Werdan, Karl; Rose-John, Stefan

2011-01-01

Abstract Inflammation is a central element of atherogenesis. Innate pathways contribute to vascular inflammation. However, the initial molecular process(es) starting atherogenesis remain elusive. The various risk factors, represented by particular compounds (activators), may cause altered cellular functions in the endothelium (e.g. vascular endothelial cell activation or -dysfunction), in invading cells (e.g. inflammatory mediator production) or in local vessel wall cells (e.g. inflammatory mediators, migration), thereby triggering the innate inflammatory process. The cellular components of innate immunology include granulocytes, natural killer cells and monocytes. Among the molecular innate constituents are innate molecules, such as the toll-like receptors or innate cytokines. Interleukin-1 (IL-1) and IL-6 are among the innate cytokines. Cytokines are potent activators of a great number of cellular functions relevant to maintain or commove homeostasis of the vessel wall. Within the vessel wall, vascular smooth muscle cells (SMCs) can significantly contribute to the cytokine-dependent inflammatory network by: (i) production of cytokines, (ii) response to cytokines and (iii) cytokine-mediated interaction with invading leucocytes. The cytokines IL-1 and IL-6 are involved in SMC-leucocyte interaction. The IL-6 effects are proposed to be mediated by trans-signalling. Dysregulated cellular functions resulting from dysregulated cytokine production may be the cause of cell accumulation, subsequent low-density lipoprotein accumulation and deposition of extracellular matrix (ECM). The deposition of ECM, increased accumulation of leucocytes and altered levels of inflammatory mediators may constitute an ‘innate-immunovascular-memory’ resulting in an ever-growing response to anew invasion. Thus, SMC-fostered inflammation, promoted by invading innate cells, may be a potent component for development and acceleration of atherosclerosis. PMID:21199323

Complex furrows in a 2D epithelial sheet code the 3D structure of a beetle horn.

PubMed

Matsuda, Keisuke; Gotoh, Hiroki; Tajika, Yuki; Sushida, Takamichi; Aonuma, Hitoshi; Niimi, Teruyuki; Akiyama, Masakazu; Inoue, Yasuhiro; Kondo, Shigeru

2017-10-24

The external organs of holometabolous insects are generated through two consecutive processes: the development of imaginal primordia and their subsequent transformation into the adult structures. During the latter process, many different phenomena at the cellular level (e.g. cell shape changes, cell migration, folding and unfolding of epithelial sheets) contribute to the drastic changes observed in size and shape. Because of this complexity, the logic behind the formation of the 3D structure of adult external organs remains largely unknown. In this report, we investigated the metamorphosis of the horn in the Japanese rhinoceros beetle Trypoxylus dichotomus. The horn primordia is essentially a 2D epithelial cell sheet with dense furrows. We experimentally unfolded these furrows using three different methods and found that the furrow pattern solely determines the 3D horn structure, indicating that horn formation in beetles occurs by two distinct processes: formation of the furrows and subsequently unfolding them. We postulate that this developmental simplicity offers an inherent advantage to understanding the principles that guide 3D morphogenesis in insects.

Quantification of substrate and cellular strains in stretchable 3D cell cultures: an experimental and computational framework.

PubMed

González-Avalos, P; Mürnseer, M; Deeg, J; Bachmann, A; Spatz, J; Dooley, S; Eils, R; Gladilin, E

2017-05-01

The mechanical cell environment is a key regulator of biological processes . In living tissues, cells are embedded into the 3D extracellular matrix and permanently exposed to mechanical forces. Quantification of the cellular strain state in a 3D matrix is therefore the first step towards understanding how physical cues determine single cell and multicellular behaviour. The majority of cell assays are, however, based on 2D cell cultures that lack many essential features of the in vivo cellular environment. Furthermore, nondestructive measurement of substrate and cellular mechanics requires appropriate computational tools for microscopic image analysis and interpretation. Here, we present an experimental and computational framework for generation and quantification of the cellular strain state in 3D cell cultures using a combination of 3D substrate stretcher, multichannel microscopic imaging and computational image analysis. The 3D substrate stretcher enables deformation of living cells embedded in bead-labelled 3D collagen hydrogels. Local substrate and cell deformations are determined by tracking displacement of fluorescent beads with subsequent finite element interpolation of cell strains over a tetrahedral tessellation. In this feasibility study, we debate diverse aspects of deformable 3D culture construction, quantification and evaluation, and present an example of its application for quantitative analysis of a cellular model system based on primary mouse hepatocytes undergoing transforming growth factor (TGF-β) induced epithelial-to-mesenchymal transition. © 2017 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Device and method for automated separation of a sample of whole blood into aliquots

DOEpatents

Burtis, Carl A.; Johnson, Wayne F.

1989-01-01

A device and a method for automated processing and separation of an unmeasured sample of whole blood into multiple aliquots of plasma. Capillaries are radially oriented on a rotor, with the rotor defining a sample chamber, transfer channels, overflow chamber, overflow channel, vent channel, cell chambers, and processing chambers. A sample of whole blood is placed in the sample chamber, and when the rotor is rotated, the blood moves outward through the transfer channels to the processing chambers where the blood is centrifugally separated into a solid cellular component and a liquid plasma component. When the rotor speed is decreased, the plasma component backfills the capillaries resulting in uniform aliquots of plasma which may be used for subsequent analytical procedures.

A Process for Manufacturing Metal-Ceramic Cellular Materials with Designed Mesostructure

NASA Astrophysics Data System (ADS)

Snelling, Dean Andrew, Jr.

The goal of this work is to develop and characterize a manufacturing process that is able to create metal matrix composites with complex cellular geometries. The novel manufacturing method uses two distinct additive manufacturing processes: i) fabrication of patternless molds for cellular metal castings and ii) printing an advanced cellular ceramic for embedding in a metal matrix. However, while the use of AM greatly improves the freedom in the design of MMCs, it is important to identify the constraints imposed by the process and its process relationships. First, the author investigates potential differences in material properties (microstructure, porosity, mechanical strength) of A356 - T6 castings resulting from two different commercially available Binder Jetting media and traditional "no-bake" silica sand. It was determined that they yielded statistically equivalent results in four of the seven tests performed: dendrite arm spacing, porosity, surface roughness, and tensile strength. They differed in sand tensile strength, hardness, and density. Additionally, two critical sources of process constraints on part geometry are examined: (i) depowdering unbound material from intricate casting channels and (ii) metal flow and solidification distances through complex mold geometries. A Taguchi Design of Experiments is used to determine the relationships of important independent variables of each constraint. For depowdering, a minimum cleaning diameter of 3 mm was determined along with an equation relating cleaning distance as a function of channel diameter. Furthermore, for metal flow, choke diameter was found to be significantly significant variable. Finally, the author presents methods to process complex ceramic structure from precursor powders via Binder Jetting AM technology to incorporate into a bonded sand mold and the subsequently casted metal matrix. Through sintering experiments, a sintering temperature of 1375°C was established for the ceramic insert (78% cordierite). Upon printing and sintering the iii ceramic, three point bend tests showed the MMCs had less strength than the matrix material likely due to the relatively high porosity developed in the body. Additionally, it was found that the ceramic metal interface had minimal mechanical interlocking and chemical bonding limiting the strength of the final MMCs.

Extracellular vesicles and their synthetic analogues in aging and age-associated brain diseases

PubMed Central

Smith, J. A.; Leonardi, T.; Huang, B.; Iraci, N.; Vega, B.; Pluchino, S.

2015-01-01

Multicellular organisms rely upon diverse and complex intercellular communications networks for a myriad of physiological processes. Disruption of these processes is implicated in the onset and propagation of disease and disorder, including the mechanisms of senescence at both cellular and organismal levels. In recent years, secreted extracellular vesicles (EVs) have been identified as a particularly novel vector by which cell-to-cell communications are enacted. EVs actively and specifically traffic bioactive proteins, nucleic acids, and metabolites between cells at local and systemic levels, modulating cellular responses in a bidirectional manner under both homeostatic and pathological conditions. EVs are being implicated not only in the generic aging process, but also as vehicles of pathology in a number of age-related diseases, including cancer and neurodegenerative and disease. Thus, circulating EVs—or specific EV cargoes—are being utilised as putative biomarkers of disease. On the other hand, EVs, as targeted intercellular shuttles of multipotent bioactive payloads, have demonstrated promising therapeutic properties, which can potentially be modulated and enhanced through cellular engineering. Furthermore, there is considerable interest in employing nanomedicinal approaches to mimic the putative therapeutic properties of EVs by employing synthetic analogues for targeted drug delivery. Herein we describe what is known about the origin and nature of EVs and subsequently review their putative roles in biology and medicine (including the use of synthetic EV analogues), with a particular focus on their role in aging and age-related brain diseases. PMID:24973266

Extracellular vesicles and their synthetic analogues in aging and age-associated brain diseases.

PubMed

Smith, J A; Leonardi, T; Huang, B; Iraci, N; Vega, B; Pluchino, S

2015-04-01

Multicellular organisms rely upon diverse and complex intercellular communications networks for a myriad of physiological processes. Disruption of these processes is implicated in the onset and propagation of disease and disorder, including the mechanisms of senescence at both cellular and organismal levels. In recent years, secreted extracellular vesicles (EVs) have been identified as a particularly novel vector by which cell-to-cell communications are enacted. EVs actively and specifically traffic bioactive proteins, nucleic acids, and metabolites between cells at local and systemic levels, modulating cellular responses in a bidirectional manner under both homeostatic and pathological conditions. EVs are being implicated not only in the generic aging process, but also as vehicles of pathology in a number of age-related diseases, including cancer and neurodegenerative and disease. Thus, circulating EVs-or specific EV cargoes-are being utilised as putative biomarkers of disease. On the other hand, EVs, as targeted intercellular shuttles of multipotent bioactive payloads, have demonstrated promising therapeutic properties, which can potentially be modulated and enhanced through cellular engineering. Furthermore, there is considerable interest in employing nanomedicinal approaches to mimic the putative therapeutic properties of EVs by employing synthetic analogues for targeted drug delivery. Herein we describe what is known about the origin and nature of EVs and subsequently review their putative roles in biology and medicine (including the use of synthetic EV analogues), with a particular focus on their role in aging and age-related brain diseases.

Molecular Genetics of Ubiquinone Biosynthesis in Animals

PubMed Central

Wang, Ying; Hekimi, Siegfried

2014-01-01

Ubiquinone (UQ), also known as coenzyme Q (CoQ), is a redox-active lipid present in all cellular membranes where it functions in a variety of cellular processes. The best known functions of UQ are to act as a mobile electron carrier in the mitochondrial respiratory chain and to serve as a lipid soluble antioxidant in cellular membranes. All eukaryotic cells synthesize their own UQ. Most of the current knowledge on the UQ biosynthetic pathway was obtained by studying Escherichia coli and S. cerevisiae UQ-deficient mutants. The orthologues of all the genes known from yeast studies to be involved in UQ biosynthesis have subsequently been found in higher organisms. Animal mutants with different genetic defects in UQ biosynthesis display very different phenotypes, despite the fact that in all these mutants the same biosynthetic pathway is affected. This review summarizes the present knowledge of the eukaryotic biosynthesis of UQ, with focus on the biosynthetic genes identified in animals, including C. elegans, rodents and humans. Moreover, we review the phenotypes of mutants in these genes and discuss the functional consequences of UQ deficiency in general. PMID:23190198

Biomechanics and Thermodynamics of Nanoparticle Interactions with Plasma and Endosomal Membrane Lipids in Cellular Uptake and Endosomal Escape

PubMed Central

2015-01-01

To be effective for cytoplasmic delivery of therapeutics, nanoparticles (NPs) taken up via endocytic pathways must efficiently transport across the cell membrane and subsequently escape from the secondary endosomes. We hypothesized that the biomechanical and thermodynamic interactions of NPs with plasma and endosomal membrane lipids are involved in these processes. Using model plasma and endosomal lipid membranes, we compared the interactions of cationic NPs composed of poly(d,l-lactide-co-glycolide) modified with the dichain surfactant didodecyldimethylammonium bromide (DMAB) or the single-chain surfactant cetyltrimethylammonium bromide (CTAB) vs anionic unmodified NPs of similar size. We validated our hypothesis in doxorubicin-sensitive (MCF-7, with relatively fluid membranes) and resistant breast cancer cells (MCF-7/ADR, with rigid membranes). Despite their cationic surface charges, DMAB- and CTAB-modified NPs showed different patterns of biophysical interaction: DMAB-modified NPs induced bending of the model plasma membrane, whereas CTAB-modified NPs condensed the membrane, thereby resisted bending. Unmodified NPs showed no effects on bending. DMAB-modified NPs also induced thermodynamic instability of the model endosomal membrane, whereas CTAB-modified and unmodified NPs had no effect. Since bending of the plasma membrane and destabilization of the endosomal membrane are critical biophysical processes in NP cellular uptake and endosomal escape, respectively, we tested these NPs for cellular uptake and drug efficacy. Confocal imaging showed that in both sensitive and resistant cells DMAB-modified NPs exhibited greater cellular uptake and escape from endosomes than CTAB-modified or unmodified NPs. Further, paclitaxel-loaded DMAB-modified NPs induced greater cytotoxicity even in resistant cells than CTAB-modified or unmodified NPs or drug in solution, demonstrating the potential of DMAB-modified NPs to overcome the transport barrier in resistant cells. In conclusion, biomechanical interactions with membrane lipids are involved in cellular uptake and endosomal escape of NPs. Biophysical interaction studies could help us better understand the role of membrane lipids in cellular uptake and intracellular trafficking of NPs. PMID:24911361

Prostate organogenesis: tissue induction, hormonal regulation and cell type specification

PubMed Central

Toivanen, Roxanne

2017-01-01

Prostate organogenesis is a complex process that is primarily mediated by the presence of androgens and subsequent mesenchyme-epithelial interactions. The investigation of prostate development is partly driven by its potential relevance to prostate cancer, in particular the apparent re-awakening of key developmental programs that occur during tumorigenesis. However, our current knowledge of the mechanisms that drive prostate organogenesis is far from complete. Here, we provide a comprehensive overview of prostate development, focusing on recent findings regarding sexual dimorphism, bud induction, branching morphogenesis and cellular differentiation. PMID:28400434

Lipid Accumulation during the Establishment of Kleptoplasty in Elysia chlorotica

PubMed Central

Pelletreau, Karen N.; Weber, Andreas P. M.; Weber, Katrin L.; Rumpho, Mary E.

2014-01-01

The establishment of kleptoplasty (retention of “stolen plastids”) in the digestive tissue of the sacoglossan Elysia chlorotica Gould was investigated using transmission electron microscopy. Cellular processes occurring during the initial exposure to plastids were observed in laboratory raised animals ranging from 1–14 days post metamorphosis (dpm). These observations revealed an abundance of lipid droplets (LDs) correlating to plastid abundance. Starvation of animals resulted in LD and plastid decay in animals <5 dpm that had not yet achieved permanent kleptoplasty. Animals allowed to feed on algal prey (Vaucheria litorea C. Agardh) for 7 d or greater retained stable plastids resistant to cellular breakdown. Lipid analysis of algal and animal samples supports that these accumulating LDs may be of plastid origin, as the often algal-derived 20∶5 eicosapentaenoic acid was found in high abundance in the animal tissue. Subsequent culturing of animals in dark conditions revealed a reduced ability to establish permanent kleptoplasty in the absence of photosynthetic processes, coupled with increased mortality. Together, these data support an important role of photosynthetic lipid production in establishing and stabilizing this unique animal kleptoplasty. PMID:24828251

Focused Metabolite Profiling for Dissecting Cellular and Molecular Processes of Living Organisms in Space Environments

NASA Technical Reports Server (NTRS)

2008-01-01

Regulatory control in biological systems is exerted at all levels within the central dogma of biology. Metabolites are the end products of all cellular regulatory processes and reflect the ultimate outcome of potential changes suggested by genomics and proteomics caused by an environmental stimulus or genetic modification. Following on the heels of genomics, transcriptomics, and proteomics, metabolomics has become an inevitable part of complete-system biology because none of the lower "-omics" alone provide direct information about how changes in mRNA or protein are coupled to changes in biological function. The challenges are much greater than those encountered in genomics because of the greater number of metabolites and the greater diversity of their chemical structures and properties. To meet these challenges, much developmental work is needed, including (1) methodologies for unbiased extraction of metabolites and subsequent quantification, (2) algorithms for systematic identification of metabolites, (3) expertise and competency in handling a large amount of information (data set), and (4) integration of metabolomics with other "omics" and data mining (implication of the information). This article reviews the project accomplishments.

Developmental consequences of cryopreservation of mammalian oocytes and embryos.

PubMed

Smith, Gary D; Silva E Silva, Cristine Ane

2004-08-01

During the last three decades, significant advances have been made in successful cryopreservation of mammalian preimplantation embryos, and more recently oocytes. The ability to cryopreserve, thaw, and establish pregnancies with supernumerary preimplantation embryos has become an important tool in fertility treatment. Human oocyte cryopreservation has practical application in preserving fertility for individuals at risk of compromised egg quality due to cancer treatments or advanced maternal age. While oocyte/embryo cryopreservation success has increased over time, there is still room for improvement. Oocytes and embryos are susceptible to cryo-damage, which collectively entails cellular damage caused by mechanical, chemical, or thermal forces during the freeze-thaw process. Basic studies focused on understanding cellular structures, their composition, and more importantly their functions, in normal cell developments will continue to be critical in assessing, understanding, and correcting oocyte/embryo cryo-damage. This review will delineate many of the oocyte/embryo intracellular and extracellular structures that are or may be compromised during cryopreservation. A global theme presented throughout this review is that many structural components of the oocyte/embryo also have essential functional roles in development. Compromising these cellular structures, and thus their cellular homeostatic functions, can deleteriously influence initial cryo-survival or compromise subsequent normal development through effects on the oocyte and/or early embryo.

Macromolecular Expression and Function: A New Paradigm for NASA Risk Assessment

NASA Technical Reports Server (NTRS)

Richmond, Robert

2003-01-01

Predicting risks in humans of either acute effects such as bone loss or muscle wasting, or late effects such as cancer, is challenging. To an approximation, this is because uncertainties of exposure to stress factors or toxic agents and the uniformity of processing subsequent damage at the cellular level within a complex set of biological variables degrade the confidence of predicting pathologic outcome. A cellular biodosimeter that simultaneously reports 1) the type of damage due to that exposure, 2) the quantity of damage incurred by that exposure, and 3) the dataset used to assess risk of developing pathologic outcome caused by that exposure would therefore be useful for predicting ultimate risks faced by an individual, such as an astronaut. It is suggested that such a biodosimeter can be based upon analyses of gene-expression and protein expression whereby large datasets of cellular response to damage are obtained and analyzed for expression-profiles correlated with established end points and molecular markers predictive for risks being assessed. The usefulness of multiparametric cellular biodosimeters could be realized by quantitatively profiling these datasets using techniques of bioinformatics. Such an approach contributes to the foundation of molecular epidemiology as a new scientific discipline, and represents a new paradigm of risk assessment.

Gaseous VOCs rapidly modify particulate matter and its biological effects – Part 1: Simple VOCs and model PM

PubMed Central

Ebersviller, S.; Lichtveld, K.; Sexton, K. G.; Zavala, J.; Lin, Y-H.; Jaspers, I.; Jeffries, H. E.

2013-01-01

This is the first of a three-part study designed to demonstrate dynamic entanglements among gaseous organic compounds (VOC), particulate matter (PM), and their subsequent potential biological effects. We study these entanglements in increasingly complex VOC and PM mixtures in urban-like conditions in a large outdoor chamber. To the traditional chemical and physical characterizations of gas and PM, we added new measurements of gas-only- and PM-only-biological effects, using cultured human lung cells as model indicators. These biological effects are assessed here as increases in cellular damage or expressed irritation (i.e., cellular toxic effects) from cells exposed to chamber air relative to cells exposed to clean air. The exposure systems permit gas-only- or PM-only-exposures from the same air stream containing both gases and PM in equilibria, i.e., there are no extractive operations prior to cell exposure. Our simple experiments in this part of the study were designed to eliminate many competing atmospheric processes to reduce ambiguity in our results. Simple volatile and semi-volatile organic gases that have inherent cellular toxic properties were tested individually for biological effect in the dark (at constant humidity). Airborne mixtures were then created with each compound and PM that has no inherent cellular toxic properties for another cellular exposure. Acrolein and p-tolualdehyde were used as model VOCs and mineral oil aerosol (MOA) was selected as a surrogate for organic-containing PM. MOA is appropriately complex in composition to represent ambient PM, and it exhibits no inherent cellular toxic effects and thus did not contribute any biological detrimental effects on its own. Chemical measurements, combined with the responses of our biological exposures, clearly demonstrate that gas-phase pollutants can modify the composition of PM (and its resulting detrimental effects on lung cells) – even if the gas-phase pollutants are not considered likely to partition to the condensed phase: the VOC-modified-PM showed significantly more damage and inflammation to lung cells than did the original PM. Because gases and PM are transported and deposited differently within the atmosphere and the lungs, these results have significant consequences. For example, current US policies for research and regulation of PM do not recognize this “effect modification” phenomena (NAS, 2004). These results present an unambiguous demonstration that – even in these simple mixtures – physical and thermal interactions alone can cause a modification of the distribution of species among the phases of airborne pollution mixtures and can result in a non-toxic phase becoming toxic due to atmospheric thermal processes only. Subsequent work extends the simple results reported here to systems with photochemical transformations of complex urban mixtures and to systems with diesel exhaust produced by different fuels. PMID:23457430

Studying Catabolism of Protein ADP-Ribosylation.

PubMed

Palazzo, Luca; James, Dominic I; Waddell, Ian D; Ahel, Ivan

2017-01-01

Protein ADP-ribosylation is a conserved posttranslational modification that regulates many major cellular functions, such as DNA repair, transcription, translation, signal transduction, stress response, cell division, aging, and cell death. Protein ADP-ribosyl transferases catalyze the transfer of an ADP-ribose (ADPr) group from the β-nicotinamide adenine dinucleotide (β-NAD + ) cofactor onto a specific target protein with the subsequent release of nicotinamide. ADP-ribosylation leads to changes in protein structure, function, stability, and localization, thus defining the appropriate cellular response. Signaling processes that are mediated by modifications need to be finely tuned and eventually silenced and one of the ways to achieve this is through the action of enzymes that remove (reverse) protein ADP-ribosylation in a timely fashion such as PARG, TARG1, MACROD1, and MACROD2. Here, we describe several basic methods used to study the enzymatic activity of de-ADP-ribosylating enzymes.

Digital gene expression analysis in hemocytes of the white shrimp Litopenaeus vannamei in response to low salinity stress.

PubMed

Zhao, Qun; Pan, Luqing; Ren, Qin; Hu, Dongxu

2015-02-01

The white shrimp Litopenaeus vannamei has been greatly impacted by low salinity stress. To gain knowledge on the immune response in L. vannamei under such stress, we investigated digital gene expression (DEG) in L. vannamei hemocytes using the deep-sequencing platform Illumina HiSeq 2000. In total, 38,155 high quality unigenes with average length 770 bp were generated; 145 and 79 genes were identified up- or down-regulated, respectively. Functional categorization and pathways of the differentially expressed genes revealed that immune signaling pathways, cellular immunity, humoral immunity, apoptosis, cellular protein synthesis, lipid transport and energy metabolism were the differentially regulated processes occurring during low salinity stress. These results will provide a resource for subsequent gene expression studies regarding environmental stress and a valuable gene information for a better understanding of immune mechanisms of L. vannamei under low salinity stress. Copyright © 2014 Elsevier Ltd. All rights reserved.

Anchored but not internalized: shape dependent endocytosis of nanodiamond

NASA Astrophysics Data System (ADS)

Zhang, Bokai; Feng, Xi; Yin, Hang; Ge, Zhenpeng; Wang, Yanhuan; Chu, Zhiqin; Raabova, Helena; Vavra, Jan; Cigler, Petr; Liu, Renbao; Wang, Yi; Li, Quan

2017-04-01

Nanoparticle-cell interactions begin with the cellular uptake of the nanoparticles, a process that eventually determines their cellular fate. In the present work, we show that the morphological features of nanodiamonds (NDs) affect both the anchoring and internalization stages of their endocytosis. While a prickly ND (with sharp edges/corners) has no trouble of anchoring onto the plasma membrane, it suffers from difficult internalization afterwards. In comparison, the internalization of a round ND (obtained by selective etching of the prickly ND) is not limited by its lower anchoring amount and presents a much higher endocytosis amount. Molecular dynamics simulation and continuum modelling results suggest that the observed difference in the anchoring of round and prickly NDs likely results from the reduced contact surface area with the cell membrane of the former, while the energy penalty associated with membrane curvature generation, which is lower for a round ND, may explain its higher probability of the subsequent internalization.

Ribose in the heart.

PubMed

Herrick, James; St Cyr, John

2008-01-01

Every cell needs energy, i.e., adenosine triphosphate (ATP), to carry out its function. Decreased oxygen levels, decreased blood flow, and other stressful conditions can drastically effect the intracellular concentrations of these energy compounds. Skeletal muscle, unlike the heart, can address this drop in ATP by employing the myokinase reaction, ultimately producing ATP with a subsequent elevation in adenosine monophosphate (AMP). Ribose, a naturally occurring 5-carbon monosaccharide, is a key component of RNA, DNA (which has deoxyribose), acetyl coenzyme A, and ATP. Each cell produces its own ribose, involved in the pentose phosphate pathway (PPP), to aid in ATP production. States of ischemia and/or hypoxia can severely lower levels of cellular energy compounds in the heart, with an associated compromise in cellular processes, ultimately reflected in altered function. Ribose appears to provide a solution to the problem in replenishing the depressed ATP levels and improving functional status of patients afflicted with cardiovascular diseases.

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

PubMed Central

Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

2015-01-01

ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm. PMID:26041286

Cellular stress responses to chronic heat shock and shell damage in temperate Mya truncata.

PubMed

Sleight, Victoria A; Peck, Lloyd S; Dyrynda, Elisabeth A; Smith, Valerie J; Clark, Melody S

2018-05-12

Acclimation, via phenotypic flexibility, is a potential means for a fast response to climate change. Understanding the molecular mechanisms underpinning phenotypic flexibility can provide a fine-scale cellular understanding of how organisms acclimate. In the last 30 years, Mya truncata populations around the UK have faced an average increase in sea surface temperature of 0.7 °C and further warming of between 1.5 and 4 °C, in all marine regions adjacent to the UK, is predicted by the end of the century. Hence, data are required on the ability of M. truncata to acclimate to physiological stresses, and most notably, chronic increases in temperature. Animals in the present study were exposed to chronic heat-stress for 2 months prior to shell damage and subsequently, only 3, out of 20 damaged individuals, were able to repair their shells within 2 weeks. Differentially expressed genes (between control and damaged animals) were functionally enriched with processes relating to cellular stress, the immune response and biomineralisation. Comparative transcriptomics highlighted genes, and more broadly molecular mechanisms, that are likely to be pivotal in this lack of acclimation. This study demonstrates that discovery-led transcriptomic profiling of animals during stress-response experiments can shed light on the complexity of biological processes and changes within organisms that can be more difficult to detect at higher levels of biological organisation.

Impact of uranium (U) on the cellular glutathione pool and resultant consequences for the redox status of U.

PubMed

Viehweger, Katrin; Geipel, Gerhard; Bernhard, Gert

2011-12-01

Uranium (U) as a redox-active heavy metal can cause various redox imbalances in plant cells. Measurements of the cellular glutathione/glutathione disulfide (GSH/GSSG) by HPLC after cellular U contact revealed an interference with this essential redox couple. The GSH content remained unaffected by 10 μM U whereas the GSSG level immediately increased. In contrast, higher U concentrations (50 μM) drastically raised both forms. Using the Nernst equation, it was possible to calculate the half-cell reduction potential of 2GSH/GSSG. In case of lower U contents the cellular redox environment shifted towards more oxidizing conditions whereas the opposite effect was obtained by higher U contents. This indicates that U contact causes a consumption of reduced redox equivalents. Artificial depletion of GSH by chlorodinitrobenzene and measuring the cellular reducing capacity by tetrazolium salt reduction underlined the strong requirement of reduced redox equivalents. An additional element of cellular U detoxification mechanisms is the complex formation between the heavy metal and carboxylic functionalities of GSH. Because two GSH molecules catalyze electron transfers each with one electron forming a dimer (GSSG) two UO(2) (2+) are reduced to each UO(2) (+) by unbound redox sensitive sulfhydryl moieties. UO(2) (+) subsequently disproportionates to UO(2) (2+) and U(4+). This explains that in vitro experiments revealed a reduction to U(IV) of only around 33% of initial U(VI). Cellular U(IV) was transiently detected with the highest level after 2 h of U contact. Hence, it can be proposed that these reducing processes are an important element of defense reactions induced by this heavy metal.

Potential coordination role between O-GlcNAcylation and epigenetics.

PubMed

Wu, Donglu; Cai, Yong; Jin, Jingji

2017-10-01

Dynamic changes of the post-translational O-GlcNAc modification (O-GlcNAcylation) are controlled by O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) and the glycoside hydrolase O-GlcNAcase (OGA) in cells. O-GlcNAcylation often occurs on serine (Ser) and threonine (Thr) residues of the specific substrate proteins via the addition of O-GlcNAc group by OGT. It has been known that O-GlcNAcylation is not only involved in many fundamental cellular processes, but also plays an important role in cancer development through various mechanisms. Recently, accumulating data reveal that O-GlcNAcylation at histones or non-histone proteins can lead to the start of the subsequent biological processes, suggesting that O-GlcNAcylation as 'protein code' or 'histone code' may provide recognition platforms or executive instructions for subsequent recruitment of proteins to carry out the specific functions. In this review, we summarize the interaction of O-GlcNAcylation and epigenetic changes, introduce recent research findings that link crosstalk between O-GlcNAcylation and epigenetic changes, and speculate on the potential coordination role of O-GlcNAcylation with epigenetic changes in intracellular biological processes.

Differential Activation of Cellular DNA Damage Responses by Replication-Defective and Replication-Competent Adenovirus Mutants

PubMed Central

Prakash, Anand; Jayaram, Sumithra

2012-01-01

Adenovirus (Ad) mutants that lack early region 4 (E4) activate the phosphorylation of cellular DNA damage response proteins. In wild-type Ad type 5 (Ad5) infections, E1b and E4 proteins target the cellular DNA repair protein Mre11 for redistribution and degradation, thereby interfering with its ability to activate phosphorylation cascades important during DNA repair. The characteristics of Ad infection that activate cellular DNA repair processes are not yet well understood. We investigated the activation of DNA damage responses by a replication-defective Ad vector (AdRSVβgal) that lacks E1 and fails to produce the immediate-early E1a protein. E1a is important for activating early gene expression from the other viral early transcription units, including E4. AdRSVβgal can deliver its genome to the cell, but it is subsequently deficient for viral early gene expression and DNA replication. We studied the ability of AdRSVβgal-infected cells to induce cellular DNA damage responses. AdRSVβgal infection does activate formation of foci containing the Mdc1 protein. However, AdRSVβgal fails to activate phosphorylation of the damage response proteins Nbs1 and Chk1. We found that viral DNA replication is important for Nbs1 phosphorylation, suggesting that this step in the viral life cycle may provide an important trigger for activating at least some DNA repair proteins. PMID:23015708

High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications.

PubMed

Stockwell, B R; Haggarty, S J; Schreiber, S L

1999-02-01

Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of gene-product alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore, it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a 'cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with small-molecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications.

Phosphoprotein profiles of candidate markers for early cellular responses to low-dose γ-radiation in normal human fibroblast cells

PubMed Central

Yim, Ji-Hye; Yun, Jung Mi; Kim, Ji Young; Lee, In Kyung; Nam, Seon Young

2017-01-01

Abstract Ionizing radiation causes biological damage that leads to severe health effects. However, the effects and subsequent health implications caused by exposure to low-dose radiation are unclear. The objective of this study was to determine phosphoprotein profiles in normal human fibroblast cell lines in response to low-dose and high-dose γ-radiation. We examined the cellular response in MRC-5 cells 0.5 h after exposure to 0.05 or 2 Gy. Using 1318 antibodies by antibody array, we observed ≥1.3-fold increases in a number of identified phosphoproteins in cells subjected to low-dose (0.05 Gy) and high-dose (2 Gy) radiation, suggesting that both radiation levels stimulate distinct signaling pathways. Low-dose radiation induced nucleic acid–binding transcription factor activity, developmental processes, and multicellular organismal processes. By contrast, high-dose radiation stimulated apoptotic processes, cell adhesion and regulation, and cellular organization and biogenesis. We found that phospho-BTK (Tyr550) and phospho-Gab2 (Tyr643) protein levels at 0.5 h after treatment were higher in cells subjected to low-dose radiation than in cells treated with high-dose radiation. We also determined that the phosphorylation of BTK and Gab2 in response to ionizing radiation was regulated in a dose-dependent manner in MRC-5 and NHDF cells. Our study provides new insights into the biological responses to low-dose γ-radiation and identifies potential candidate markers for monitoring exposure to low-dose ionizing radiation. PMID:28122968

The pore-forming bacterial effector, VopQ, halts autophagic turnover.

PubMed

Sreelatha, Anju; Orth, Kim; Starai, Vincent J

2013-12-01

Vibrio parahemolyticus Type III effector VopQ is both necessary and sufficient to induce autophagy within one hour of infection. We demonstrated that VopQ interacts with the Vo domain of the conserved vacuolar H(+)-ATPase. Membrane-associated VopQ subsequently forms pores in the membranes of acidic compartments, resulting in immediate release of protons without concomitant release of lumenal protein contents. These studies show how a bacterial pathogen can compromise host ion potentials using a gated pore-forming effector to equilibrate levels of small molecules found in endolysosomal compartments and disrupt cellular processes such as autophagy.

Aptamers as tools for target prioritization and lead identification.

PubMed

Burgstaller, Petra; Girod, Anne; Blind, Michael

2002-12-15

The increasing number of potential drug target candidates has driven the development of novel technologies designed to identify functionally important targets and enhance the subsequent lead discovery process. Highly specific synthetic nucleic acid ligands--also known as aptamers--offer a new exciting route in the drug discovery process by linking target validation directly with HTS. Recently, aptamers have proven to be valuable tools for modulating the function of endogenous cellular proteins in their natural environment. A set of technologies has been developed to use these sophisticated ligands for the validation of potential drug targets in disease models. Moreover, aptamers that are specific antagonists of protein function can act as substitute interaction partners in HTS assays to facilitate the identification of small-molecule lead compounds.

Molecular Processes that Drive Cigarette Smoke–Induced Epithelial Cell Fate of the Lung

PubMed Central

Nyunoya, Toru; Mebratu, Yohannes; Contreras, Amelia; Delgado, Monica; Chand, Hitendra S.

2014-01-01

Cigarette smoke contains numerous chemical compounds, including abundant reactive oxygen/nitrogen species and aldehydes, and many other carcinogens. Long-term cigarette smoking significantly increases the risk of various lung diseases, including chronic obstructive pulmonary disease and lung cancer, and contributes to premature death. Many in vitro and in vivo studies have elucidated mechanisms involved in cigarette smoke–induced inflammation, DNA damage, and autophagy, and the subsequent cell fates, including cell death, cellular senescence, and transformation. In this Translational Review, we summarize the known pathways underlying these processes in airway epithelial cells to help reveal future challenges and describe possible directions of research that could lead to better management and treatment of these diseases. PMID:24111585

The evolution of structural and chemical heterogeneity during rapid solidification at gas atomization

NASA Astrophysics Data System (ADS)

Golod, V. M.; Sufiiarov, V. Sh

2017-04-01

Gas atomization is a high-performance process for manufacturing superfine metal powders. Formation of the powder particles takes place primarily through the fragmentation of alloy melt flow with high-pressure inert gas, which leads to the formation of non-uniform sized micron-scale particles and subsequent their rapid solidification due to heat exchange with gas environment. The article presents results of computer modeling of crystallization process, simulation and experimental studies of the cellular-dendrite structure formation and microsegregation in different size particles. It presents results of adaptation of the approach for local nonequilibrium solidification to conditions of crystallization at gas atomization, detected border values of the particle size at which it is possible a manifestation of diffusionless crystallization.

Adaptive response in animals exposed to non-ionizing radiofrequency fields: some underlying mechanisms.

PubMed

Cao, Yi; Tong, Jian

2014-04-22

During the last few years, our research group has been investigating the phenomenon of adaptive response in animals exposed to non-ionizing radiofrequency fields. The results from several separate studies indicated a significant increase in survival, decreases in genetic damage as well as oxidative damage and, alterations in several cellular processes in mice pre-exposed to radiofrequency fields and subsequently subjected to sub-lethal or lethal doses of γ-radiation or injected with bleomycin, a radiomimetic chemical mutagen. These observations indicated the induction of adaptive response providing the animals the ability to resist subsequent damage. Similar studies conducted by independent researchers in mice and rats have supported our observation on increased survival. In this paper, we have presented a brief review of all of our own and other independent investigations on radiofrequency fields-induced adaptive response and some underlying mechanisms discussed.

Adaptive Response in Animals Exposed to Non-Ionizing Radiofrequency Fields: Some Underlying Mechanisms

PubMed Central

Cao, Yi; Tong, Jian

2014-01-01

During the last few years, our research group has been investigating the phenomenon of adaptive response in animals exposed to non-ionizing radiofrequency fields. The results from several separate studies indicated a significant increase in survival, decreases in genetic damage as well as oxidative damage and, alterations in several cellular processes in mice pre-exposed to radiofrequency fields and subsequently subjected to sub-lethal or lethal doses of γ-radiation or injected with bleomycin, a radiomimetic chemical mutagen. These observations indicated the induction of adaptive response providing the animals the ability to resist subsequent damage. Similar studies conducted by independent researchers in mice and rats have supported our observation on increased survival. In this paper, we have presented a brief review of all of our own and other independent investigations on radiofrequency fields-induced adaptive response and some underlying mechanisms discussed. PMID:24758897

The influenza virus NS1 protein as a therapeutic target.

PubMed

Engel, Daniel A

2013-09-01

Nonstructural protein 1 (NS1) of influenza A virus plays a central role in virus replication and blockade of the host innate immune response, and is therefore being considered as a potential therapeutic target. The primary function of NS1 is to dampen the host interferon (IFN) response through several distinct molecular mechanisms that are triggered by interactions with dsRNA or specific cellular proteins. Sequestration of dsRNA by NS1 results in inhibition of the 2'-5' oligoadenylate synthetase/RNase L antiviral pathway, and also inhibition of dsRNA-dependent signaling required for new IFN production. Binding of NS1 to the E3 ubiquitin ligase TRIM25 prevents activation of RIG-I signaling and subsequent IFN induction. Cellular RNA processing is also targeted by NS1, through recognition of cleavage and polyadenylation specificity factor 30 (CPSF30), leading to inhibition of IFN-β mRNA processing as well as that of other cellular mRNAs. In addition NS1 binds to and inhibits cellular protein kinase R (PKR), thus blocking an important arm of the IFN system. Many additional proteins have been reported to interact with NS1, either directly or indirectly, which may serve its anti-IFN and additional functions, including the regulation of viral and host gene expression, signaling pathways and viral pathogenesis. Many of these interactions are potential targets for small-molecule intervention. Structural, biochemical and functional studies have resulted in hypotheses for drug discovery approaches that are beginning to bear experimental fruit, such as targeting the dsRNA-NS1 interaction, which could lead to restoration of innate immune function and inhibition of virus replication. This review describes biochemical, cell-based and nucleic acid-based approaches to identifying NS1 antagonists. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

The influenza virus NS1 protein as a therapeutic target

PubMed Central

Engel, Daniel A.

2015-01-01

Nonstructural protein 1 (NS1) of influenza A virus plays a central role in virus replication and blockade of the host innate immune response, and is therefore being considered as a potential therapeutic target. The primary function of NS1 is to dampen the host interferon (IFN) response through several distinct molecular mechanisms that are triggered by interactions with dsRNA or specific cellular proteins. Sequestration of dsRNA by NS1 results in inhibition of the 2’-5’ oligoadenylate synthetase/RNase L antiviral pathway, and also inhibition of dsRNA-dependent signaling required for new IFN production. Binding of NS1 to the E3 ubiquitin ligase TRIM25 prevents activation of RIG-I signaling and subsequent IFN induction. Cellular RNA processing is also targeted by NS1, through recognition of cleavage and polyadenylation specificity factor 30 (CPSF30), leading to inhibition of IFN- mRNA processing as well as that of other cellular mRNAs. In addition NS1 binds to and inhibits cellular protein kinase R (PKR), thus blocking an important arm of the IFN system. Many additional proteins have been reported to interact with NS1, either directly or indirectly, which may serve its anti-IFN and additional functions, including the regulation of viral and host gene expression, signaling pathways and viral pathogenesis. Many of these interactions are potential targets for small-molecule intervention. Structural, biochemical and functional studies have resulted in hypotheses for drug discovery approaches that are beginning to bear experimental fruit, such as targeting the dsRNA-NS1 interaction, which could lead to restoration of innate immune function and inhibition of virus replication. This review describes biochemical, cell-based and nucleic acid-based approaches to identifying NS1 antagonists. PMID:23796981

Inflammatory etiopathogenesis of systemic lupus erythematosus: an update

PubMed Central

Podolska, Malgorzata J; Biermann, Mona HC; Maueröder, Christian; Hahn, Jonas; Herrmann, Martin

2015-01-01

The immune system struggles every day between responding to foreign antigens and tolerating self-antigens to delicately maintain tissue homeostasis. If self-tolerance is broken, the development of autoimmunity can be the consequence, as it is in the case of the chronic inflammatory autoimmune disease systemic lupus erythematosus (SLE). SLE is considered to be a multifactorial disease comprising various processes and cell types that act abnormally and in a harmful way. Oxidative stress, infections, or, in general, tissue injury are accompanied by massive cellular demise. Several processes such as apoptosis, necrosis, or NETosis (formation of Neutrophil Extracellular Traps [NETs]) may occur alone or in combination. If clearance of dead cells is insufficient, cellular debris may accumulate and trigger inflammation and leakage of cytoplasmic and nuclear autoantigens like ribonucleoproteins, DNA, or histones. Inadequate removal of cellular remnants in the germinal centers of secondary lymphoid organs may result in the presentation of autoantigens by follicular dendritic cells to autoreactive B cells that had been generated by chance during the process of somatic hypermutation (loss of peripheral tolerance). The improper exposure of nuclear autoantigens in this delicate location is consequently prone to break self-tolerance to nuclear autoantigens. Indeed, the germline variants of autoantibodies often do not show autoreactivity. The subsequent production of autoantibodies plays a critical role in the development of the complex immunological disorder fostering SLE. Immune complexes composed of cell-derived autoantigens and autoantibodies are formed and get deposited in various tissues, such as the kidney, leading to severe organ damage. Alternatively, they may also be formed in situ by binding to planted antigens of circulating autoantibodies. Here, we review current knowledge about the etiopathogenesis of SLE including the involvement of different types of cell death, serving as the potential source of autoantigens, and impaired clearance of cell remnants, causing accumulation of cellular debris. PMID:26316795

The functional interplay between the HIF pathway and the ubiquitin system - more than a one-way road.

PubMed

Günter, Julia; Ruiz-Serrano, Amalia; Pickel, Christina; Wenger, Roland H; Scholz, Carsten C

2017-07-15

The hypoxia inducible factor (HIF) pathway and the ubiquitin system represent major cellular processes that are involved in the regulation of a plethora of cellular signaling pathways and tissue functions. The ubiquitin system controls the ubiquitination of proteins, which is the covalent linkage of one or several ubiquitin molecules to specific targets. This ubiquitination is catalyzed by approximately 1000 different E3 ubiquitin ligases and can lead to different effects, depending on the type of internal ubiquitin chain linkage. The best-studied function is the targeting of proteins for proteasomal degradation. The activity of E3 ligases is antagonized by proteins called deubiquitinases (or deubiquitinating enzymes), which negatively regulate ubiquitin chains. This is performed in most cases by the catalytic removal of these chains from the targeted protein. The HIF pathway is regulated in an oxygen-dependent manner by oxygen-sensing hydroxylases. Covalent modification of HIFα subunits leads to the recruitment of an E3 ligase complex via the von Hippel-Lindau (VHL) protein and the subsequent polyubiquitination and proteasomal degradation of HIFα subunits, demonstrating the regulation of the HIF pathway by the ubiquitin system. This unidirectional effect of an E3 ligase on the HIF pathway is the best-studied example for the interplay between these two important cellular processes. However, additional regulatory mechanisms of the HIF pathway through the ubiquitin system are emerging and, more recently, also the reciprocal regulation of the ubiquitin system through components of the HIF pathway. Understanding these mechanisms and their relevance for the activity of each other is of major importance for the comprehensive elucidation of the oxygen-dependent regulation of cellular processes. This review describes the current knowledge of the functional bidirectional interplay between the HIF pathway and the ubiquitin system on the protein level. Copyright © 2017 Elsevier Inc. All rights reserved.

c-di-AMP: An Essential Molecule in the Signaling Pathways that Regulate the Viability and Virulence of Gram-Positive Bacteria

PubMed Central

Fahmi, Tazin; Port, Gary C.

2017-01-01

Signal transduction pathways enable organisms to monitor their external environment and adjust gene regulation to appropriately modify their cellular processes. Second messenger nucleotides including cyclic adenosine monophosphate (c-AMP), cyclic guanosine monophosphate (c-GMP), cyclic di-guanosine monophosphate (c-di-GMP), and cyclic di-adenosine monophosphate (c-di-AMP) play key roles in many signal transduction pathways used by prokaryotes and/or eukaryotes. Among the various second messenger nucleotides molecules, c-di-AMP was discovered recently and has since been shown to be involved in cell growth, survival, and regulation of virulence, primarily within Gram-positive bacteria. The cellular level of c-di-AMP is maintained by a family of c-di-AMP synthesizing enzymes, diadenylate cyclases (DACs), and degradation enzymes, phosphodiesterases (PDEs). Genetic manipulation of DACs and PDEs have demonstrated that alteration of c-di-AMP levels impacts both growth and virulence of microorganisms. Unlike other second messenger molecules, c-di-AMP is essential for growth in several bacterial species as many basic cellular functions are regulated by c-di-AMP including cell wall maintenance, potassium ion homeostasis, DNA damage repair, etc. c-di-AMP follows a typical second messenger signaling pathway, beginning with binding to receptor molecules to subsequent regulation of downstream cellular processes. While c-di-AMP binds to specific proteins that regulate pathways in bacterial cells, c-di-AMP also binds to regulatory RNA molecules that control potassium ion channel expression in Bacillus subtilis. c-di-AMP signaling also occurs in eukaryotes, as bacterially produced c-di-AMP stimulates host immune responses during infection through binding of innate immune surveillance proteins. Due to its existence in diverse microorganisms, its involvement in crucial cellular activities, and its stimulating activity in host immune responses, c-di-AMP signaling pathway has become an attractive antimicrobial drug target and therefore has been the focus of intensive study in several important pathogens. PMID:28783096

The adenovirus E4-ORF3 protein functions as a SUMO E3 ligase for TIF-1γ sumoylation and poly-SUMO chain elongation.

PubMed

Sohn, Sook-Young; Hearing, Patrick

2016-06-14

The adenovirus (Ad) early region 4 (E4)-ORF3 protein regulates diverse cellular processes to optimize the host environment for the establishment of Ad replication. E4-ORF3 self-assembles into multimers to form a nuclear scaffold in infected cells and creates distinct binding interfaces for different cellular target proteins. Previous studies have shown that the Ad5 E4-ORF3 protein induces sumoylation of multiple cellular proteins and subsequent proteasomal degradation of some of them, but the detailed mechanism of E4-ORF3 function remained unknown. Here, we investigate the role of E4-ORF3 in the sumoylation process by using transcription intermediary factor (TIF)-1γ as a substrate. Remarkably, we discovered that purified E4-ORF3 protein stimulates TIF-1γ sumoylation in vitro, demonstrating that E4-ORF3 acts as a small ubiquitin-like modifier (SUMO) E3 ligase. Furthermore, E4-ORF3 significantly increases poly-SUMO3 chain formation in vitro in the absence of substrate, showing that E4-ORF3 has SUMO E4 elongase activity. An E4-ORF3 mutant, which is defective in protein multimerization, exhibited severely decreased activity, demonstrating that E4-ORF3 self-assembly is required for these activities. Using a SUMO3 mutant, K11R, we found that E4-ORF3 facilitates the initial acceptor SUMO3 conjugation to TIF-1γ as well as poly-SUMO chain elongation. The E4-ORF3 protein displays no SUMO-targeted ubiquitin ligase activity in our assay system. These studies reveal the mechanism by which E4-ORF3 targets specific cellular proteins for sumoylation and proteasomal degradation and provide significant insight into how a small viral protein can play a role as a SUMO E3 ligase and E4-like SUMO elongase to impact a variety of cellular responses.

Cellular Precipitates Of Iron Oxide in Olivine in a Stratospheric Interplanetary Dust Particle

NASA Technical Reports Server (NTRS)

Rietmeijer, Frans J. M.

1996-01-01

The petrology of a massive olivine-sulphide interplanetary dust particle shows melting of Fe,Ni-sulphide plus complete loss of sulphur and subsequent quenching to a mixture of iron-oxides and Fe,Ni-metal. Oxidation of the fayalite component in olivine produced maghemite discs and cellular intergrowths with olivine and rare andradite-rich garnet. Cellular reactions require no long-range solid-state diffusion and are kinetically favourable during pyrometamorphic oxidation. Local melting of the cellular intergrowths resulted in three dimensional symplectic textures. Dynamic pyrometamorphism of this asteroidal particle occurred at approx. 1100 C during atmospheric entry flash (5-15 s) heating.

Examination of laser microbeam cell lysis in a PDMS microfluidic channel using time-resolved imaging

PubMed Central

Quinto-Su, Pedro A.; Lai, Hsuan-Hong; Yoon, Helen H.; Sims, Christopher E.; Allbritton, Nancy L.; Venugopalan, Vasan

2008-01-01

We use time-resolved imaging to examine the lysis dynamics of non-adherent BAF-3 cells within a microfluidic channel produced by the delivery of single highly-focused 540 ps duration laser pulses at λ = 532 nm. Time-resolved bright-field images reveal that the delivery of the pulsed laser microbeam results in the formation of a laser-induced plasma followed by shock wave emission and cavitation bubble formation. The confinement offered by the microfluidic channel constrains substantially the cavitation bubble expansion and results in significant deformation of the PDMS channel walls. To examine the cell lysis and dispersal of the cellular contents, we acquire time-resolved fluorescence images of the process in which the cells were loaded with a fluorescent dye. These fluorescence images reveal cell lysis to occur on the nanosecond to microsecond time scale by the plasma formation and cavitation bubble dynamics. Moreover, the time-resolved fluorescence images show that while the cellular contents are dispersed by the expansion of the laser-induced cavitation bubble, the flow associated with the bubble collapse subsequently re-localizes the cellular contents to a small region. This capacity of pulsed laser microbeam irradiation to achieve rapid cell lysis in microfluidic channels with minimal dilution of the cellular contents has important implications for their use in lab-on-a-chip applications. PMID:18305858

Dynamics of Cellular Responses to Radiation

PubMed Central

Wodarz, Dominik; Sorace, Ron; Komarova, Natalia L.

2014-01-01

Understanding the consequences of exposure to low dose ionizing radiation is an important public health concern. While the risk of low dose radiation has been estimated by extrapolation from data at higher doses according to the linear non-threshold model, it has become clear that cellular responses can be very different at low compared to high radiation doses. Important phenomena in this respect include radioadaptive responses as well as low-dose hyper-radiosensitivity (HRS) and increased radioresistance (IRR). With radioadaptive responses, low dose exposure can protect against subsequent challenges, and two mechanisms have been suggested: an intracellular mechanism, inducing cellular changes as a result of the priming radiation, and induction of a protected state by inter-cellular communication. We use mathematical models to examine the effect of these mechanisms on cellular responses to low dose radiation. We find that the intracellular mechanism can account for the occurrence of radioadaptive responses. Interestingly, the same mechanism can also explain the existence of the HRS and IRR phenomena, and successfully describe experimentally observed dose-response relationships for a variety of cell types. This indicates that different, seemingly unrelated, low dose phenomena might be connected and driven by common core processes. With respect to the inter-cellular communication mechanism, we find that it can also account for the occurrence of radioadaptive responses, indicating redundancy in this respect. The model, however, also suggests that the communication mechanism can be vital for the long term survival of cell populations that are continuously exposed to relatively low levels of radiation, which cannot be achieved with the intracellular mechanism in our model. Experimental tests to address our model predictions are proposed. PMID:24722167

A Technical Review of Cellular Radio and Analysis of a Possible Protocol

DTIC Science & Technology

1992-09-01

9 1. The Pioneers ............. ................................... 9 2. Time line of Radio Evolution...cellular telephone. Advances in low-power radio transmission and the speed with which modern computers can aid in frequency management and signal...lecturer at the Royal Institution in London. He subsequently worked his way up to lecturer and devoted ever increasing amounts of time to experiments

P53 protein in proliferation, repair and apoptosis of cells.

PubMed

Wawryk-Gawda, Ewelina; Chylińska-Wrzos, Patrycja; Lis-Sochocka, Marta; Chłapek, Katarzyna; Bulak, Kamila; Jędrych, Marian; Jodłowska-Jędrych, Barbara

2014-05-01

The p53 protein is an important factor of many intra- and extracellular processes. This protein regulates the repair of cellular DNA and induces apoptosis. It is also responsible for the regulation of the senescence and the cell entering the subsequent stages of the cellular cycle. The protein p53 is also involved in inhibiting angiogenesis and the induction of oxidative shock. In our study, we examined the activity of p53 protein in the uterine epithelial cells in rats treated with cladribine. Its action is mainly based on apoptosis induction. We compared the activity of p53 protein in cells with a high apoptosis index and in cells with active repair mechanisms and high proliferation index. We observed stronger p53 protein expression in the epithelial cells of the materials taken 24 h after the last dose of 2-CdA associated with the active process of apoptosis and inhibition of proliferation. After 4 weeks from the last dose of cladribine, the stronger expression of p53 protein was associated with both the existing changes in the cell's genome, the effects of the ongoing repair mechanisms, as well as the high proliferation activity.

Emerging connections between RNA and autophagy.

PubMed

Frankel, Lisa B; Lubas, Michal; Lund, Anders H

2017-01-02

Macroautophagy/autophagy is a key catabolic process, essential for maintaining cellular homeostasis and survival through the removal and recycling of unwanted cellular material. Emerging evidence has revealed intricate connections between the RNA and autophagy research fields. While a majority of studies have focused on protein, lipid and carbohydrate catabolism via autophagy, accumulating data supports the view that several types of RNA and associated ribonucleoprotein complexes are specifically recruited to phagophores (precursors to autophagosomes) and subsequently degraded in the lysosome/vacuole. Moreover, recent studies have revealed a substantial number of novel autophagy regulators with RNA-related functions, indicating roles for RNA and associated proteins not only as cargo, but also as regulators of this process. In this review, we discuss widespread evidence of RNA catabolism via autophagy in yeast, plants and animals, reviewing the molecular mechanisms and biological importance in normal physiology, stress and disease. In addition, we explore emerging evidence of core autophagy regulation mediated by RNA-binding proteins and noncoding RNAs, and point to gaps in our current knowledge of the connection between RNA and autophagy. Finally, we discuss the pathological implications of RNA-protein aggregation, primarily in the context of neurodegenerative disease.

Dynamic formation of ER–PM junctions presents a lipid phosphatase to regulate phosphoinositides

PubMed Central

Jensen, Jill B.; Vivas, Oscar; Kruse, Martin; Traynor-Kaplan, Alexis E.; Hille, Bertil

2016-01-01

Endoplasmic reticulum–plasma membrane (ER–PM) contact sites play an integral role in cellular processes such as excitation–contraction coupling and store-operated calcium entry (SOCE). Another ER–PM assembly is one tethered by the extended synaptotagmins (E-Syt). We have discovered that at steady state, E-Syt2 positions the ER and Sac1, an integral ER membrane lipid phosphatase, in discrete ER–PM junctions. Here, Sac1 participates in phosphoinositide homeostasis by limiting PM phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2. Activation of G protein–coupled receptors that deplete PM PI(4,5)P2 disrupts E-Syt2–mediated ER–PM junctions, reducing Sac1’s access to the PM and permitting PM PI(4)P and PI(4,5)P2 to recover. Conversely, depletion of ER luminal calcium and subsequent activation of SOCE increases the amount of Sac1 in contact with the PM, depleting PM PI(4)P. Thus, the dynamic presence of Sac1 at ER–PM contact sites allows it to act as a cellular sensor and controller of PM phosphoinositides, thereby influencing many PM processes. PMID:27044890

Electromagnetic Interference Assessment of CDMA and GSM Wireless Phones to Aircraft Navigation Radios

NASA Technical Reports Server (NTRS)

Ely, Jay J.; Nguyen, Truong X.; Koppen, Sandra V.; Salud, M. Theresa

2002-01-01

To address the concern for cellular phone electromagnetic interference (EMI) to aircraft radios, a radiated emission measurement process for CDMA (IS-95) and GSM (ETSI GSM 11.22) wireless handsets was developed. Spurious radiated emissions were efficiently characterized from devices tested in either a semi-anechoic or reverberation chamber, in terms of effective isotropic radiated power. Eight representative handsets (4 GSM, 4 CDMA) were commanded to operate while varying their radio transmitter parameters (power, modulation, etc.). This report provides a detailed description of the measurement process and resulting data, which may subsequently be used by others as a basis of consistent evaluation for cellular/PCS phones, Bluetooth, IEEE802.11b, IEEE802.11a, FRS/GMRS radios, and other portable transmitters. Aircraft interference path loss (IPL) and navigation radio interference threshold data from numerous reference documents, standards, and NASA partnerships were compiled. Using this data, a preliminary risk assessment is provided for CDMA and GSM wireless phone interference to aircraft localizer, Glideslope, VOR, and GPS radio receivers on typical transport airplanes. The report identifies where existing data for device emissions, IPL, and navigation radio interference thresholds needs to be extended for an accurate risk assessment for wireless transmitters in aircraft.

Amyloid-linked cellular toxicity triggered by bacterial inclusion bodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gonzalez-Montalban, Nuria; Departament de Genetica i de Microbiologia, Universitat Autonoma de Barcelona, Bellaterra, 08193 Barcelona; Ciber de Bioingenieria, Biomateriales y Nanomedicina

The aggregation of proteins in the form of amyloid fibrils and plaques is the characteristic feature of some pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. The mechanisms by which the aggregation processes result in cell damage are under intense investigation but recent data indicate that prefibrillar aggregates are the most proximate mediators of toxicity rather than mature fibrils. Since it has been shown that prefibrillar forms of the nondisease-related misfolded proteins are highly toxic to cultured mammalian cells we have studied the cytoxicity associated to bacterial inclusion bodies that have been recently described as protein deposits presenting amyloid-likemore » structures. We have proved that bacterial inclusion bodies composed by a misfolding-prone {beta}-galactosidase fusion protein are clearly toxic for mammalian cells but the {beta}-galactosidase wild type enzyme forming more structured thermal aggregates does not impair cell viability, despite it also binds and enter into the cells. These results are in the line that the most cytotoxic aggregates are early prefibrilar assemblies but discard the hypothesis that the membrane destabilization is Key event to subsequent disruption of cellular processes, such as ion balance, oxidative state and the eventually cell death.« less

Aspartate β-hydroxylase modulates cellular senescence via glycogen synthase kinase 3β in hepatocellular carcinoma

PubMed Central

Iwagami, Yoshifumi; Huang, Chiung-Kuei; Olsen, Mark J.; Thomas, John-Michael; Jang, Grace; Kim, Miran; Lin, Qiushi; Carlson, Rolf I.; Wagner, Carl E.; Dong, Xiaoqun; Wands, Jack R.

2015-01-01

Background & Aims Aspartate β-hydroxylase (ASPH) is an enzyme overexpressed in human hepatocellular carcinoma (HCC) tumors and participates in the malignant transformation process. We determined if ASPH was a therapeutic target by exerting effects on cellular senescence to retard HCC progression. Methods ASPH knockdown or knockout was achieved by shRNAs or CRISPR/Cas9 system, respectively, whereas enzymatic inhibition was rendered by a potent 2nd generation small molecule inhibitor (SMI) of ASPH. Alterations of cell proliferation, colony formation and cellular senescence were evaluated in human HCC cell lines. The potential mechanisms for activating cellular senescence were explored using murine subcutaneous and orthotopic xenograft models. Results Inhibition of ASPH expression and enzymatic activity significantly reduced cell proliferation and colony formation, but induced tumor cell senescence. Following inhibition of ASPH activity, phosphorylation of GSK3β and p16 expression were increased to promote senescence whereas cyclin D1 and PCNA were decreased to reduce cell proliferation. The mechanisms involved demonstrate that ASPH binds to GSK3β and inhibits its subsequent interactions with AKT and p38 upstream kinases as shown by co-immunoprecipitation. In vivo experiments demonstrated that the SMI treatment of HCC bearing mice resulted in significant dose-dependent reduced tumor growth, induced phosphorylation of GSK3β, enhanced p16 expression in tumor cells and promoted cellular senescence. Conclusions We have identified a new mechanism that promotes HCC growth and progression by modulating senescence of tumor cells. These findings suggest that ASPH enzymatic activity is a novel therapeutic target for HCC. PMID:26683595

Temperature Treatment of Highly Porous Zirconium-Containing Metal-Organic Frameworks Extends Drug Delivery Release.

PubMed

Teplensky, Michelle H; Fantham, Marcus; Li, Peng; Wang, Timothy C; Mehta, Joshua P; Young, Laurence J; Moghadam, Peyman Z; Hupp, Joseph T; Farha, Omar K; Kaminski, Clemens F; Fairen-Jimenez, David

2017-06-07

Utilizing metal-organic frameworks (MOFs) as a biological carrier can lower the amount of the active pharmaceutical ingredient (API) required in cancer treatments to provide a more efficacious therapy. In this work, we have developed a temperature treatment process for delaying the release of a model drug compound from the pores of NU-1000 and NU-901, while taking care to utilize these MOFs' large pore volume and size to achieve exceptional model drug loading percentages over 35 wt %. Video-rate super-resolution microscopy reveals movement of MOF particles when located outside of the cell boundary, and their subsequent immobilization when taken up by the cell. Through the use of optical sectioning structured illumination microscopy (SIM), we have captured high-resolution 3D images showing MOF uptake by HeLa cells over a 24 h period. We found that addition of a model drug compound into the MOF and the subsequent temperature treatment process does not affect the rate of MOF uptake by the cell. Endocytosis analysis revealed that MOFs are internalized by active transport and that inhibiting the caveolae-mediated pathway significantly reduced cellular uptake of MOFs. Encapsulation of an anticancer therapeutic, alpha-cyano-4-hydroxycinnamic acid (α-CHC), and subsequent temperature treatment produced loadings of up to 81 wt % and demonstrated efficacy at killing cells beyond the burst release effect.

A murine retrovirus co-Opts YB-1, a translational regulator and stress granule-associated protein, to facilitate virus assembly.

PubMed

Bann, Darrin V; Beyer, Andrea R; Parent, Leslie J

2014-04-01

The Gag protein of the murine retrovirus mouse mammary tumor virus (MMTV) orchestrates the assembly of immature virus particles in the cytoplasm which are subsequently transported to the plasma membrane for release from the cell. The morphogenetic pathway of MMTV assembly is similar to that of Saccharomyces cerevisiae retrotransposons Ty1 and Ty3, which assemble virus-like particles (VLPs) in intracytoplasmic ribonucleoprotein (RNP) complexes. Assembly of Ty1 and Ty3 VLPs depends upon cellular mRNA processing factors, prompting us to examine whether MMTV utilizes a similar set of host proteins to facilitate viral capsid assembly. Our data revealed that MMTV Gag colocalized with YB-1, a translational regulator found in stress granules and P bodies, in intracytoplasmic foci. The association of MMTV Gag and YB-1 in cytoplasmic granules was not disrupted by cycloheximide treatment, suggesting that these sites were not typical stress granules. However, the association of MMTV Gag and YB-1 was RNA dependent, and an MMTV RNA reporter construct colocalized with Gag and YB-1 in cytoplasmic RNP complexes. Knockdown of YB-1 resulted in a significant decrease in MMTV particle production, indicating that YB-1 plays a role in MMTV capsid formation. Analysis by live-cell imaging with fluorescence recovery after photobleaching (FRAP) revealed that the population of Gag proteins localized within YB-1 complexes was relatively immobile, suggesting that Gag forms stable complexes in association with YB-1. Together, our data imply that the formation of intracytoplasmic Gag-RNA complexes is facilitated by YB-1, which promotes MMTV virus assembly. Cellular mRNA processing factors regulate the posttranscriptional fates of mRNAs, affecting localization and utilization of mRNAs under normal conditions and in response to stress. RNA viruses such as retroviruses interact with cellular mRNA processing factors that accumulate in ribonucleoprotein complexes known as P bodies and stress granules. This report shows for the first time that mouse mammary tumor virus (MMTV), a mammalian retrovirus that assembles intracytoplasmic virus particles, commandeers the cellular factor YB-1, a key regulator of translation involved in the cellular stress response. YB-1 is essential for the efficient production of MMTV particles, a process directed by the viral Gag protein. We found that Gag and YB-1 localize together in cytoplasmic granules. Functional studies of Gag/YB-1 granules suggest that they may be sites where virus particles assemble. These studies provide significant insights into the interplay between mRNA processing factors and retroviruses.

Iterons, fractals and computations of automata

NASA Astrophysics Data System (ADS)

Siwak, Paweł

1999-03-01

Processing of strings by some automata, when viewed on space-time (ST) diagrams, reveals characteristic soliton-like coherent periodic objects. They are inherently associated with iterations of automata mappings thus we call them the iterons. In the paper we present two classes of one-dimensional iterons: particles and filtrons. The particles are typical for parallel (cellular) processing, while filtrons, introduced in (32) are specific for serial processing of strings. In general, the images of iterated automata mappings exhibit not only coherent entities but also the fractals, and quasi-periodic and chaotic dynamics. We show typical images of such computations: fractals, multiplication by a number, and addition of binary numbers defined by a Turing machine. Then, the particles are presented as iterons generated by cellular automata in three computations: B/U code conversion (13, 29), majority classification (9), and in discrete version of the FPU (Fermi-Pasta-Ulam) dynamics (7, 23). We disclose particles by a technique of combinational recoding of ST diagrams (as opposed to sequential recoding). Subsequently, we recall the recursive filters based on FCA (filter cellular automata) window operators, and considered by Park (26), Ablowitz (1), Fokas (11), Fuchssteiner (12), Bruschi (5) and Jiang (20). We present the automata equivalents to these filters (33). Some of them belong to the class of filter automata introduced in (30). We also define and illustrate some properties of filtrons. Contrary to particles, the filtrons interact nonlocally in the sense that distant symbols may influence one another. Thus their interactions are very unusual. Some examples have been given in (32). Here we show new examples of filtron phenomena: multifiltron solitonic collisions, attracting and repelling filtrons, trapped bouncing filtrons (which behave like a resonance cavity) and quasi filtrons.

Macrophage and T cell dynamics during the development and disintegration of mycobacterial granulomas.

PubMed

Egen, Jackson G; Rothfuchs, Antonio Gigliotti; Feng, Carl G; Winter, Nathalie; Sher, Alan; Germain, Ronald N

2008-02-01

Granulomas play a key role in host protection against mycobacterial pathogens, with their breakdown contributing to exacerbated disease. To better understand the initiation and maintenance of these structures, we employed both high-resolution multiplex static imaging and intravital multiphoton microscopy of Mycobacterium bovis BCG-induced liver granulomas. We found that Kupffer cells directly capture blood-borne bacteria and subsequently nucleate formation of a nascent granuloma by recruiting both uninfected liver-resident macrophages and blood-derived monocytes. Within the mature granuloma, these myeloid cell populations formed a relatively immobile cellular matrix that interacted with a highly dynamic effector T cell population. The efficient recruitment of these T cells was highly dependent on TNF-alpha-derived signals, which also maintained the granuloma structure through preferential effects on uninfected macrophage populations. By characterizing the migration of both innate and adaptive immune cells throughout the process of granuloma development, these studies provide a new perspective on the cellular events involved in mycobacterial containment and escape.

DNA ends alter the molecular composition and localization of Ku multicomponent complexes.

PubMed

Adelmant, Guillaume; Calkins, Anne S; Garg, Brijesh K; Card, Joseph D; Askenazi, Manor; Miron, Alex; Sobhian, Bijan; Zhang, Yi; Nakatani, Yoshihiro; Silver, Pamela A; Iglehart, J Dirk; Marto, Jarrod A; Lazaro, Jean-Bernard

2012-08-01

The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.

Optimal placement of fast cut back units based on the theory of cellular automata and agent

NASA Astrophysics Data System (ADS)

Yan, Jun; Yan, Feng

2017-06-01

The thermal power generation units with the function of fast cut back could serve power for auxiliary system and keep island operation after a major blackout, so they are excellent substitute for the traditional black-start power sources. Different placement schemes for FCB units have different influence on the subsequent restoration process. Considering the locality of the emergency dispatching rules, the unpredictability of specific dispatching instructions and unexpected situations like failure of transmission line energization, a novel deduction model for network reconfiguration based on the theory of cellular automata and agent is established. Several indexes are then defined for evaluating the placement schemes for FCB units. The attribute weights determination method based on subjective and objective integration and grey relational analysis are combinatorically used to determine the optimal placement scheme for FCB unit. The effectiveness of the proposed method is validated by the test results on the New England 10-unit 39-bus power system.

Molecular variation of the nonribosomal peptide-polyketide siderophore yersiniabactin through biosynthetic and metabolic engineering.

PubMed

Ahmadi, Mahmoud Kamal; Fawaz, Samar; Fang, Lei; Yu, Zhipeng; Pfeifer, Blaine A

2016-05-01

The production of the mixed nonribosomal peptide-polyketide natural product yersiniabactin (Ybt) has been established using E. coli as a heterologous host. In this study, precursor-directed biosynthesis was used to generate five new analogs of Ybt, demonstrating the flexibility of the heterologous system and the biosynthetic process in allowing compound diversity. A combination of biosynthetic and cellular engineering was then used to influence the production metrics of the resulting analogs. First, the cellular levels and activity of FadL, a hydrocarbon transport protein, were tested for subsequent influence upon exogenous precursor uptake and Ybt analog production with a positive correlation observed between FadL over-production and analog formation. Next, a Ybt biosynthetic editing enzyme was removed from the heterologous system which decreased native compound production but increased analog formation. A final series of experiments enhanced endogenous anthranilate towards complete pathway formation of the associated analog which showed a selective ability to bind gold. © 2015 Wiley Periodicals, Inc.

POLYELECTROLYTE MULTILAYER STAMPING IN AQUEOUS PHASE AND NON-CONTACT MODE

PubMed Central

Mehrotra, Sumit; Lee, Ilsoon; Liu, Chun; Chan, Christina

2011-01-01

Polyelectrolyte multilayer (PEM) transfer printing has been previously achieved by stamping under dry conditions. Here, we show for the first time, that PEM can be transferred from a stamp to the base substrate under aqueous conditions whereby the two surfaces are in a non-contact mode. Degradable multilayers of (PAA/PEG)10.5 followed by non-degradable multilayers of (PDAC/SPS)80.5 were fabricated under acidic pH conditions on either PDMS or glass (stamp), and subsequently transferred over top of another multilayer prepared on a different substrate (base substrate), with a spacing of ~ 200 μm between the stamping surface and the base substrate. This multilayer transfer was performed under physiological pH conditions. This process is referred to herein as non-contact, aqueous-phase multilayer (NAM) transfer. NAM transfer can be useful for applications such as fabricating three-dimensional (3-D) cellular scaffolds. We attempted to create a 3-D cellular scaffold using NAM transfer, and characterized the scaffolds with conventional and fluorescence microscopy. PMID:21860540

The increasing diversity of functions attributed to the SAFB family of RNA-/DNA-binding proteins.

PubMed

Norman, Michael; Rivers, Caroline; Lee, Youn-Bok; Idris, Jalilah; Uney, James

2016-12-01

RNA-binding proteins play a central role in cellular metabolism by orchestrating the complex interactions of coding, structural and regulatory RNA species. The SAFB (scaffold attachment factor B) proteins (SAFB1, SAFB2 and SAFB-like transcriptional modulator, SLTM), which are highly conserved evolutionarily, were first identified on the basis of their ability to bind scaffold attachment region DNA elements, but attention has subsequently shifted to their RNA-binding and protein-protein interactions. Initial studies identified the involvement of these proteins in the cellular stress response and other aspects of gene regulation. More recently, the multifunctional capabilities of SAFB proteins have shown that they play crucial roles in DNA repair, processing of mRNA and regulatory RNA, as well as in interaction with chromatin-modifying complexes. With the advent of new techniques for identifying RNA-binding sites, enumeration of individual RNA targets has now begun. This review aims to summarise what is currently known about the functions of SAFB proteins. © 2016 The Author(s).

Chemical Sintering Generates Uniform Porous Hyaluronic Acid Hydrogels

PubMed Central

Cam, Cynthia; Segura, Tatiana

2014-01-01

Implantation of scaffolds for tissue repair has been met with limited success primarily due to the inability to achieve vascularization within the construct. Many strategies have shifted to incorporate pores into these scaffolds to encourage rapid cellular infiltration and subsequent vascular ingrowth. We utilized an efficient chemical sintering technique to create a uniform network of polymethyl methacrylate (PMMA) microspheres for porous hyaluronic acid hydrogel formation. The porous hydrogels generated from chemical sintering possessed comparable pore uniformity and interconnectivity as the commonly used non- and heat sintering techniques. Moreover, similar cell response to the porous hydrogels generated from each sintering approach was observed in cell viability, spreading, proliferation in vitro, as well as, cellular invasion in vivo. We propose chemical sintering of PMMA microspheres using a dilute acetone solution as an alternative method to generating porous hyaluronic acid hydrogels since it requires equal or ten-fold less processing time as the currently used non-sintering or heat sintering technique, respectively. PMID:24120847

Ultra-localized single cell electroporation using silicon nanowires.

PubMed

Jokilaakso, Nima; Salm, Eric; Chen, Aaron; Millet, Larry; Guevara, Carlos Duarte; Dorvel, Brian; Reddy, Bobby; Karlstrom, Amelie Eriksson; Chen, Yu; Ji, Hongmiao; Chen, Yu; Sooryakumar, Ratnasingham; Bashir, Rashid

2013-02-07

Analysis of cell-to-cell variation can further the understanding of intracellular processes and the role of individual cell function within a larger cell population. The ability to precisely lyse single cells can be used to release cellular components to resolve cellular heterogeneity that might be obscured when whole populations are examined. We report a method to position and lyse individual cells on silicon nanowire and nanoribbon biological field effect transistors. In this study, HT-29 cancer cells were positioned on top of transistors by manipulating magnetic beads using external magnetic fields. Ultra-rapid cell lysis was subsequently performed by applying 600-900 mV(pp) at 10 MHz for as little as 2 ms across the transistor channel and the bulk substrate. We show that the fringing electric field at the device surface disrupts the cell membrane, leading to lysis from irreversible electroporation. This methodology allows rapid and simple single cell lysis and analysis with potential applications in medical diagnostics, proteome analysis and developmental biology studies.

Liposome encapsulation of chelating agents

DOEpatents

Rahman, Yueh Erh

1976-01-13

A method for transferring a chelating agent across a cellular membrane by encapsulating the charged chelating agent within liposomes and carrying the liposome-encapsulated chelating agent to the cellular membrane where the liposomes containing the chelating agent will be taken up by the cells, thereby transferring the chelating agent across the cellular membrane. A chelating agent can be introduced into the interior of a cell of a living organism wherein the liposomes will be decomposed, releasing the chelating agent to the interior of the cell. The released chelating agent will complex intracellularly deposited toxic heavy metals, permitting the more soluble metal complex to transfer across the cellular membrane from the cell and subsequently be removed from the living organism.

Structural properties of fracture haematoma: current status and future clinical implications.

PubMed

Wang, Xin; Friis, Thor; Glatt, Vaida; Crawford, Ross; Xiao, Yin

2017-10-01

Blood clots (haematomas) that form immediately following a bone fracture have been shown to be vital for the subsequent healing process. During the clotting process, a number of factors can influence the fibrin clot structure, such as fibrin polymerization, growth factor binding, cellular infiltration (including platelet retraction), protein concentrations and cytokines. The modulation of the fibrin clot structure within the fracture site has important clinical implications and could result in the development of multifunctional scaffolds that mimic the natural structure of a haematoma. Artificial haematoma structures such as these can be created from the patient's own blood and can therefore act as an ideal bone defect filling material for potential clinical application to accelerate bone regeneration. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Nuclear Exodus: Herpesviruses Lead the Way

PubMed Central

Bigalke, Janna M.; Heldwein, Ekaterina E.

2016-01-01

Most DNA viruses replicate in the nucleus and exit it either by passing through the nuclear pores or by rupturing the nuclear envelope. Unusually, herpesviruses have evolved a complex mechanism of nuclear escape whereby nascent capsids bud at the inner nuclear membrane to form perinuclear virions that subsequently fuse with the outer nuclear membrane, releasing capsids into the cytosol. Although this general scheme is accepted in the field, the players and their roles are still debated. Recent studies illuminated critical mechanistic features of this enigmatic process and uncovered surprising parallels with a novel cellular nuclear export process. This review summarizes our current understanding of nuclear egress in herpesviruses, examines the experimental evidence and models, and outlines outstanding questions with the goal of stimulating new research in this area. PMID:27482898

Small Talk: Cell-to-Cell Communication in Bacteria

ScienceCinema

Bassler, Bonnie [Princeton University, Princeton, New Jersey, United States

2017-12-09

Cell-cell communication in bacteria involves the production, release, and subsequent detection of chemical signaling molecules called autoinducers. This process, called quorum sensing, allows bacteria to regulate gene expression on a population-wide scale. Processes controlled by quorum sensing are usually ones that are unproductive when undertaken by an individual bacterium but become effective when undertaken by the group. For example, quorum sensing controls bioluminescence, secretion of virulence factors, biofilm formation, sporulation, and the exchange of DNA. Thus, quorum sensing is a mechanism that allows bacteria to function as multi-cellular organisms. Bacteria make, detect, and integrate information from multiple autoinducers, some of which are used exclusively for intra-species communication while others enable communication between species. Research is now focused on the development of therapies that interfere with quorum sensing to control bacterial virulence.

Roles of F-box proteins in human digestive system tumors (Review).

PubMed

Gong, Jian; Lv, Liang; Huo, Jirong

2014-12-01

F-box proteins (FBPs), the substrate-recognition subunit of E3 ubiquitin (Ub) ligase, are the important components of Ub proteasome system (UPS). FBPs are involved in multiple cellular processes through ubiquitylation and subsequent degradation of their target proteins. Many studies have described the roles of FBPs in human cancers. Digestive system tumors account for a large proportion of all the tumors, and their mortality is very high. This review summarizes for the first time the roles of FBPs in digestive system tumorige-nesis and tumor progression, aiming at finding new routes for the rational design of targeted anticancer therapies in digestive system tumors.

Struvite crystallization

NASA Technical Reports Server (NTRS)

Barak, Phillip W. (Inventor); Tabanpour, Menachem E. (Inventor); Meyer, Juliane M. (Inventor); Avila-Segura, Mauricio (Inventor)

2007-01-01

The present invention provides a method and apparatus for removing phosphorus from phosphorus containing waste. In one embodiment, the method is preferably carried out by contacting the phosphorus containing waste with a non-cellular membrane and precipitating phosphorus from the waste as struvite. Another aspect of the invention includes a method of removing phosphorus from phosphorus containing sewage comprising filtrates and biosolids. The removal of phosphorus as struvite occurs in two stages as primary and secondary removal. In the primary removal process, the sewage from a dewatering unit is contacted with a first polymeric membrane reactor and the phosphorus is removed as primary struvite. Subsequently Mg is added so as promote struvite formation and the secondary removal process of struvite. In the secondary removal process, the sewage from GBT Filtrate well or Centrifuge Liquor well is contacted with a second monomolecular membrane and the phosphorus is removed as secondary struvite.

Control of Meiotic Crossovers: From Double-Strand Break Formation to Designation

PubMed Central

Gray, Stephen

2017-01-01

Meiosis, the mechanism of creating haploid gametes, is a complex cellular process observed across sexually reproducing organisms. Fundamental to meiosis is the process of homologous recombination, whereby DNA double-strand breaks are introduced into the genome and are subsequently repaired to generate either noncrossovers or crossovers. Although homologous recombination is essential for chromosome pairing during prophase I, the resulting crossovers are critical for maintaining homolog interactions and enabling accurate segregation at the first meiotic division. Thus, the placement, timing, and frequency of crossover formation must be exquisitely controlled. In this review, we discuss the proteins involved in crossover formation, the process of their formation and designation, and the rules governing crossovers, all within the context of the important landmarks of prophase I. We draw together crossover designation data across organisms, analyze their evolutionary divergence, and propose a universal model for crossover regulation. PMID:27648641

Zn2+ at a cellular crossroads

PubMed Central

Liang, Xiaomeng; Dempski, Robert E.; Burdette, Shawn C.

2016-01-01

Zinc is an essential micronutrient for cellular homeostasis. Initially proposed to only contribute to cellular viability through structural roles and non-redox catalysis, advances in quantifying changes in nM and pM quantities of Zn2+ have elucidated increasing functions as an important signaling molecule. This includes Zn2+-mediated regulation of transcription factors and subsequent protein expression, storage and release of intracellular compartments of zinc quanta into the extracellular space which modulates plasma membrane protein function, as well as intracellular signaling pathways which contribute to the immune response. This review highlights some recent advances in our understanding of zinc signaling. PMID:27010344

Cellular Microcultures: Programming Mechanical and Physicochemical Properties of 3D Hydrogel Cellular Microcultures via Direct Ink Writing (Adv. Healthcare Mater. 9/2016).

PubMed

McCracken, Joselle M; Badea, Adina; Kandel, Mikhail E; Gladman, A Sydney; Wetzel, David J; Popescu, Gabriel; Lewis, Jennifer A; Nuzzo, Ralph G

2016-05-01

R. Nuzzo and co-workers show on page 1025 how compositional differences in hydrogels are used to tune their cellular compliance by controlling their polymer mesh properties and subsequent uptake of the protein poly-l-lysine (green spheres in circled inset). The cover image shows pyramid micro-scaffolds prepared using direct ink writing (DIW) that differentially direct fibroblast and preosteoblast growth in 3D, depending on cell motility and surface treatment. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

STAT3 Potentiates SIAH-1 Mediated Proteasomal Degradation of β-Catenin in Human Embryonic Kidney Cells.

PubMed

Shin, Minkyung; Yi, Eun Hee; Kim, Byung-Hak; Shin, Jae-Cheon; Park, Jung Youl; Cho, Chung-Hyun; Park, Jong-Wan; Choi, Kang-Yell; Ye, Sang-Kyu

2016-11-30

The β-catenin functions as an adhesion molecule and a component of the Wnt signaling pathway. In the absence of the Wnt ligand, β-catenin is constantly phosphorylated, which designates it for degradation by the APC complex. This process is one of the key regulatory mechanisms of β-catenin. The level of β-catenin is also controlled by the E3 ubiquitin protein ligase SIAH-1 via a phosphorylation-independent degradation pathway. Similar to β-catenin, STAT3 is responsible for various cellular processes, such as survival, proliferation, and differentiation. However, little is known about how these molecules work together to regulate diverse cellular processes. In this study, we investigated the regulatory relationship between STAT3 and β-catenin in HEK293T cells. To our knowledge, this is the first study to report that β-catenin-TCF-4 transcriptional activity was suppressed by phosphorylated STAT3; furthermore, STAT3 inactivation abolished this effect and elevated activated β-catenin levels. STAT3 also showed a strong interaction with SIAH-1, a regulator of active β-catenin via degradation, which stabilized SIAH-1 and increased its interaction with β-catenin. These results suggest that activated STAT3 regulates active β-catenin protein levels via stabilization of SIAH-1 and the subsequent ubiquitin-dependent proteasomal degradation of β-catenin in HEK293T cells.

Tubing-Electrospinning: A One-Step Process for Fabricating Fibrous Matrices with Spatial, Chemical, and Mechanical Gradients.

PubMed

Kim, Jung-Suk; Im, Byung Gee; Jin, Gyuhyung; Jang, Jae-Hyung

2016-08-31

Guiding newly generated tissues in a gradient pattern, thereby precisely mimicking inherent tissue morphology and subsequently arranging the intimate networks between adjacent tissues, is essential to raise the technical levels of tissue engineering and facilitate its transition into the clinic. In this study, a straightforward electrospinning method (the tubing-electrospinning technique) was developed to create fibrous matrices readily with diverse gradient patterns and to induce patterned cellular responses. Gradient fibrous matrices can be produced simply by installing a series of polymer-containing lengths of tubing into an electrospinning circuit and sequentially processing polymers without a time lag. The loading of polymer samples with different characteristics, including concentration, wettability, and mechanical properties, into the tubing system enabled unique features in fibrous matrices, such as longitudinal gradients in fiber density, surface properties, and mechanical stiffness. The resulting fibrous gradients were shown to arrange cellular migration and residence in a gradient manner, thereby offering efficient cues to mediate patterned tissue formation. The one-step process using tubing-electrospinning apparatus can be used without significant modifications regardless of the type of fibrous gradient. Hence, the tubing-electrospinning system can serve as a platform that can be readily used by a wide-range of users to induce patterned tissue formation in a gradient manner, which will ultimately improve the functionality of tissue engineering scaffolds.

Charting the travels of copper in eukaryotes from yeast to mammals

PubMed Central

Nevitt, Tracy; Öhrvik, Helena; Thiele, Dennis J.

2012-01-01

Throughout evolution, all organisms have harnessed the redox properties of copper (Cu) and iron (Fe) as a cofactor or structural determinant of proteins that perform critical functions in biology. At its most sobering stance to Earth’s biome, Cu biochemistry allows photosynthetic organisms to harness solar energy and convert it into the organic energy that sustains the existence of all nonphotosynthetic life forms. The conversion of organic energy, in the form of nutrients that include carbohydrates, amino acids and fatty acids, is subsequently released during cellular respiration, itself a Cu-dependent process, and stored as ATP that is used to drive a myriad of critical biological processes such as enzyme-catalyzed biosynthetic processes, transport of cargo around cells and across membranes, and protein degradation. The life-supporting properties of Cu incur a significant challenge to cells that must not only exquisitely balance intracellular Cu concentrations, but also chaperone this redox-active metal from its point of cellular entry to its ultimate destination so as to avert the potential for inappropriate biochemical interactions or generation of damaging reactive oxidative species (ROS). In this review we chart the travels of Cu from the extracellular milieu of fungal and mammalian cells, its path within the cytosol as inferred by the proteins and ligands that escort and deliver Cu to intracellular organelles and protein targets, and its journey throughout the body of mammals. PMID:22387373

Development And Use Of Advanced Microfabricated Traction Force Sensing Substrates To Study The Effect of Nanosilver On Human Macrophages

NASA Astrophysics Data System (ADS)

Stark, Daniel Thomas

While nanoparticles are a natural byproduct of combustion and a number of natural processes, engineered nanoparticles have only recently entered the consumer market. This motivates the development of methods for studying their effects on human cells, thereby indicating how larger models such as animals and humans might react to them. This research develops a method to mechanically characterize cellular traction forces as a measure of exposure to nanoparticles. To do this, 1microm micropillar molds were fabricated in silicon wafers using smooth sidewall reactive ion plasma etching technologies. Polydimethylsiloxane (PDMS), was cured inside the silicon molds, subsequently treated for cell culture and used to measure cellular traction forces over time in live cell time-lapse experiments. For the first time, transmitted light was used to visualize the PDMS micropillars; a force resolution of 5.6 +/-2.1nN was achieved across all experiments using a standard Olympus IX81 confocal microscope affixed with a 60x NA2.1 objective. To initiate cellular movement, monocyte chemoattractant protein (MCP-1) was conjugated to 1microm latex beads. The effects of 40nm silver nanoparticle exposures were quantified using the micropillar array. Changes in cellular behavior between the control group and cells exposed to nanosilver were not significant, although a comparison between the 5microg/ml and 10microg/ml nanosilver concentrations yielded strong significance using a 2 sided Students t test.

Compound immobilization and drug-affinity chromatography.

PubMed

Rix, Uwe; Gridling, Manuela; Superti-Furga, Giulio

2012-01-01

Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to understanding its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identification of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobilization, which therefore needs to be not only efficient, but also mild. The subsequent affinity chromatography step needs to preserve molecular and conformational integrity of both bait compound and proteins in order to result in the desired specific enrichment while ensuring a high level of compatibility with downstream analysis by mass spectrometry. Thus, the right choice of detergent, buffer, and protease inhibitors is also essential. This chapter describes a widely applicable procedure for the immobilization of small molecule drugs and for drug-affinity chromatography with subsequent protein identification by mass spectrometry.

Au-rich filamentary behavior and associated subband gap optical absorption in hyperdoped Si

NASA Astrophysics Data System (ADS)

Yang, W.; Akey, A. J.; Smillie, L. A.; Mailoa, J. P.; Johnson, B. C.; McCallum, J. C.; Macdonald, D.; Buonassisi, T.; Aziz, M. J.; Williams, J. S.

2017-12-01

Au-hyperdoped Si, synthesized by ion implantation and pulsed laser melting, is known to exhibit a strong sub-band gap photoresponse that scales monotonically with the Au concentration. However, there is thought to be a limit to this behavior since ultrahigh Au concentrations (>1 ×1020c m-3 ) are expected to induce cellular breakdown during the rapid resolidification of Si, a process that is associated with significant lateral impurity precipitation. This work shows that the cellular morphology observed in Au-hyperdoped Si differs from that in conventional, steady-state cellular breakdown. In particular, Rutherford backscattering spectrometry combined with channeling and transmission electron microscopy revealed an inhomogeneous Au distribution and a subsurface network of Au-rich filaments, within which the Au impurities largely reside on substitutional positions in the crystalline Si lattice, at concentrations as high as ˜3 at. %. The measured substitutional Au dose, regardless of the presence of Au-rich filaments, correlates strongly with the sub-band gap optical absorptance. Upon subsequent thermal treatment, the supersaturated Au forms precipitates, while the Au substitutionality and the sub-band gap optical absorption both decrease. These results offer insight into a metastable filamentary regime in Au-hyperdoped Si that has important implications for Si-based infrared optoelectronics.

Dynamic photopatterning of cells in situ by Q-switched neodymium-doped yttrium ortho-vanadate laser.

PubMed

Deka, Gitanjal; Okano, Kazunori; Kao, Fu-Jen

2014-01-01

Cellular micropattering has been increasingly adopted in quantitative biological experiments. A Q-switched pulsed neodymium-doped yttrium ortho-vanadate (Nd∶YVO4) laser directed in-situ microfabrication technique for cell patterning is presented. A platform is designed uniquely to achieve laser ablation. The platform is comprised of thin gold coating over a glass surface that functions as a thermal transducer and is over-layered by a cell repellant polymer layer. Micropatterns are engraved on the platform, subsequently exposing specific cell adhesive micro-domains by ablating the gold-polymer coating photothermally. Experimental results indicate that the proposed approach is applicable under culture conditions, viable toward cells, and has a higher engraving speed. Possible uses in arraying isolated single cells on the platform are also shown. Additionally, based on those micro-patterns, dynamic cellular morphological changes and migrational speed in response to geometrical barriers are studied to demonstrate the potential applications of the proposed approach. Our results further demonstrate that cells in narrower geometry had elongated shapes and higher migrational speed than those in wider geometry. Importantly, the proposed approach will provide a valuable reference for efforts to study single cell dynamics and cellular migration related processes for areas such as cell division, wound healing, and cancer invasion.

Quality controls in cellular immunotherapies: rapid assessment of clinical grade dendritic cells by gene expression profiling.

PubMed

Castiello, Luciano; Sabatino, Marianna; Zhao, Yingdong; Tumaini, Barbara; Ren, Jiaqiang; Ping, Jin; Wang, Ena; Wood, Lauren V; Marincola, Francesco M; Puri, Raj K; Stroncek, David F

2013-02-01

Cell-based immunotherapies are among the most promising approaches for developing effective and targeted immune response. However, their clinical usefulness and the evaluation of their efficacy rely heavily on complex quality control assessment. Therefore, rapid systematic methods are urgently needed for the in-depth characterization of relevant factors affecting newly developed cell product consistency and the identification of reliable markers for quality control. Using dendritic cells (DCs) as a model, we present a strategy to comprehensively characterize manufactured cellular products in order to define factors affecting their variability, quality and function. After generating clinical grade human monocyte-derived mature DCs (mDCs), we tested by gene expression profiling the degrees of product consistency related to the manufacturing process and variability due to intra- and interdonor factors, and how each factor affects single gene variation. Then, by calculating for each gene an index of variation we selected candidate markers for identity testing, and defined a set of genes that may be useful comparability and potency markers. Subsequently, we confirmed the observed gene index of variation in a larger clinical data set. In conclusion, using high-throughput technology we developed a method for the characterization of cellular therapies and the discovery of novel candidate quality assurance markers.

Targeting dendritic cells through gold nanoparticles: A review on the cellular uptake and subsequent immunological properties.

PubMed

Ahmad, Suhana; Zamry, Anes Ateqah; Tan, Hern-Tze Tina; Wong, Kah Keng; Lim, JitKang; Mohamud, Rohimah

2017-11-01

Gold nanoparticles (NPs) have been proposed as a highly potential tool in immunotherapies due to its advantageous properties including customizable size and shapes, surface functionality and biocompatibility. Dendritic cells (DCs), the sentinels of immune response, have been of interest to be manipulated by using gold NPs for targeted delivery of immunotherapeutic agent. Researches done especially in human DCs showed a variation of gold NPs effects on cellular uptake and internalization, DC maturation and subsequent T cells priming as well as cytotoxicity. In this review, we describe the synthesis and physiochemical properties of gold NPs as well as the importance of gold NPs in immunotherapies through their actions on human DCs. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Receptor trafficking via the perinuclear recycling compartment accompanied by cell division is necessary for permanent neurotensin cell sensitization and leads to chronic mitogen-activated protein kinase activation.

PubMed

Toy-Miou-Leong, Mireille; Cortes, Catherine Llorens; Beaudet, Alain; Rostène, William; Forgez, Patricia

2004-03-26

Most G protein-coupled receptors are internalized after interaction with their respective ligand, a process that subsequently contributes to cell desensitization, receptor endocytosis, trafficking, and finally cell resensitization. Although cellular mechanisms leading to cell desensitization have been widely studied, those responsible for cell resensitization are still poorly understood. We examined here the traffic of the high affinity neurotensin receptor (NT1 receptor) following prolonged exposure to high agonist concentration. Fluorescence and confocal microscopy of Chinese hamster ovary, human neuroblastoma (CHP 212), and murine neuroblastoma (N1E-115) cells expressing green fluorescent protein-tagged NT1 receptor revealed that under prolonged treatment with saturating concentrations of neurotensin (NT) agonist, NT1 receptor and NT transiently accumulated in the perinuclear recycling compartment (PNRC). During this cellular event, cell surface receptors remained markedly depleted as detected by both confocal microscopy and (125)I-NT binding assays. In dividing cells, we observed that following prolonged NT agonist stimulation, NT1 receptors were removed from the PNRC, accumulated in dispersed vesicles inside the cytoplasm, and subsequently reappeared at the cell surface. This NT binding recovery allowed for constant cell sensitization and led to a chronic activation of mitogen-activated protein kinases p42 and p44. Under these conditions, the constant activation of NT1 receptor generates an oncogenic regulation. These observations support the potent role for neuropeptides, such as NT, in cancer progression.

Candida albicans-Induced Epithelial Damage Mediates Translocation through Intestinal Barriers

PubMed Central

2018-01-01

ABSTRACT Life-threatening systemic infections often occur due to the translocation of pathogens across the gut barrier and into the bloodstream. While the microbial and host mechanisms permitting bacterial gut translocation are well characterized, these mechanisms are still unclear for fungal pathogens such as Candida albicans, a leading cause of nosocomial fungal bloodstream infections. In this study, we dissected the cellular mechanisms of translocation of C. albicans across intestinal epithelia in vitro and identified fungal genes associated with this process. We show that fungal translocation is a dynamic process initiated by invasion and followed by cellular damage and loss of epithelial integrity. A screen of >2,000 C. albicans deletion mutants identified genes required for cellular damage of and translocation across enterocytes. Correlation analysis suggests that hypha formation, barrier damage above a minimum threshold level, and a decreased epithelial integrity are required for efficient fungal translocation. Translocation occurs predominantly via a transcellular route, which is associated with fungus-induced necrotic epithelial damage, but not apoptotic cell death. The cytolytic peptide toxin of C. albicans, candidalysin, was found to be essential for damage of enterocytes and was a key factor in subsequent fungal translocation, suggesting that transcellular translocation of C. albicans through intestinal layers is mediated by candidalysin. However, fungal invasion and low-level translocation can also occur via non-transcellular routes in a candidalysin-independent manner. This is the first study showing translocation of a human-pathogenic fungus across the intestinal barrier being mediated by a peptide toxin. PMID:29871918

Developmental Progression in the Coral Acropora digitifera Is Controlled by Differential Expression of Distinct Regulatory Gene Networks

PubMed Central

Reyes-Bermudez, Alejandro; Villar-Briones, Alejandro; Ramirez-Portilla, Catalina; Hidaka, Michio; Mikheyev, Alexander S.

2016-01-01

Corals belong to the most basal class of the Phylum Cnidaria, which is considered the sister group of bilaterian animals, and thus have become an emerging model to study the evolution of developmental mechanisms. Although cell renewal, differentiation, and maintenance of pluripotency are cellular events shared by multicellular animals, the cellular basis of these fundamental biological processes are still poorly understood. To understand how changes in gene expression regulate morphogenetic transitions at the base of the eumetazoa, we performed quantitative RNA-seq analysis during Acropora digitifera’s development. We collected embryonic, larval, and adult samples to characterize stage-specific transcription profiles, as well as broad expression patterns. Transcription profiles reconstructed development revealing two main expression clusters. The first cluster grouped blastula and gastrula and the second grouped subsequent developmental time points. Consistently, we observed clear differences in gene expression between early and late developmental transitions, with higher numbers of differentially expressed genes and fold changes around gastrulation. Furthermore, we identified three coexpression clusters that represented discrete gene expression patterns. During early transitions, transcriptional networks seemed to regulate cellular fate and morphogenesis of the larval body. In late transitions, these networks seemed to play important roles preparing planulae for switch in lifestyle and regulation of adult processes. Although developmental progression in A. digitifera is regulated to some extent by differential coexpression of well-defined gene networks, stage-specific transcription profiles appear to be independent entities. While negative regulation of transcription is predominant in early development, cell differentiation was upregulated in larval and adult stages. PMID:26941230

Hydrodynamic size-dependent cellular uptake of aqueous QDs probed by fluorescence correlation spectroscopy.

PubMed

Dong, Chaoqing; Irudayaraj, Joseph

2012-10-11

Aqueous quantum dots (QDs) directly synthesized with various thiol ligands have been investigated as imaging probes in living cells. However, the effect of the surface chemistry of these ligands on QDs' cellular uptakes and their intracellular fate remains poorly understood. In this work, four CdTe QDs were directly synthesized under aqueous conditions using four different thiols as stabilizers and their interactions with cells were investigated. Fluorescence correlation spectroscopy (FCS), X-ray photoelectron spectroscopy (XPS), and zeta potential measurements on QDs primarily show that the surface structure of these QDs is highly dependent on the thiol ligands used in the preparation of QDs' precursors, including its layer thicknesses, densities, and surface charges. Subsequently, FCS integrated with the maximum-entropy-method-based FCS (MEMFCS) was used to investigate the concentration distribution and dynamics of these QDs in living A-427 cells. Our findings indicate that QDs' surface characteristics affect cell membrane adsorption and subsequent internalization. More critically, we show that the cellular uptake of aqueous QDs is dependent on their hydrodynamic diameter and might have the potential to escape trapped environments to accumulate in the cytoplasm.

Is there a role for organic trace element supplements in transition cow health?

PubMed

Andrieu, S

2008-04-01

Optimal transition cow health is the key to success of the subsequent lactation, and increasing attention has been focused on management and nutritional practices that support it. Physiological stress during the transition period alters the efficiency of the immune system, making the lactating dairy cow more susceptible to infectious diseases, such as mastitis and metritis, with subsequent impairment of reproductive performance. Trace elements have a specific role in free radical control at the cellular level and influence the anti-oxidant/free radical balance. Dietary trace elements must be available for absorption throughout the whole of the digestive process until they reach the final site of absorption in the small intestine. Negative interactions between minerals can occur and, as the intestinal environment lowers the absorption of ionic minerals, chelation technology has been developed to increase mineral bioavailability. Organic trace elements have been used in dairy cow experiments, resulting in significant improvements in udder health, lameness and reproductive performance.

Quantitative 4D analyses of epithelial folding during Drosophila gastrulation.

PubMed

Khan, Zia; Wang, Yu-Chiun; Wieschaus, Eric F; Kaschube, Matthias

2014-07-01

Understanding the cellular and mechanical processes that underlie the shape changes of individual cells and their collective behaviors in a tissue during dynamic and complex morphogenetic events is currently one of the major frontiers in developmental biology. The advent of high-speed time-lapse microscopy and its use in monitoring the cellular events in fluorescently labeled developing organisms demonstrate tremendous promise in establishing detailed descriptions of these events and could potentially provide a foundation for subsequent hypothesis-driven research strategies. However, obtaining quantitative measurements of dynamic shapes and behaviors of cells and tissues in a rapidly developing metazoan embryo using time-lapse 3D microscopy remains technically challenging, with the main hurdle being the shortage of robust imaging processing and analysis tools. We have developed EDGE4D, a software tool for segmenting and tracking membrane-labeled cells using multi-photon microscopy data. Our results demonstrate that EDGE4D enables quantification of the dynamics of cell shape changes, cell interfaces and neighbor relations at single-cell resolution during a complex epithelial folding event in the early Drosophila embryo. We expect this tool to be broadly useful for the analysis of epithelial cell geometries and movements in a wide variety of developmental contexts. © 2014. Published by The Company of Biologists Ltd.

Mouse Norovirus infection promotes autophagy induction to facilitate replication but prevents final autophagosome maturation

DOE Office of Scientific and Technical Information (OSTI.GOV)

O’Donnell, Tanya B.; Hyde, Jennifer L.; Mintern, Justine D.

Autophagy is a cellular process used to eliminate intracellular pathogens. Many viruses however are able to manipulate this cellular process for their own advantage. Here we demonstrate that Mouse Norovirus (MNV) infection induces autophagy but does not appear to utilise the autophagosomal membrane for establishment and formation of the viral replication complex. We have observed that MNV infection results in lipidation and recruitment of LC3 to the autophagosome membrane but prevents subsequent fusion of the autophagosomes with lysosomes, as SQSTM1 (an autophagy receptor) accumulates and Lysosome-Associated Membrane Protein1 is sequestered to the MNV replication complex (RC) rather than to autophagosomes.more » We have additionally observed that chemical modulation of autophagy differentially affects MNV replication. From this study we can conclude that MNV infection induces autophagy, however suppresses the final maturation step of this response, indicating that autophagy induction contributes to MNV replication independently of RC biogenesis. - Highlights: • MNV induces autophagy in infected murine macrophages. • MNV does not utilise autophagosomal membranes for replication. • The MNV-induced autophagosomes do not fuse with lysosomes. • MNV sequesters SQSTM1 to prevent autophagy degradation and turnover. • Chemical modulation of autophagy enhances MNV replication.« less

Live-Cell Imaging of Mitochondria and the Actin Cytoskeleton in Budding Yeast.

PubMed

Higuchi-Sanabria, Ryo; Swayne, Theresa C; Boldogh, Istvan R; Pon, Liza A

2016-01-01

Maintenance and regulation of proper mitochondrial dynamics and functions are necessary for cellular homeostasis. Numerous diseases, including neurodegeneration and muscle myopathies, and overall cellular aging are marked by declining mitochondrial function and subsequent loss of multiple other cellular functions. For these reasons, optimized protocols are needed for visualization and quantification of mitochondria and their function and fitness. In budding yeast, mitochondria are intimately associated with the actin cytoskeleton and utilize actin for their movement and inheritance. This chapter describes optimal approaches for labeling mitochondria and the actin cytoskeleton in living budding yeast cells, for imaging the labeled cells, and for analyzing the resulting images.

Mitochondria targeting by environmental stressors: Implications for redox cellular signaling.

PubMed

Blajszczak, Chuck; Bonini, Marcelo G

2017-11-01

Mitochondria are cellular powerhouses as well as metabolic and signaling hubs regulating diverse cellular functions, from basic physiology to phenotypic fate determination. It is widely accepted that reactive oxygen species (ROS) generated in mitochondria participate in the regulation of cellular signaling, and that some mitochondria chronically operate at a high ROS baseline. However, it is not completely understood how mitochondria adapt to persistently high ROS states and to environmental stressors that disturb the redox balance. Here we will review some of the current concepts regarding how mitochondria resist oxidative damage, how they are replaced when excessive oxidative damage compromises function, and the effect of environmental toxicants (i.e. heavy metals) on the regulation of mitochondrial ROS (mtROS) production and subsequent impact. Copyright © 2017 Elsevier B.V. All rights reserved.

In utero exposure to Onchocerca volvulus: relationship to subsequent infection intensity and cellular immune responsiveness.

PubMed Central

Elson, L H; Days, A; Calvopiña, M; Paredes, W; Araujo, E; Guderian, R H; Bradley, J E; Nutman, T B

1996-01-01

Afro-Ecuadorian individuals from an area where Onchocerca volvulus is hyperendemic have been monitored for infection over the past 16 years. To determine whether in utero exposure to O. volvulus biases a child's subsequent immune responses, children (9 to 16 years old) for whom the mother's infection status was known were chosen for study. Children of infected mothers (n = 19) had significantly higher levels of skin microfilariae than children of uninfected mothers (n = 13; P = 0.021). While the serum levels of O. volvulus-specific immunoglobulin G (IgG), IgG subclasses, and IgE showed no significant differences between the two groups of children, peripheral blood mononuclear cells of children of infected mothers produced higher levels of Th2-type cytokines to several parasite antigens and lower levels of Th1-type cytokines to nonparasite antigens than those of children of uninfected mothers. Thus, in utero exposure to O. volvulus has a long-term effect on the child's subsequent cellular immune response that may render the child more susceptible to O. volvulus infection postnatally. PMID:8945547

Imaging of Posttraumatic Arthritis, Avascular Necrosis, Septic Arthritis, Complex Regional Pain Syndrome, and Cancer Mimicking Arthritis.

PubMed

Rupasov, Andrey; Cain, Usa; Montoya, Simone; Blickman, Johan G

2017-09-01

This article focuses on the imaging of 5 discrete entities with a common end result of disability: posttraumatic arthritis, a common form of secondary osteoarthritis that results from a prior insult to the joint; avascular necrosis, a disease of impaired osseous blood flow, leading to cellular death and subsequent osseous collapse; septic arthritis, an infectious process leading to destructive changes within the joint; complex regional pain syndrome, a chronic limb-confined painful condition arising after injury; and cases of cancer mimicking arthritis, in which the initial findings seem to represent arthritis, despite a more insidious cause. Copyright © 2017 Elsevier Inc. All rights reserved.

Probing cellular heterogeneity in cytokine-secreting immune cells using droplet-based microfluidics.

PubMed

Chokkalingam, Venkatachalam; Tel, Jurjen; Wimmers, Florian; Liu, Xin; Semenov, Sergey; Thiele, Julian; Figdor, Carl G; Huck, Wilhelm T S

2013-12-21

Here, we present a platform to detect cytokine (IL-2, IFN-γ, TNF-α) secretion of single, activated T-cells in droplets over time. We use a novel droplet-based microfluidic approach to encapsulate cells in monodisperse agarose droplets together with functionalized cytokine-capture beads for subsequent binding and detection of secreted cytokines from single cells. This method allows high-throughput detection of cellular heterogeneity and maps subsets within cell populations with specific functions.

CoCr wear particles generated from CoCr alloy metal-on-metal hip replacements, and cobalt ions stimulate apoptosis and expression of general toxicology-related genes in monocyte-like U937 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Posada, Olga M., E-mail: O.M.PosadaEstefan@leeds.ac.uk; Gilmour, Denise; Tate, Rothwelle J., E-mail: r.j.tate@strath.ac.uk

Cobalt-chromium (CoCr) particles in the nanometre size range and their concomitant release of Co and Cr ions into the patients' circulation are produced by wear at the articulating surfaces of metal-on-metal (MoM) implants. This process is associated with inflammation, bone loss and implant loosening and led to the withdrawal from the market of the DePuy ASR™ MoM hip replacements in 2010. Ions released from CoCr particles derived from a resurfacing implant in vitro and their subsequent cellular up-take were measured by ICP-MS. Moreover, the ability of such metal debris and Co ions to induce both apoptosis was evaluated with bothmore » FACS and immunoblotting. qRT-PCR was used to assess the effects on the expression of lymphotoxin alpha (LTA), BCL2-associated athanogene (BAG1), nitric oxide synthase 2 inducible (NOS2), FBJ murine osteosarcoma viral oncogene homolog (FOS), growth arrest and DNA-damage-inducible alpha (GADD45A). ICP-MS showed that the wear debris released significant (p < 0.05) amounts of Co and Cr ions into the culture medium, and significant (p < 0.05) cellular uptake of both ions. There was also an increase (p < 0.05) in apoptosis after a 48 h exposure to wear debris. Analysis of qRT-PCR results found significant up-regulation (p < 0.05) particularly of NOS2 and BAG1 in Co pre-treated cells which were subsequently exposed to Co ions + debris. Metal debris was more effective as an inducer of apoptosis and gene expression when cells had been pre-treated with Co ions. This suggests that if a patient receives sequential bilateral CoCr implants, the second implant may be more likely to produce adverse effects than the first one. - Highlights: • Effects of CoCr nanoparticles and Co ions on U937 cells were investigated. • Ions released from wear debris play an important role in cellular response, • Toxicity of Co ions could be related to NO metabolic processes and apoptosis. • CoCr particles were a more effective inducer of apoptosis after cell priming. • CoCr particles were a more effective inducer of gene expression after cell priming.« less

Discovery and Characterization of Novel Nonsubstrate and Substrate NAMPT Inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilsbacher, Julie L.; Cheng, Min; Cheng, Dong

Cancer cells are highly reliant on NAD+-dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD+, and subsequently ATP, and produce cell deathmore » when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236–45.« less

Extraterrestrial Organic Chemistry: From the Interstellar Medium to the Origins of Life

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; DeVincenzi, Donald L. (Technical Monitor)

2000-01-01

Extraterrestrially delivered organics in the origin of cellular life. Various processes leading to the emergence of cellular life from organics delivered from space to earth or other planetary bodies in the solar system will be reviewed. The focus will be on: (1) self-assembly of amphiphilic material to vesicles and other structures, such as micelles and multilayers, and its role in creating environments suitable for chemical catalysis, (2) a possible role of extraterrestrial delivery of organics in the formation of the simplest bioenergetics (3) mechanisms leading from amino acids or their precursors to simple peptides and, subsequently, to the evolution of metabolism. These issues will be discussed from two opposite points of view: (1) Which molecules could have been particularly useful in the protobiological evolution; this may provide focus for searching for these molecules in interstellar media. (2) Assuming that a considerable part of the inventory of organic matter on the early earth was delivered extraterrestrially, what does relative abundance of different organics in space tell us about the scenario leading to the origin of life.

Feasibility study of a mini fuel cell to detect interference from a cellular phone

NASA Astrophysics Data System (ADS)

Abdullah, M. O.; Gan, Y. K.

Fuel cells produce electricity without involving combustion processes. They generate no noise, vibration or air pollution and are therefore suitable for use in many vibration-free power-generating applications. In this study, a mini alkaline fuel cell signal detector system has been designed, constructed and tested. The initial results have shown the applicability of such system for used as an indicator of signal disturbance from cellular phones. A small disturbance even at 4 mV cm -1, corresponding to an amplitude of 12-18 mG in terms of electromagnetic field, can be well detected by such a device. Subsequently, a thermodynamics model has been developed to provide a parametric study by simulation to show the likely performance of the fuel cell alone in other environments. As such the model can provide many useful generic design data for alkaline fuel cells. Two general conclusions can be drawn from the present theoretical study: (i) fuel cell performance increases with temperature, pressure and correction factor, C f; (ii) the temperature factor (E/ T) increases with increasing temperature and with increasing pressure factor.

Neurobiological Mechanisms for the Regulation of Mammalian Sleep-Wake Behavior: Reinterpretation of Historical Evidence and Inclusion of Contemporary Cellular and Molecular Evidence

PubMed Central

Datta, Subimal; MacLean, Robert Ross

2007-01-01

At its most basic level, the function of mammalian sleep can be described as a restorative process of the brain and body; recently, however, progressive research has revealed a host of vital functions to which sleep is essential. Although many excellent reviews on sleep behavior have been published, none have incorporated contemporary studies examining the molecular mechanisms that govern the various stages of sleep. Utilizing a holistic approach, this review is focused on the basic mechanisms involved in the transition from wakefulness, initiation of sleep and the subsequent generation of slow-wave sleep and rapid eye movement (REM) sleep. Additionally, using recent molecular studies and experimental evidence that provides a direct link to sleep as a behavior, we have developed a new model, the Cellular-Molecular-Network model, explaining the mechanisms responsible for regulating REM sleep. By analyzing the fundamental neurobiological mechanisms responsible for the generation and maintenance of sleep-wake behavior in mammals, we intend to provide a broader understanding of our present knowledge in the field of sleep research. PMID:17445891

Exploring the interactome: microfluidic isolation of proteins and interacting partners for quantitative analysis by electron microscopy.

PubMed

Giss, Dominic; Kemmerling, Simon; Dandey, Venkata; Stahlberg, Henning; Braun, Thomas

2014-05-20

Multimolecular protein complexes are important for many cellular processes. However, the stochastic nature of the cellular interactome makes the experimental detection of complex protein assemblies difficult and quantitative analysis at the single molecule level essential. Here, we present a fast and simple microfluidic method for (i) the quantitative isolation of endogenous levels of untagged protein complexes from minute volumes of cell lysates under close to physiological conditions and (ii) the labeling of specific components constituting these complexes. The method presented uses specific antibodies that are conjugated via a photocleavable linker to magnetic beads that are trapped in microcapillaries to immobilize the target proteins. Proteins are released by photocleavage, eluted, and subsequently analyzed by quantitative transmission electron microscopy at the single molecule level. Additionally, before photocleavage, immunogold can be employed to label proteins that interact with the primary target protein. Thus, the presented method provides a new way to study the interactome and, in combination with single molecule transmission electron microscopy, to structurally characterize the large, dynamic, heterogeneous multimolecular protein complexes formed.

Optical control and study of biological processes at the single-cell level in a live organism

NASA Astrophysics Data System (ADS)

Feng, Zhiping; Zhang, Weiting; Xu, Jianmin; Gauron, Carole; Ducos, Bertrand; Vriz, Sophie; Volovitch, Michel; Jullien, Ludovic; Weiss, Shimon; Bensimon, David

2013-07-01

Living organisms are made of cells that are capable of responding to external signals by modifying their internal state and subsequently their external environment. Revealing and understanding the spatio-temporal dynamics of these complex interaction networks is the subject of a field known as systems biology. To investigate these interactions (a necessary step before understanding or modelling them) one needs to develop means to control or interfere spatially and temporally with these processes and to monitor their response on a fast timescale (< minute) and with single-cell resolution. In 2012, an EMBO workshop on ‘single-cell physiology’ (organized by some of us) was held in Paris to discuss those issues in the light of recent developments that allow for precise spatio-temporal perturbations and observations. This review will be largely based on the investigations reported there. We will first present a non-exhaustive list of examples of cellular interactions and developmental pathways that could benefit from these new approaches. We will review some of the novel tools that have been developed for the observation of cellular activity and then discuss the recent breakthroughs in optical super-resolution microscopy that allow for optical observations beyond the diffraction limit. We will review the various means to photo-control the activity of biomolecules, which allow for local perturbations of physiological processes. We will end up this review with a report on the current status of optogenetics: the use of photo-sensitive DNA-encoded proteins as sensitive reporters and efficient actuators to perturb and monitor physiological processes.

Processing Characteristics and Properties of the Cellular Products Made by Using Special Foaming Agents

NASA Astrophysics Data System (ADS)

Garbacz, Tomasz; Dulebova, Ludmila

2012-12-01

The paper describes the manufacturing process of extruded products by the cellular extrusion method, and presents specifications of the blowing agents used in the extrusion process as well as process conditions. The process of cellular extrusion of thermoplastic materials is aimed at obtaining cellular shapes and coats with reduced density, presenting no hollows on the surface of extruder product and displaying minimal contraction under concurrent maintenance of properties similar to properties of products extruded by means of the conventional method. In order to obtain cellular structure, the properties of extruded product are modified by applying suitable plastic or inserting auxiliary agents.

Female gametophyte development and double fertilization in Balsas teosinte, Zea mays subsp. parviglumis (Poaceae).

PubMed

Wu, Chi-Chih; Diggle, Pamela K; Friedman, William E

2011-09-01

Over the course of maize evolution, domestication played a major role in the structural transition of the vegetative and reproductive characteristics that distinguish it from its closest wild relative, Zea mays subsp. parviglumis (Balsas teosinte). Little is known, however, about impacts of the domestication process on the cellular features of the female gametophyte and the subsequent reproductive events after fertilization, even though they are essential components of plant sexual reproduction. In this study, we investigated the developmental and cellular features of the Balsas teosinte female gametophyte and early developing seed in order to unravel the key structural and evolutionary transitions of the reproductive process associated with the domestication of the ancestor of maize. Our results show that the female gametophyte of Balsas teosinte is a variation of the Polygonum type with proliferative antipodal cells and is similar to that of maize. The fertilization process of Balsas teosinte also is basically similar to domesticated maize. In contrast to maize, many events associated with the development of the embryo and endosperm appear to be initiated earlier in Balsas teosinte. Our study suggests that the pattern of female gametophyte development with antipodal proliferation is common among species and subspecies of Zea and evolved before maize domestication. In addition, we propose that the relatively longer duration of the free nuclear endosperm phase in maize is correlated with the development of a larger fruit (kernel or caryopsis) and with a bigger endosperm compared with Balsas teosinte.

Aging with a traumatic brain injury: Could behavioral morbidities and endocrine symptoms be influenced by microglial priming?

PubMed

Ziebell, Jenna M; Rowe, Rachel K; Muccigrosso, Megan M; Reddaway, Jack T; Adelson, P David; Godbout, Jonathan P; Lifshitz, Jonathan

2017-01-01

A myriad of factors influence the developmental and aging process and impact health and life span. Mounting evidence indicates that brain injury, even moderate injury, can lead to lifetime of physical and mental health symptoms. Therefore, the purpose of this mini-review is to discuss how recovery from traumatic brain injury (TBI) depends on age-at-injury and how aging with a TBI affects long-term recovery. TBI initiates pathophysiological processes that dismantle circuits in the brain. In response, reparative and restorative processes reorganize circuits to overcome the injury-induced damage. The extent of circuit dismantling and subsequent reorganization depends as much on the initial injury parameters as other contributing factors, such as genetics and age. Age-at-injury influences the way the brain is able to repair itself, as a result of developmental status, extent of cellular senescence, and injury-induced inflammation. Moreover, endocrine dysfunction can occur with TBI. Depending on the age of the individual at the time of injury, endocrine dysfunction may disrupt growth, puberty, influence social behaviors, and possibly alter the inflammatory response. In turn, activation of microglia, the brain's immune cells, after injury may continue to fuel endocrine dysfunction. With age, the immune system develops and microglia become primed to subsequent challenges. Sustained inflammation and microglial activation can continue for weeks to months post-injury. This prolonged inflammation can influence developmental processes, behavioral performance and age-related decline. Overall, brain injury may influence the aging process and expedite glial and neuronal alterations that impact mental health. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of a Honeycomb-Like Scaffold With Dielectrophoresis-Based Patterning for Tissue Engineering.

PubMed

Huan, Zhijie; Chu, Henry K; Yang, Jie; Sun, Dong

2017-04-01

Seeding and patterning of cells with an engineered scaffold is a critical process in artificial tissue construction and regeneration. To date, many engineered scaffolds exhibit simple intrinsic designs, which fail to mimic the geometrical complexity of native tissues. In this study, a novel scaffold that can automatically seed cells into multilayer honeycomb patterns for bone tissue engineering application was designed and examined. The scaffold incorporated dielectrophoresis for noncontact manipulation of cells and intrinsic honeycomb architectures were integrated in each scaffold layer. When a voltage was supplied to the stacked scaffold layers, three-dimensional electric fields were generated, thereby manipulating cells to form into honeycomb-like cellular patterns for subsequent culture. The biocompatibility of the scaffold material was confirmed through the cell viability test. Experiments were conducted to evaluate the cell viability during DEP patterning at different voltage amplitudes, frequencies, and manipulating time. Three different mammalian cells were examined and the effects of the cell size and the cell concentration on the resultant cellular patterns were evaluated. Results showed that the proposed scaffold structure was able to construct multilayer honeycomb cellular patterns in a manner similar to the natural tissue. This honeycomb-like scaffold and the dielectrophoresis-based patterning technique examined in this study could provide the field with a promising tool to enhance seeding and patterning of a wide range of cells for the development of high-quality artificial tissues.

Elixir of Life: Thwarting Aging With Regenerative Reprogramming.

PubMed

Beyret, Ergin; Martinez Redondo, Paloma; Platero Luengo, Aida; Izpisua Belmonte, Juan Carlos

2018-01-05

All living beings undergo systemic physiological decline after ontogeny, characterized as aging. Modern medicine has increased the life expectancy, yet this has created an aged society that has more predisposition to degenerative disorders. Therefore, novel interventions that aim to extend the healthspan in parallel to the life span are needed. Regeneration ability of living beings maintains their biological integrity and thus is the major leverage against aging. However, mammalian regeneration capacity is low and further declines during aging. Therefore, modalities that reinforce regeneration can antagonize aging. Recent advances in the field of regenerative medicine have shown that aging is not an irreversible process. Conversion of somatic cells to embryonic-like pluripotent cells demonstrated that the differentiated state and age of a cell is not fixed. Identification of the pluripotency-inducing factors subsequently ignited the idea that cellular features can be reprogrammed by defined factors that specify the desired outcome. The last decade consequently has witnessed a plethora of studies that modify cellular features including the hallmarks of aging in addition to cellular function and identity in a variety of cell types in vitro. Recently, some of these reprogramming strategies have been directly used in animal models in pursuit of rejuvenation and cell replacement. Here, we review these in vivo reprogramming efforts and discuss their potential use to extend the longevity by complementing or augmenting the regenerative capacity. © 2017 American Heart Association, Inc.

Transfer of Free Polymannose-type Oligosaccharides from the Cytosol to Lysosomes in Cultured Human Hepatocellular Carcinoma HEPG2 Cells

PubMed Central

Saint-Pol, Agnès; Bauvy, Chantal; Codogno, Patrice; Moore, Stuart E.H.

1997-01-01

Large, free polymannose oligosaccharides generated during glycoprotein biosynthesis rapidly appear in the cytosol of HepG2 cells where they undergo processing by a cytosolic endo H–like enzyme and a mannosidase to yield the linear isomer of Man5GlcNAc (Man[α1-2]Man[α1-2]Man[α1-3][Man α1-6]Man[β14]GlcNAc). Here we have examined the fate of these partially trimmed oligosaccharides in intact HepG2 cells. Subsequent to pulse–chase incubations with d-[2- 3H]mannose followed by permeabilization of cells with streptolysin O free oligosaccharides were isolated from the resulting cytosolic and membrane-bound compartments. Control pulse–chase experiments revealed that total cellular free oligosaccharides are lost from HepG2 cells with a half-life of 3–4 h. In contrast use of the vacuolar H+/ATPase inhibitor, concanamycin A, stabilized total cellular free oligosaccharides and enabled us to demonstrate a translocation of partially trimmed oligosaccharides from the cytosol into a membrane-bound compartment. This translocation process was unaffected by inhibitors of autophagy but inhibited if cells were treated with either 100 μM swainsonine, which provokes a cytosolic accumulation of large free oligosaccharides bearing 8-9 residues of mannose, or agents known to reduce cellular ATP levels which lead to the accumulation of the linear isomer of Man5GlcNAc in the cytosol. Subcellular fractionation studies on Percoll density gradients revealed that the cytosol-generated linear isomer of Man5GlcNAc is degraded in a membrane-bound compartment that cosediments with lysosomes. PMID:9008702

Transfer of free polymannose-type oligosaccharides from the cytosol to lysosomes in cultured human hepatocellular carcinoma HepG2 cells.

PubMed

Saint-Pol, A; Bauvy, C; Codogno, P; Moore, S E

1997-01-13

Large, free polymannose oligosaccharides generated during glycoprotein biosynthesis rapidly appear in the cytosol of HepG2 cells where they undergo processing by a cytosolic endo H-like enzyme and a mannosidase to yield the linear isomer of Man5GlcNAc (Man[alpha 1-2]Man[alpha 1-2]Man[alpha 1-3][Man alpha 1-6]Man[beta 1-4] GlcNAc). Here we have examined the fate of these partially trimmed oligosaccharides in intact HepG2 cells. Subsequent to pulse-chase incubations with D-[2-3H]mannose followed by permeabilization of cells with streptolysin O free oligosaccharides were isolated from the resulting cytosolic and membrane-bound compartments. Control pulse-chase experiments revealed that total cellular free oligosaccharides are lost from HepG2 cells with a half-life of 3-4 h. In contrast use of the vacuolar H+/ATPase inhibitor, concanamycin A, stabilized total cellular free oligosaccharides and enabled us to demonstrate a translocation of partially trimmed oligosaccharides from the cytosol into a membrane-bound compartment. This translocation process was unaffected by inhibitors of autophagy but inhibited if cells were treated with either 100 microM swainsonine, which provokes a cytosolic accumulation of large free oligosaccharides bearing 8-9 residues of mannose, or agents known to reduce cellular ATP levels which lead to the accumulation of the linear isomer of Man5GlcNAc in the cytosol. Subcellular fractionation studies on Percoll density gradients revealed that the cytosol-generated linear isomer of Man5GlcNAc is degraded in a membrane-bound compartment that cosediments with lysosomes.

Adaptation response of Pseudomonas fragi on refrigerated solid matrix to a moderate electric field.

PubMed

Chen, Wenbo; Hu, Honghai; Zhang, Chunjiang; Huang, Feng; Zhang, Dequan; Zhang, Hong

2017-02-10

Moderate electric field (MEF) technology is a promising food preservation strategy since it relies on physical properties-rather than chemical additives-to preserve solid cellular foods during storage. However, the effectiveness of long-term MEF exposure on the psychrotrophic microorganisms responsible for the food spoilage at cool temperatures remains unclear. The spoilage-associated psychrotroph Pseudomonas fragi MC16 was obtained from pork samples stored at 7 °C. Continuous MEF treatment attenuated growth and resulted in subsequent adaptation of M16 cultured on nutrient agar plates at 7 °C, compared to the control cultures, as determined by biomass analysis and plating procedures. Moreover, intracellular dehydrogenase activity and ATP levels also indicated an initial effect of MEF treatment followed by cellular recovery, and extracellular β-galactosidase activity assays indicated no obvious changes in cell membrane permeability. Furthermore, microscopic observations using scanning and transmission electron microscopy revealed that MEF induced sublethal cellular injury during early treatment stages, but no notable changes in morphology or cytology on subsequent days. Our study provides direct evidence that psychrotrophic P. fragi MC16 cultured on nutrient agar plates at 7 °C are capable of adapting to MEF treatment.

Therapeutics incorporating blood constituents.

PubMed

Charoenphol, Phapanin; Oswalt, Katie; Bishop, Corey J

2018-04-05

Blood deficiency and dysfunctionality can result in adverse events, which can primarily be treated by transfusion of blood or the re-introduction of properly functioning sub-components. Blood constituents can be engineered on the sub-cellular (i.e., DNA recombinant technology) and cellular level (i.e., cellular hitchhiking for drug delivery) for supplementing and enhancing therapeutic efficacy, in addition to rectifying dysfunctioning mechanisms (i.e., clotting). Herein, we report the progress of blood-based therapeutics, with an emphasis on recent applications of blood transfusion, blood cell-based therapies and biomimetic carriers. Clinically translated technologies and commercial products of blood-based therapeutics are subsequently highlighted and perspectives on challenges and future prospects are discussed. Blood-based therapeutics is a burgeoning field and has advanced considerably in recent years. Blood and its constituents, with and without modification (i.e., combinatorial), have been utilized in a broad spectrum of pre-clinical and clinically-translated treatments. This review article summarizes the most up-to-date progress of blood-based therapeutics in the following contexts: synthetic blood substitutes, acellular/non-recombinant therapies, cell-based therapies, and therapeutic sub-components. The article subsequently discusses clinically-translated technologies and future prospects thereof. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Biosynthetic processing of the oligosaccharide chains of cellular fibronectin.

PubMed

Olden, K; Hunter, V A; Yamada, K M

1980-10-15

We have examined the maturation or processing of the oligosaccharides of cellular fibronectin in cultured chick embryo fibroblasts. Fibronectin was pulse-labeled with [2-3H]mannose of [35S]methionine, and the turnover rates of carbohydrate and polypeptide portions of immunoprecipitated fibronectin were compared. The oligosaccharides on fibronectin were analyzed by gel electrophoresis for alterations in sensitivity to the enzyme endo-beta-N-acetylgluosaminidase H, which specifically cleaves the 'high-mannose' class of asparagine-linked oligosaccharide. Incorporated mannose was removed only at early time points, suggesting that the structure of fibronectin oligosaccharides was altered due to processing. This possibility was confirmed by the analysis of glycopeptides generated by exhaustive pronase digestion. Two major glycopeptide structures were detected; their properties correspond to a 'high-mannose' oligosaccharide precursor and a 'complex' carbohydrate product. The precursor-product relationship of these two forms of oligosaccharide chains was demonstrated by pulse-chase labeling experiments. The precursor glycopeptide had an apparent size (Mr 2100) comparable to (Man)9GlcNAc (Mr 2080), and was sensitive to endo-beta-N-acetylglucosaminidase H; nearly all of the labeled mannose incorporated in a 10 min pulse was released from fibronectin glycopeptides by this enzyme. During a 90 min chase period, the glycopeptides became larger and increasingly resistant to endo-beta-N-acetylglucosaminidase H cleavage. The final 'complex' or processed oligosaccharide structure contained approximately two-thirds less [3H]mannose, was insensitive to endo-beta-N-acetylglucosaminidase H and had an apparent Mr of 2300 as estimated by gel filtration. We conclude that the carbohydrate portion of fibronectin is synthesized as a 'high-mannose' intermediate and is subsequently processed to give the characteristic 'complex' oligosaccharide chains of fibronectin.

Cellular structure of lean hydrogen flames in microgravity

NASA Technical Reports Server (NTRS)

Patnaik, G.; Kailasanath, K.

1990-01-01

Detailed, time-dependent, two-dimensional numerical simulations of premixed laminar flames have been used to study the initiation and subsequent development of cellular structures in lean hydrogen-air flames. The model includes detailed hydrogen-oxygen combustion with 24 elementary reactions of eight reactive species and a nitrogen diluent, molecular diffusion of all species, thermal conduction, viscosity, and convection. This model has been used to study the nonlinear evolution of cellular flame structure and shows that cell splitting, as observed in experiments, can be predicted numerically for sufficiently reactive mixtures. The structures that evolved also resembled the cellular structures observed in experiments. The present study shows that the 'cell-split limit' postulated from experimental observations is an intrinsic property of the mixture and that external factors such as heat losses are not necessary to cause this limit.

Grb2 mediates semaphorin-4D-dependent RhoA inactivation.

PubMed

Sun, Tianliang; Krishnan, Rameshkumar; Swiercz, Jakub M

2012-08-01

Signaling through the semaphorin 4D (Sema4D) receptor plexin-B1 is modulated by its interaction with tyrosine kinases ErbB-2 and Met. In cells expressing the plexin-B1-ErbB-2 receptor complex, ligand stimulation results in the activation of small GTPase RhoA and stimulation of cellular migration. By contrast, in cells expressing plexin-B1 and Met, ligand stimulation results in an association with the RhoGTPase-activating protein p190 RhoGAP and subsequent RhoA inactivation--a process that involves the tyrosine phosphorylation of plexin-B1 by Met. Inactivation of RhoA is necessary for Sema4D-mediated inhibition of cellular migration. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGAP interaction and activity. Here we show that the activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation by Met creates a docking site for the SH2 domain of growth factor receptor bound-2 (Grb2). Grb2 is thereby recruited into the plexin-B1 receptor complex and, through its SH3 domain, interacts with p190 RhoGAP and mediates RhoA deactivation. Phosphorylation of plexin-B1 by Met and the recruitment of Grb2 have no effect on the R-RasGAP activity of plexin-B1, but are required for Sema4D-induced, RhoA-dependent antimigratory effects of Sema4D on breast cancer cells. These data show Grb2 as a direct link between plexin and p190-RhoGAP-mediated downstream signaling.

Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian Schmidtea mediterranea.

PubMed

Bansal, Dhiru; Kulkarni, Jahnavi; Nadahalli, Kavana; Lakshmanan, Vairavan; Krishna, Srikar; Sasidharan, Vidyanand; Geo, Jini; Dilipkumar, Shilpa; Pasricha, Renu; Gulyani, Akash; Raghavan, Srikala; Palakodeti, Dasaradhi

2017-09-01

Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown of smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including zfp-1 , are translationally regulated by SMED-PABPC2 . Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration. © 2017. Published by The Company of Biologists Ltd.

Theory and Modeling of Liquid Explosive Detonation

NASA Astrophysics Data System (ADS)

Tarver, Craig M.; Urtiew, Paul A.

2010-10-01

The current understanding of the detonation reaction zones of liquid explosives is discussed in this article. The physical and chemical processes that precede and follow exothermic chemical reaction within the detonation reaction zone are discussed within the framework of the nonequilibrium Zeldovich-von Neumann-Doring (NEZND) theory of self-sustaining detonation. Nonequilibrium chemical and physical processes cause finite time duration induction zones before exothermic chemical energy release occurs. This separation between the leading shock wave front and the chemical energy release needed to sustain it results in shock wave amplification and the subsequent formation of complex three-dimensional cellular structures in all liquid detonation waves. To develop a practical Zeldovich-von Neumann-Doring (ZND) reactive flow model for liquid detonation, experimental data on reaction zone structure, confined failure diameter, unconfined failure diameter, and failure wave velocity in the Dremin-Trofimov test for detonating nitromethane are calculated using the ignition and growth reactive flow model.

Convergence and Extrusion Are Required for Normal Fusion of the Mammalian Secondary Palate

PubMed Central

Kim, Seungil; Lewis, Ace E.; Singh, Vivek; Ma, Xuefei; Adelstein, Robert; Bush, Jeffrey O.

2015-01-01

The fusion of two distinct prominences into one continuous structure is common during development and typically requires integration of two epithelia and subsequent removal of that intervening epithelium. Using confocal live imaging, we directly observed the cellular processes underlying tissue fusion, using the secondary palatal shelves as a model. We find that convergence of a multi-layered epithelium into a single-layer epithelium is an essential early step, driven by cell intercalation, and is concurrent to orthogonal cell displacement and epithelial cell extrusion. Functional studies in mice indicate that this process requires an actomyosin contractility pathway involving Rho kinase (ROCK) and myosin light chain kinase (MLCK), culminating in the activation of non-muscle myosin IIA (NMIIA). Together, these data indicate that actomyosin contractility drives cell intercalation and cell extrusion during palate fusion and suggest a general mechanism for tissue fusion in development. PMID:25848986

Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian Schmidtea mediterranea

PubMed Central

Bansal, Dhiru; Kulkarni, Jahnavi; Nadahalli, Kavana; Lakshmanan, Vairavan; Krishna, Srikar; Sasidharan, Vidyanand; Dilipkumar, Shilpa; Gulyani, Akash; Raghavan, Srikala

2017-01-01

Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown of smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including zfp-1, are translationally regulated by SMED-PABPC2. Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration. PMID:28807897

Mechanisms of bacterial DNA replication restart

PubMed Central

Windgassen, Tricia A; Wessel, Sarah R; Bhattacharyya, Basudeb

2018-01-01

Abstract Multi-protein DNA replication complexes called replisomes perform the essential process of copying cellular genetic information prior to cell division. Under ideal conditions, replisomes dissociate only after the entire genome has been duplicated. However, DNA replication rarely occurs without interruptions that can dislodge replisomes from DNA. Such events produce incompletely replicated chromosomes that, if left unrepaired, prevent the segregation of full genomes to daughter cells. To mitigate this threat, cells have evolved ‘DNA replication restart’ pathways that have been best defined in bacteria. Replication restart requires recognition and remodeling of abandoned replication forks by DNA replication restart proteins followed by reloading of the replicative DNA helicase, which subsequently directs assembly of the remaining replisome subunits. This review summarizes our current understanding of the mechanisms underlying replication restart and the proteins that drive the process in Escherichia coli (PriA, PriB, PriC and DnaT). PMID:29202195

3D patterned substrates for bioartificial blood vessels - The effect of hydrogels on aligned cells on a biomaterial surface.

PubMed

Zhao, Xinxin; Irvine, Scott Alexander; Agrawal, Animesh; Cao, Ye; Lim, Pei Qi; Tan, Si Ying; Venkatraman, Subbu S

2015-10-01

The optimal bio-artificial blood vessel construct is one that has a compliant tubular core with circumferentially aligned smooth muscle cells (SMCs). Obtaining this well-aligned pattern of SMCs on a scaffold is highly beneficial as this cellular orientation preserves the SMC contractile phenotype. We used 3D patterning to create channels on a polycaprolactone (PCL) scaffold; SMCs were then found to be aligned within the microchannels. To preserve this alignment, and to provide a protective coating that could further incorporate cells, we evaluated the use of two hydrogels, one based on poly(ethylene glycol) diacrylate (PEGDA) and the other based on gelatin. Hydrogels were either physically coated on the PCL surfaces or covalently linked via suitable surface modification of PCL. For covalent immobilization of PEGDA hydrogel, alkene groups were introduced on PCL, while for gelatin covalent linkage, serum proteins were introduced. It is, however, crucial that the hydrogel coating does not disrupt the cellular patterning and distribution. We show in this work that both the process of coating as well as the nature of the coating are critical to preservation of the aligned SMCs. The covalent coating methods involving the crosslinking of hydrogels with the surface of PCL films promoted hydrogel retention time on the film as compared with physical deposition. Furthermore, subsequent hydrogel degradation is affected by the components of the cell culture medium, hinting at a possible route to in vivo biodegradation. Surface features control cellular orientation and subsequently influence their functionality, a useful effect for cellularized biomedical devices. Such devices also can benefit from protective and cell friendly hydrogel coatings. However, literature is lacking on the fate of cells that have endured hydrogel coating whilst orientated on a biomaterial surface. In particular, elucidation of the cells ability to remain adherent and orientated post hydrogel addition. Coating requires two procedures that may be deleterious to the orientated cells: the surface pretreatment for gel binding and the hydrogel crosslinking reaction. We compare transglutaminase gelatin crosslinking and UV initiated PEGDA crosslinking, coated onto smooth muscle cells orientated on patterned PCL surfaces. This original study will be of considerable use to the wider biomaterials community. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Controlling human corneal stromal stem cell contraction to mediate rapid cell and matrix organization of real architecture for 3-dimensional tissue equivalents.

PubMed

Mukhey, Dev; Phillips, James B; Daniels, Julie T; Kureshi, Alvena K

2018-02-01

The architecture of the human corneal stroma consists of a highly organized extracellular matrix (ECM) interspersed with keratocytes. Their progenitor cells; corneal stromal stem cells (CSSC) are located at the periphery, in the limbal stroma. A highly organized corneal ECM is critical for effective transmission of light but this structure may be compromised during injury or disease, resulting in loss of vision. Re-creating normal organization in engineered tissue equivalents for transplantation often involves lengthy culture times that are inappropriate for clinical use or utilisation of synthetic substrates that bring complications such as corneal melting. CSSC have great therapeutic potential owing to their ability to reorganize a disorganized matrix, restoring transparency in scarred corneas. We examined CSSC contractile behavior to assess whether this property could be exploited to rapidly generate cell and ECM organization in Real Architecture For 3D Tissues (RAFT) tissue equivalents (TE) for transplantation. Free-floating collagen gels were characterized to assess contractile behavior of CSSC and establish optimum cell density and culture times. To mediate cell and collagen organization, tethered collagen gels seeded with CSSC were cultured and subsequently stabilized with the RAFT process. We demonstrated rapid creation of biomimetic RAFT TE with tunable structural properties. These displayed three distinct regions of varying degrees of cellular and collagen organization. Interestingly, increased organization coincided with a dramatic loss of PAX6 expression in CSSC, indicating rapid differentiation into keratocytes. The organized RAFT TE system could be a useful bioengineering tool to rapidly create an organized ECM while simultaneously controlling cell phenotype. For the first time, we have demonstrated that human CSSC exhibit the phenomenon of cellular self-alignment in tethered collagen gels. We found this mediated rapid co-alignment of collagen fibrils and thus subsequently exploited this property in vitro to improve the architecture of engineered RAFT tissue equivalents of the corneal stroma. Existing techniques are extremely lengthy and carry significant risk and cost for GMP manufacture. This rapid and tunable technique takes just 8 h of culture and is therefore ideal for clinical manufacture, creating biomimetic tissue equivalents with both cellular and ECM organization. Thus, cellular self-alignment can be a useful bioengineering tool for the development of organized tissue equivalents in a variety of applications. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Phagocytosis: studies by optical tweezers and time-resolved microspectrofluorometry

NASA Astrophysics Data System (ADS)

Schneckenburger, Herbert; Sailer, Reinhard; Hendinger, Anita; Gschwend, Michael H.; Bauer, Manfred; Strauss, Wolfgang S. L.

1999-01-01

Cellular uptake of transparent Latex particles by J774A.1 mouse macrophages has been studied: First, single beads were kept within an optical light trap and located in close vicinity to individual cells. Uptake of the beads was visualized, and intrinsic fluorescence was detected in the spectral range of 420 - 530 nm. Second, time-gated fluorescence spectra of single cells were recorded at pre- selected times during one hour after cellular uptake. A rapid increase of autofluorescence and a subsequent decrease to the level of control cells within about 10 min. was measured within a time gate of 0 - 5 ns after the exciting laser pulses, and attributed to the 'free' coenzyme NAD(P)H. In contrast, fluorescence increase of NAD(P)H bound to proteins (measured within time gates of 5 - 10 ns or 10 - 15 ns) was less pronounced, and the subsequent decrease occurred within a longer period (about one hour).

Differential effects of the extracellular microenvironment on human embryonic stem cell differentiation into keratinocytes and their subsequent replicative life span.

PubMed

Movahednia, Mohammad Mehdi; Kidwai, Fahad Karim; Zou, Yu; Tong, Huei Jinn; Liu, Xiaochen; Islam, Intekhab; Toh, Wei Seong; Raghunath, Michael; Cao, Tong

2015-04-01

Culture microenvironment plays a critical role in the propagation and differentiation of human embryonic stem cells (hESCs) and their differentiated progenies. Although high efficiency of hESC differentiation to keratinocytes (hESC-Kert) has been achieved, little is known regarding the effects of early culture microenvironment and pertinent extracellular matrix (ECM) interactions during epidermal commitment on subsequent proliferative capacity of hESC-Kert. The aim of this study is to evaluate the effects of the different ECM microenvironments during hESC differentiation on subsequent replicative life span of hESC-Kert. In doing so, H1-hESCs were differentiated to keratinocytes (H1-Kert) in two differentiation systems. The first system employed autologous fibroblast feeder support, in which keratinocytes (H1-Kert(ACC)) were derived by coculture of hESCs with hESC-derived fibroblasts (H1-ebFs). The second system employed a novel decellularized matrix from H1-ebFs to create a dermoepidermal junction-like (DEJ) matrix. H1-Kert(AFF) were derived by differentiation of hESCs on the feeder-free system employing the DEJ matrix. Our study indicated that the feeder-free system with the use of DEJ matrix was more efficient in differentiation of hESCs toward epidermal progenitors. However, the feeder-free system was not sufficient to support the subsequent replicative capacity of differentiated keratinocytes. Of note, H1-Kert(AFF) showed limited replicative capacity with reduced telomere length and early cellular senescence. We further showed that the lack of cell-cell interactions during epidermal commitment led to heightened production of TGF-β1 by hESC-Kert during extended culture, which in turn was responsible for resulting in the limited replicative life span with cellular senescence of hESC-Kert derived under the feeder-free culture system. This study highlights for the first time the importance of the culture microenvironment and cell-ECM interactions during differentiation of hESCs on subsequent replicative life span and cellular senescence of the differentiated keratinocytes, with implications for use of these cells for applications in tissue engineering and regenerative medicine.

Mechanisms for Variation of Cellular P Stoichiometry: Diverse Cellular Phosphorus Allocation Strategies Across Microbial Groups from the Sargasso Sea

NASA Astrophysics Data System (ADS)

Popendorf, K.; Duhamel, S.

2016-02-01

Phosphorus is the least abundant of the three major macronutrients that define the canonical Redfield ratio, but its place in the backbone of nucleic acids and as an energy trafficking molecule lays a lower bound of cellular phosphorus content that is essential for all life. In addition to forming DNA, RNA, and adenosine triphosphate (ATP), significant amounts of cellular phosphorus may also be allocated to the production of phospholipids and polyphosphate. These latter two biochemicals in particular may occur in significant but highly variable amounts across different microbial groups, and the variation in cellular allocation to these biochemicals may be a contributing factor in defining the elemental stoichiometry of microbes. We investigated this variation in cellular phosphorus allocation across the most abundant microbial groups in the P-depleted Sargasso Sea: Prochlorococcus, Synechococcus, and heterotrophic bacteria. By coupling radioisotope tracing of phosphate and ATP with cell sorting flow cytometry and subsequent biochemical extractions, we made novel measurements of the P allocation to DNA, phospholipids, and polyphosphate in individual microbial groups from environmental populations. These results provide new insights into the cellular mechanisms of variation in stoichiometry and different microbial strategies for adaptation to low-P environments.

MagR Alone Is Insufficient to Confer Cellular Calcium Responses to Magnetic Stimulation

PubMed Central

Pang, Keliang; You, He; Chen, Yanbo; Chu, Pengcheng; Hu, Meiqin; Shen, Jianying; Guo, Wei; Xie, Can; Lu, Bai

2017-01-01

Magnetic manipulation of cell activity offers advantages over optical manipulation but an ideal tool remains elusive. The MagR protein was found through its interaction with cryptochrome (Cry) and the protein in solution appeared to respond to magnetic stimulation (MS). After we initiated an investigation on the specific role of MagR in cellular response to MS, a subsequent study claimed that MagR expression alone could achieve cellular activation by MS. Here we report that despite systematically testing different ways of measuring intracellular calcium and different MS protocols, it was not possible to detect any cellular or neuronal responses to MS in MagR-expressing HEK cells or primary neurons from the dorsal root ganglion and the hippocampus. By contrast, in neurons co-expressing MagR and channelrhodopin, optical but not MS increased calcium influx in hippocampal neurons. Our results indicate that MagR alone is not sufficient to confer cellular magnetic responses. PMID:28360843

Cellular dynamics of bovine aortic smooth muscle cells measured using MEMS force sensors

NASA Astrophysics Data System (ADS)

Tsukagoshi, Takuya; Nguyen, Thanh-Vinh; Hirayama Shoji, Kayoko; Takahashi, Hidetoshi; Matsumoto, Kiyoshi; Shimoyama, Isao

2018-04-01

Adhesive cells perceive the mechanical properties of the substrates to which they adhere, adjusting their cellular mechanical forces according to their biological characteristics. This mechanical interaction subsequently affects the growth, locomotion, and differentiation of the cell. However, little is known about the detailed mechanism that underlies this interaction between adherent cells and substrates because dynamically measuring mechanical phenomena is difficult. Here, we utilize microelectromechamical systems force sensors that can measure cellular traction forces with high temporal resolution (~2.5 µs) over long periods (~3 h). We found that the cellular dynamics reflected physical phenomena with time scales from milliseconds to hours, which contradicts the idea that cellular motion is slow. A single focal adhesion (FA) generates an average force of 7 nN, which disappears in ms via the action of trypsin-ethylenediaminetetraacetic acid. The force-changing rate obtained from our measurements suggests that the time required for an FA to decompose was nearly proportional to the force acting on the FA.

EPS in Environmental Microbial Biofilms as Examined by Advanced Imaging Techniques

NASA Astrophysics Data System (ADS)

Neu, T. R.; Lawrence, J. R.

2006-12-01

Biofilm communities are highly structured associations of cellular and polymeric components which are involved in biogenic and geogenic environmental processes. Furthermore, biofilms are also important in medical (infection), industrial (biofouling) and technological (biofilm engineering) processes. The interfacial microbial communities in a specific habitat are highly dynamic and change according to the environmental parameters affecting not only the cellular but also the polymeric constituents of the system. Through their EPS biofilms interact with dissolved, colloidal and particulate compounds from the bulk water phase. For a long time the focus in biofilm research was on the cellular constituents in biofilms and the polymer matrix in biofilms has been rather neglected. The polymer matrix is produced not only by different bacteria and archaea but also by eukaryotic micro-organisms such as algae and fungi. The mostly unidentified mixture of EPS compounds is responsible for many biofilm properties and is involved in biofilm functionality. The chemistry of the EPS matrix represents a mixture of polymers including polysaccharides, proteins, nucleic acids, neutral polymers, charged polymers, amphiphilic polymers and refractory microbial polymers. The analysis of the EPS may be done destructively by means of extraction and subsequent chemical analysis or in situ by means of specific probes in combination with advanced imaging. In the last 15 years laser scanning microscopy (LSM) has been established as an indispensable technique for studying microbial communities. LSM with 1-photon and 2-photon excitation in combination with fluorescence techniques allows 3-dimensional investigation of fully hydrated, living biofilm systems. This approach is able to reveal data on biofilm structural features as well as biofilm processes and interactions. The fluorescent probes available allow the quantitative assessment of cellular as well as polymer distribution. For this purpose lectin-binding- analysis has been suggested as a suitable approach to image glycoconjugates within the polymer matrix of biofilm communities. More recently synchrotron radiation is increasingly recognized as a powerful tool for studying biological samples. Hard X-ray excitation can be used to map elemental composition whereas IR imaging allows examination of biological macromolecules. A further technique called soft X-ray scanning transmission microscopy (STXM) has the advantage of both techniques and may be employed to detect elements as well as biomolecules. Using the appropriate spectra, near edge X-ray absorption fine structure (NEXAFS) microscopy allows quantitative chemical mapping at 50 nm resolution. In this presentation the applicability of LSM and STXM will be demonstrated using several examples of different environmental biofilm systems. The techniques in combination provide a new view of complex microbial communities and their interaction with the environment. These advanced imaging techniques offer the possibility to study the spatial structure of cellular and polymeric compounds in biofilms as well as biofilm microhabitats, biofilm functionality and biofilm processes.

Cellular Immune Response to Cytomegalovirus Infection After Renal Transplantation

PubMed Central

Linnemann, Calvin C.; Kauffman, Carol A.; First, M. Roy; Schiff, Gilbert M.; Phair, John P.

1978-01-01

A prospective study of 15 patients who received renal transplants defined the effect of renal transplantation on the cellular immune response to cytomegalovirus infection. Of 15 patients, 14 developed cytomegalovirus infection, usually in the first 2 months after transplantation, and all infections were accompanied by a normal humoral immune response. After the initiation of immunosuppressive therapy and transplantation, there was a general depression of lymphocyte transformation, as reflected in the response to phytohemagglutinin, accompanied by a specific defect in cellular immunity, as indicated by lymphocyte transformation to cytomegalovirus antigen. Eleven patients had cellular immunity to cytomegalovirus before transplantation, and all of these became negative in the first month after transplantation. In subsequent months, only 6 of the 14 study patients with cytomegalovirus infection developed specific cellular immune responses to cytomegalovirus. This occurred most often in patients who had severe febrile illnesses in association with infection. The specific cellular immune response which developed in the posttransplant period did not persist in three of the patients. This study demonstrates the dissociation of the humoral and cellular immune response to cytomegalovirus infection in renal transplant patients and indicates the importance of the loss of cellular immunity in the appearance of infection. Previously infected patients lost their cell-mediated immunity and had reactivation infections despite the presence of serum antibody. PMID:215541

Hybrid biosynthetic gene therapy vector development and dual engineering capacity.

PubMed

Jones, Charles H; Ravikrishnan, Anitha; Chen, Mingfu; Reddinger, Ryan; Kamal Ahmadi, Mahmoud; Rane, Snehal; Hakansson, Anders P; Pfeifer, Blaine A

2014-08-26

Genetic vaccines offer a treatment opportunity based upon successful gene delivery to specific immune cell modulators. Driving the process is the vector chosen for gene cargo packaging and subsequent delivery to antigen-presenting cells (APCs) capable of triggering an immune cascade. As such, the delivery process must successfully navigate a series of requirements and obstacles associated with the chosen vector and target cell. In this work, we present the development and assessment of a hybrid gene delivery vector containing biological and biomaterial components. Each component was chosen to design and engineer gene delivery separately in a complimentary and fundamentally distinct fashion. A bacterial (Escherichia coli) inner core and a biomaterial [poly(beta-amino ester)]-coated outer surface allowed the simultaneous application of molecular biology and polymer chemistry to address barriers associated with APC gene delivery, which include cellular uptake and internalization, phagosomal escape, and intracellular cargo concentration. The approach combined and synergized normally disparate vector properties and tools, resulting in increased in vitro gene delivery beyond individual vector components or commercially available transfection agents. Furthermore, the hybrid device demonstrated a strong, efficient, and safe in vivo humoral immune response compared with traditional forms of antigen delivery. In summary, the flexibility, diversity, and potential of the hybrid design were developed and featured in this work as a platform for multivariate engineering at the vector and cellular scales for new applications in gene delivery immunotherapy.

Lipolysis - a highly regulated multi-enzyme complex mediates the catabolism of cellular fat stores.

PubMed

Lass, Achim; Zimmermann, Robert; Oberer, Monika; Zechner, Rudolf

2011-01-01

Lipolysis is the biochemical pathway responsible for the catabolism of triacylglycerol (TAG) stored in cellular lipid droplets. The hydrolytic cleavage of TAG generates non-esterified fatty acids, which are subsequently used as energy substrates, essential precursors for lipid and membrane synthesis, or mediators in cell signaling processes. Consistent with its central importance in lipid and energy homeostasis, lipolysis occurs in essentially all tissues and cell types, it is most abundant, however, in white and brown adipose tissue. Over the last 5years, important enzymes and regulatory protein factors involved in lipolysis have been identified. These include an essential TAG hydrolase named adipose triglyceride lipase (ATGL) [annotated as patatin-like phospholipase domain-containing protein A2], the ATGL activator comparative gene identification-58 [annotated as α/β hydrolase containing protein 5], and the ATGL inhibitor G0/G1 switch gene 2. Together with the established hormone-sensitive lipase [annotated as lipase E] and monoglyceride lipase, these proteins constitute the basic "lipolytic machinery". Additionally, a large number of hormonal signaling pathways and lipid droplet-associated protein factors regulate substrate access and the activity of the "lipolysome". This review summarizes the current knowledge concerning the enzymes and regulatory processes governing lipolysis of fat stores in adipose and non-adipose tissues. Special emphasis will be given to ATGL, its regulation, and physiological function. Copyright © 2010 Elsevier Ltd. All rights reserved.

Comparison between the early cellular response to electron radiation and the production of tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sinclair, Ian P.

1972-11-01

The aim of the study is to quantitate radiation-induced cell population changes in various epithelial components of rat skin and to correlate them, if possible with the subsequent production of tumors.

``Sheddable'' PEG-lipid to balance the contradiction of PEGylation between long circulation and poor uptake

NASA Astrophysics Data System (ADS)

Zhao, Caiyan; Deng, Hongzhang; Xu, Jing; Li, Shuyi; Zhong, Lin; Shao, Leihou; Wu, Yan; Liang, Xing-Jie

2016-05-01

PEGylated lipids confer longer systemic circulation and tumor accumulation via the enhanced permeability and retention (EPR) effect. However, PEGylation inhibits cellular uptake and subsequent endosomal escape. In order to balance the contradiction between the advantages of long circulation and the disadvantages of poor uptake of PEGylated lipids, we prepared a ``sheddable'' PEG-lipid micelle system based on the conjugation of PEG and phosphatidyl ethanolamine (DSPE) with a pH sensitive benzoic imine bond. In a physiological environment, the PEG-protected micelles were not readily taken up by the reticuloendothelial system (RES) and could be successfully delivered to tumor tissue by the EPR effect. In a tumor acidic microenvironment, the PEG chains detached from the surfaces of the micelles while the degree of linker cleavage could not cause a significant particle size change, which facilitated the carrier binding to tumor cells and improved the cellular uptake. Subsequently, the ``sheddable'' PEG-lipid micelles easily internalized into cells and the increased acidity in the lysosomes further promoted drug release. Thus, this ``sheddable'' PEG-lipid nanocarrier could be a good candidate for effective intracellular drug delivery in cancer chemotherapy.PEGylated lipids confer longer systemic circulation and tumor accumulation via the enhanced permeability and retention (EPR) effect. However, PEGylation inhibits cellular uptake and subsequent endosomal escape. In order to balance the contradiction between the advantages of long circulation and the disadvantages of poor uptake of PEGylated lipids, we prepared a ``sheddable'' PEG-lipid micelle system based on the conjugation of PEG and phosphatidyl ethanolamine (DSPE) with a pH sensitive benzoic imine bond. In a physiological environment, the PEG-protected micelles were not readily taken up by the reticuloendothelial system (RES) and could be successfully delivered to tumor tissue by the EPR effect. In a tumor acidic microenvironment, the PEG chains detached from the surfaces of the micelles while the degree of linker cleavage could not cause a significant particle size change, which facilitated the carrier binding to tumor cells and improved the cellular uptake. Subsequently, the ``sheddable'' PEG-lipid micelles easily internalized into cells and the increased acidity in the lysosomes further promoted drug release. Thus, this ``sheddable'' PEG-lipid nanocarrier could be a good candidate for effective intracellular drug delivery in cancer chemotherapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr02174c

Cross-Linked Micellar Spherical Nucleic Acids from Thermoresponsive Templates

PubMed Central

2017-01-01

A one-pot synthesis of micellar spherical nucleic acid (SNA) nanostructures using Pluronic F127 as a thermoresponsive template is reported. These novel constructs are synthesized in a chemically straightforward process that involves intercalation of the lipid tails of DNA amphiphiles (CpG motifs for TLR-9 stimulation) into the hydrophobic regions of Pluronic F127 micelles, followed by chemical cross-linking and subsequent removal of non-cross-linked structures. The dense nucleic acid shell of the resulting cross-linked micellar SNA enhances their stability in physiological media and facilitates their rapid cellular internalization, making them effective TLR-9 immunomodulatory agents. These constructs underscore the potential of SNAs in regulating immune response and address the relative lack of stability of noncovalent constructs. PMID:28207251

Morphogenesis and gravity in a whole amphibian embryo and in isolated blastomeres of sea urchins.

PubMed

Izumi-Kurotani, Akemi; Kiyomoto, Masato

2003-01-01

Fertilization and subsequent embryogenesis of newts occurred normally under microgravity in two Astronewt flight experiments. By accumulation of the results from the amphibian flight experiments including 'Astronewt', it is considered that gravity has rather small effects on the early development of amphibian eggs. However, some temporary abnormalities, which recover in the course of the further developmental process, have been observed. Some regulations may occur in whole embryos. For a thorough knowledge about the role of gravity in morphogenesis, we need to investigate the gravitational effects on a single cell in a whole embryo. We propose a new experimental system with sea urchin embryos and micromeres for further studies at a cellular level of the effects of gravity on morphogenesis.

Potential of proton-pumping rhodopsins: engineering photosystems into microorganisms.

PubMed

Claassens, Nico J; Volpers, Michael; dos Santos, Vitor A P Martins; van der Oost, John; de Vos, Willem M

2013-11-01

A wide range of proton-pumping rhodopsins (PPRs) have been discovered in recent years. Using a synthetic biology approach, PPR photosystems with different features can be easily introduced in nonphotosynthetic microbial hosts. PPRs can provide hosts with the ability to harvest light and drive the sustainable production of biochemicals or biofuels. PPRs use light energy to generate an outward proton flux, and the resulting proton motive force can subsequently power cellular processes. Recently, the introduction of PPRs in microbial production hosts has successfully led to light-driven biotechnological conversions. In this review, we discuss relevant features of natural PPRs, evaluate reported biotechnological applications of microbial production hosts equipped with PPRs, and provide an outlook on future developments. Copyright © 2013 Elsevier Ltd. All rights reserved.

Modulation of Dendritic Cell Activation and Subsequent Th1 Cell Polarization by Lidocaine

PubMed Central

Chung, Yeonseok

2015-01-01

Dendritic cells play an essential role in bridging innate and adaptive immunity by recognizing cellular stress including pathogen- and damage-associated molecular patterns and by shaping the types of antigen-specific T cell immunity. Although lidocaine is widely used in clinical settings that trigger cellular stress, it remains unclear whether such treatment impacts the activation of innate immune cells and subsequent differentiation of T cells. Here we showed that lidocaine inhibited the production of IL–6, TNFα and IL–12 from dendritic cells in response to toll-like receptor ligands including lipopolysaccharide, poly(I:C) and R837 in a dose-dependent manner. Notably, the differentiation of Th1 cells was significantly suppressed by the addition of lidocaine while the same treatment had little effect on the differentiation of Th17, Th2 and regulatory T cells in vitro. Moreover, lidocaine suppressed the ovalbumin-specific Th1 cell responses in vivo induced by the adoptive transfer of ovalbumin-pulsed dendritic cells. These results demonstrate that lidocaine inhibits the activation of dendritic cells in response to toll-like receptor signals and subsequently suppresses the differentiation of Th1 cell responses. PMID:26445366

A scale‐down mimic for mapping the process performance of centrifugation, depth and sterile filtration

PubMed Central

Joseph, Adrian; Kenty, Brian; Mollet, Michael; Hwang, Kenneth; Rose, Steven; Goldrick, Stephen; Bender, Jean; Farid, Suzanne S.

2016-01-01

ABSTRACT In the production of biopharmaceuticals disk‐stack centrifugation is widely used as a harvest step for the removal of cells and cellular debris. Depth filters followed by sterile filters are often then employed to remove residual solids remaining in the centrate. Process development of centrifugation is usually conducted at pilot‐scale so as to mimic the commercial scale equipment but this method requires large quantities of cell culture and significant levels of effort for successful characterization. A scale‐down approach based upon the use of a shear device and a bench‐top centrifuge has been extended in this work towards a preparative methodology that successfully predicts the performance of the continuous centrifuge and polishing filters. The use of this methodology allows the effects of cell culture conditions and large‐scale centrifugal process parameters on subsequent filtration performance to be assessed at an early stage of process development where material availability is limited. Biotechnol. Bioeng. 2016;113: 1934–1941. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26927621

The impact of ischemia-reperfusion injuries on skin resident murine dendritic cells.

PubMed

Goh, Chi Ching; Evrard, Maximilien; Chong, Shu Zhen; Tan, Yingrou; Tan, Leonard De Li; Teng, Karen Wei Weng; Weninger, Wolfgang; Becker, David Laurence; Tey, Hong Liang; Newell, Evan William; Liu, Bin; Ng, Lai Guan

2018-06-01

Pressure ulcers are a chronic problem for patients or the elderly who require extended periods of bed rest. The formation of ulcers is due to repeated cycles of ischemia-reperfusion (IR), which initiates an inflammatory response. Advanced ulcers disrupt the skin barrier, resulting in further complications. To date, the immunological aspect of skin IR has been understudied, partly due to the complexity of the skin immune cells. Through a combination of mass cytometry, confocal imaging and intravital multiphoton imaging, this study establishes a workflow for multidimensionality single cell analysis of skin myeloid cell responses in the context of IR injury with high spatiotemporal resolution. The data generated has provided us with previously uncharacterized insights into the distinct cellular behavior of resident dendritic cells (DCs) and recruited neutrophils post IR. Of interest, we observed a drop in DDC numbers in the IR region, which was subsequently replenished 48h post IR. More importantly, in these cells, we observe an attenuated response to repeated injuries, which may have implications in the subsequent wound healing process. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cellular therapy in bone-tendon interface regeneration

PubMed Central

Rothrauff, Benjamin B; Tuan, Rocky S

2014-01-01

The intrasynovial bone-tendon interface is a gradual transition from soft tissue to bone, with two intervening zones of uncalcified and calcified fibrocartilage. Following injury, the native anatomy is not restored, resulting in inferior mechanical properties and an increased risk of re-injury. Recent in vivo studies provide evidence of improved healing when surgical repair of the bone-tendon interface is augmented with cells capable of undergoing chondrogenesis. In particular, cellular therapy in bone-tendon healing can promote fibrocartilage formation and associated improvements in mechanical properties. Despite these promising results in animal models, cellular therapy in human patients remains largely unexplored. This review highlights the development and structure-function relationship of normal bone-tendon insertions. The natural healing response to injury is discussed, with subsequent review of recent research on cellular approaches for improved healing. Finally, opportunities for translating in vivo findings into clinical practice are identified. PMID:24326955

Micro-thermocouple probe for measurement of cellular thermal responses.

PubMed

Watanabe, M; Kakuta, N; Mabuchi, K; Yamada, Y

2005-01-01

We have produced micro-thermocouple probes for the measurement of cellular thermal responses. Cells generate heat with their metabolisms and more heat with reactions to a certain physical or chemical stimulation. Therefore, the analysis of the cellular thermal responses would provide new physiological information. However, a real-time thermal measurement technique on a target of a single cell has not been established. In this study, glass micropipettes, which are widely used in bioengineering and medicine, are used for the base of the thermocouple probes. Using microfabrication techniques, the junction of two different metal films is formed at the micropipette tip with a diameter of 1 μm. This probe can inject a chemical substance into a cell and to detect its subsequent temperature changes simultaneously.

Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence

PubMed Central

Sohel, Md. Mahmodul Hasan; Hoelker, Michael; Noferesti, Sina Seifi; Salilew-Wondim, Dessie; Tholen, Ernst; Looft, Christian; Rings, Franca; Uddin, Muhammad Jasim; Spencer, Thomas E.; Schellander, Karl; Tesfaye, Dawit

2013-01-01

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment. PMID:24223816

Fabrication of viable and functional pre-vascularized modular bone tissues by coculturing MSCs and HUVECs on microcarriers in spinner flasks.

PubMed

Zhang, Songjie; Zhou, Min; Ye, Zhaoyang; Zhou, Yan; Tan, Wen-Song

2017-08-01

Slow vascularization often impedes the viability and function of engineered bone replacements. Prevascularization is a promising way to solve this problem. In this study, a new process was developed by integrating microcarrier culture and coculture to fabricate pre-vascularized bone microtissues with mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs). Initially, coculture medium and cell ratio between MSCs and HUVECs were optimized in tissue culture plates concerning cell proliferation, osteogenesis and angiogenesis. Subsequently, cells were seeded onto CultiSpher S microcarriers in spinner flasks and subjected to a two-stage (proliferative-osteogenic) culture process for four weeks. Both cells proliferated and functioned well in chosen medium and a 1 : 1 ratio between MSCs and HUVECs was chosen for better angiogenesis. After four weeks of culture in spinner flasks, the microtissues were formed with high cellularity, evenly distributed cells and tube formation ability. While coculture with HUVECs exerted an inhibitory effect on osteogenic differentiation of MSCs, with downregulated alkaline phosphatase activity, mineralization and gene expression of COLI, RUNX2 and OCN, this could be attenuated by employing a delayed seeding strategy of HUVECs against MSCs during the microtissue fabrication process. Collectively, this work established an effective method to fabricate pre-vascularized bone microtissues, which would lay a solid foundation for subsequent development of vascularized tissue grafts for bone regeneration. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A hybrid finite-element and cellular-automaton framework for modeling 3D microstructure of Ti–6Al–4V alloy during solid–solid phase transformation in additive manufacturing

NASA Astrophysics Data System (ADS)

Chen, Shaohua; Xu, Yaopengxiao; Jiao, Yang

2018-06-01

Additive manufacturing such as selective laser sintering and electron beam melting has become a popular technique which enables one to build near-net-shape product from packed powders. The performance and properties of the manufactured product strongly depends on its material microstructure, which is in turn determined by the processing conditions including beam power density, spot size, scanning speed and path etc. In this paper, we develop a computational framework that integrates the finite element method (FEM) and cellular automaton (CA) simulation to model the 3D microstructure of additively manufactured Ti–6Al–4V alloy, focusing on the β → α + β transition pathway in a consolidated alloy region as the power source moves away from this region. Specifically, the transient temperature field resulted from a scanning laser/electron beam following a zig-zag path is first obtained by solving nonlinear heat transfer equations using the FEM. Next, a CA model for the β → α + β phase transformation in the consolidated alloy is developed which explicitly takes into account the temperature dependent heterogeneous nucleation and anisotropic growth of α grains from the parent β phase field. We verify our model by reproducing the overall transition kinetics predicted by the Johnson–Mehl–Avrami–Kolmogorov theory under a typical processing condition and by quantitatively comparing our simulation results with available experimental data. The utility of the model is further demonstrated by generating large-field realistic 3D alloy microstructures for subsequent structure-sensitive micro-mechanical analysis. In addition, we employ our model to generate a wide spectrum of alloy microstructures corresponding to different processing conditions for establishing quantitative process-structure relations for the system.

Towards the prediction of essential genes by integration of network topology, cellular localization and biological process information

PubMed Central

2009-01-01

Background The identification of essential genes is important for the understanding of the minimal requirements for cellular life and for practical purposes, such as drug design. However, the experimental techniques for essential genes discovery are labor-intensive and time-consuming. Considering these experimental constraints, a computational approach capable of accurately predicting essential genes would be of great value. We therefore present here a machine learning-based computational approach relying on network topological features, cellular localization and biological process information for prediction of essential genes. Results We constructed a decision tree-based meta-classifier and trained it on datasets with individual and grouped attributes-network topological features, cellular compartments and biological processes-to generate various predictors of essential genes. We showed that the predictors with better performances are those generated by datasets with integrated attributes. Using the predictor with all attributes, i.e., network topological features, cellular compartments and biological processes, we obtained the best predictor of essential genes that was then used to classify yeast genes with unknown essentiality status. Finally, we generated decision trees by training the J48 algorithm on datasets with all network topological features, cellular localization and biological process information to discover cellular rules for essentiality. We found that the number of protein physical interactions, the nuclear localization of proteins and the number of regulating transcription factors are the most important factors determining gene essentiality. Conclusion We were able to demonstrate that network topological features, cellular localization and biological process information are reliable predictors of essential genes. Moreover, by constructing decision trees based on these data, we could discover cellular rules governing essentiality. PMID:19758426

Nuclear deterrents: Intrinsic regulators of IL-1β-induced effects on hippocampal neurogenesis.

PubMed

O'Léime, Ciarán S; Cryan, John F; Nolan, Yvonne M

2017-11-01

Hippocampal neurogenesis, the process by which new neurons are born and develop into the host circuitry, begins during embryonic development and persists throughout adulthood. Over the last decade considerable insights have been made into the role of hippocampal neurogenesis in cognitive function and the cellular mechanisms behind this process. Additionally, an increasing amount of evidence exists on the impact of environmental factors, such as stress and neuroinflammation on hippocampal neurogenesis and subsequent impairments in cognition. Elevated expression of the pro-inflammatory cytokine interleukin-1β (IL-1β) in the hippocampus is established as a significant contributor to the neuronal demise evident in many neurological and psychiatric disorders and is now known to negatively regulate hippocampal neurogenesis. In order to prevent the deleterious effects of IL-1β on neurogenesis it is necessary to identify signalling pathways and regulators of neurogenesis within neural progenitor cells that can interact with IL-1β. Nuclear receptors are ligand regulated transcription factors that are involved in modulating a large number of cellular processes including neurogenesis. In this review we focus on the signalling mechanisms of specific nuclear receptors involved in regulating neurogenesis (glucocorticoid receptors, peroxisome proliferator activated receptors, estrogen receptors, and nuclear receptor subfamily 2 group E member 1 (NR2E1 or TLX)). We propose that these nuclear receptors could be targeted to inhibit neuroinflammatory signalling pathways associated with IL-1β. We discuss their potential to be therapeutic targets for neuroinflammatory disorders affecting hippocampal neurogenesis and associated cognitive function. Copyright © 2017 Elsevier Inc. All rights reserved.

Tube formation by complex cellular processes in Ciona intestinalis notochord.

PubMed

Dong, Bo; Horie, Takeo; Denker, Elsa; Kusakabe, Takehiro; Tsuda, Motoyuki; Smith, William C; Jiang, Di

2009-06-15

In the course of embryogenesis multicellular structures and organs are assembled from constituent cells. One structural component common to many organs is the tube, which consists most simply of a luminal space surrounded by a single layer of epithelial cells. The notochord of ascidian Ciona forms a tube consisting of only 40 cells, and serves as a hydrostatic "skeleton" essential for swimming. While the early processes of convergent extension in ascidian notochord development have been extensively studied, the later phases of development, which include lumen formation, have not been well characterized. Here we used molecular markers and confocal imaging to describe tubulogenesis in the developing Ciona notochord. We found that during tubulogenesis each notochord cell established de novo apical domains, and underwent a mesenchymal-epithelial transition to become an unusual epithelial cell with two opposing apical domains. Concomitantly, extracellular luminal matrix was produced and deposited between notochord cells. Subsequently, each notochord cell simultaneously executed two types of crawling movements bi-directionally along the anterior/posterior axis on the inner surface of notochordal sheath. Lamellipodia-like protrusions resulted in cell lengthening along the anterior/posterior axis, while the retraction of trailing edges of the same cell led to the merging of the two apical domains. As a result, the notochord cells acquired endothelial-like shape and formed the wall of the central lumen. Inhibition of actin polymerization prevented the cell movement and tube formation. Ciona notochord tube formation utilized an assortment of common and fundamental cellular processes including cell shape change, apical membrane biogenesis, cell/cell adhesion remodeling, dynamic cell crawling, and lumen matrix secretion.

A branching process model for the analysis of abortive colony size distributions in carbon ion-irradiated normal human fibroblasts.

PubMed

Sakashita, Tetsuya; Hamada, Nobuyuki; Kawaguchi, Isao; Hara, Takamitsu; Kobayashi, Yasuhiko; Saito, Kimiaki

2014-05-01

A single cell can form a colony, and ionizing irradiation has long been known to reduce such a cellular clonogenic potential. Analysis of abortive colonies unable to continue to grow should provide important information on the reproductive cell death (RCD) following irradiation. Our previous analysis with a branching process model showed that the RCD in normal human fibroblasts can persist over 16 generations following irradiation with low linear energy transfer (LET) γ-rays. Here we further set out to evaluate the RCD persistency in abortive colonies arising from normal human fibroblasts exposed to high-LET carbon ions (18.3 MeV/u, 108 keV/µm). We found that the abortive colony size distribution determined by biological experiments follows a linear relationship on the log-log plot, and that the Monte Carlo simulation using the RCD probability estimated from such a linear relationship well simulates the experimentally determined surviving fraction and the relative biological effectiveness (RBE). We identified the short-term phase and long-term phase for the persistent RCD following carbon-ion irradiation, which were similar to those previously identified following γ-irradiation. Taken together, our results suggest that subsequent secondary or tertiary colony formation would be invaluable for understanding the long-lasting RCD. All together, our framework for analysis with a branching process model and a colony formation assay is applicable to determination of cellular responses to low- and high-LET radiation, and suggests that the long-lasting RCD is a pivotal determinant of the surviving fraction and the RBE.

Transcriptomic profiling of adaptive responses to ocean acidification.

PubMed

Goncalves, Priscila; Jones, David B; Thompson, Emma L; Parker, Laura M; Ross, Pauline M; Raftos, David A

2017-11-01

Some populations of marine organisms appear to have inherent tolerance or the capacity for acclimation to stressful environmental conditions, including those associated with climate change. Sydney rock oysters from the B2 breeding line exhibit resilience to ocean acidification (OA) at the physiological level. To understand the molecular basis of this physiological resilience, we analysed the gill transcriptome of B2 oysters that had been exposed to near-future projected ocean pH over two consecutive generations. Our results suggest that the distinctive performance of B2 oysters in the face of OA is mediated by the selective expression of genes involved in multiple cellular processes. Subsequent high-throughput qPCR revealed that some of these transcriptional changes are exclusive to B2 oysters and so may be associated with their resilience to OA. The intracellular processes mediated by the differentially abundant genes primarily involve control of the cell cycle and maintenance of cellular homeostasis. These changes may enable B2 oysters to prevent apoptosis resulting from oxidative damage or to alleviate the effects of apoptosis through regulation of the cell cycle. Comparative analysis of the OA conditioning effects across sequential generations supported the contention that B2 and wild-type oysters have different trajectories of changing gene expression and responding to OA. Our findings reveal the broad set of molecular processes underlying transgenerational conditioning and potential resilience to OA in a marine calcifier. Identifying the mechanisms of stress resilience can uncover the intracellular basis for these organisms to survive and thrive in a rapidly changing ocean. © 2017 John Wiley & Sons Ltd.

From morphology to clinical pathophysiology: multiphoton fluorescence lifetime imaging at patients' bedside

NASA Astrophysics Data System (ADS)

Mess, Christian; Zens, Katharina; Gorzelanny, Christian; Metze, Dieter; Luger, Thomas A.; König, Karsten; Schneider, Stefan W.; Huck, Volker

2017-02-01

Application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of skin diseases. By means of multiphoton excitation, endogenous biomolecules like NADH, collagen or elastin show autofluorescence or second harmonic generation. Thus, these molecules provide information about the subcellular morphology, epidermal architecture and physiological condition of the skin. To gain a deeper understanding of the linkage between cellular structure and physiological processes, non-invasive multiphotonbased intravital tomography (MPT) and fluorescence lifetime imaging (FLIM) were combined within the scopes of inflammatory skin, chronic wounds and drug delivery in clinical application. The optical biopsies generated via MPT were morphologically analyzed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Independent morphometric algorithms reliably showed a perinuclear accumulation in lesional skin in contrast to an even distribution in healthy skin. Confirmatively, MPT-FLIM showed an obvious metabolic shift in lesions. Moreover, detection of the onset and progression of inflammatory processes could be achieved. The feasibility of primary in vivo tracking of applied therapeutic agents further broadened our scope: We examined the permeation and subsequent distribution of agents directly visualized in patientś skin in short-term repetitive measurements. Furthermore, we performed MPT-FLIM follow-up investigations in the long-term course of therapy. Therefore, clinical MPT-FLIM application offers new insights into the pathophysiology and the individual therapeutic course of skin diseases, facilitating a better understanding of the processes of inflammation and wound healing.

Upregulation of cytosolic NADP+-dependent isocitrate dehydrogenase by hyperglycemia protects renal cells against oxidative stress.

PubMed

Lee, Soh-Hyun; Ha, Sun-Ok; Koh, Ho-Jin; Kim, KilSoo; Jeon, Seon-Min; Choi, Myung-Sook; Kwon, Oh-Shin; Huh, Tae-Lin

2010-02-28

Hyperglycemia-induced oxidative stress is widely recognized as a key mediator in the pathogenesis of diabetic nephropathy, a complication of diabetes. We found that both expression and enzymatic activity of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) were upregulated in the renal cortexes of diabetic rats and mice. Similarly, IDPc was induced in murine renal proximal tubular OK cells by high hyperglycemia, while it was abrogated by co-treatment with the antioxidant N-Acetyl-Cysteine (NAC). In OK cells, increased expression of IDPc by stable transfection prevented hyperglycemia-mediated reactive oxygen species (ROS) production, subsequent cellular oxidative stress and extracellular matrix accumulation, whereas these processes were all stimulated by decreased IDPc expression. In addition, production of NADPH and GSH in the cytosol was positively correlated with the expression level of IDPc in OK cells. These results together indicate that upregulation of IDPc in response to hyperglycemia might play an essential role in preventing the progression of diabetic nephropathy, which is accompanied by ROS-induced cellular damage and fibrosis, by providing NADPH, the reducing equivalent needed for recycling reduced glutathione and low molecular weight antioxidant thiol proteins.

Photoactivatable protein labeling by singlet oxygen mediated reactions.

PubMed

To, Tsz-Leung; Medzihradszky, Katalin F; Burlingame, Alma L; DeGrado, William F; Jo, Hyunil; Shu, Xiaokun

2016-07-15

Protein-protein interactions regulate many biological processes. Identification of interacting proteins is thus an important step toward molecular understanding of cell signaling. The aim of this study was to investigate the use of photo-generated singlet oxygen and a small molecule for proximity labeling of interacting proteins in cellular environment. The protein of interest (POI) was fused with a small singlet oxygen photosensitizer (miniSOG), which generates singlet oxygen ((1)O2) upon irradiation. The locally generated singlet oxygen then activated a biotin-conjugated thiol molecule to form a covalent bond with the proteins nearby. The labeled proteins can then be separated and subsequently identified by mass spectrometry. To demonstrate the applicability of this labeling technology, we fused the miniSOG to Skp2, an F-box protein of the SCF ubiquitin ligase, and expressed the fusion protein in mammalian cells and identified that the surface cysteine of its interacting partner Skp1 was labeled by the biotin-thiol molecule. This photoactivatable protein labeling method may find important applications including identification of weak and transient protein-protein interactions in the native cellular context, as well as spatial and temporal control of protein labeling. Copyright © 2016 Elsevier Ltd. All rights reserved.

The GARP Complex Is Involved in Intracellular Cholesterol Transport via Targeting NPC2 to Lysosomes.

PubMed

Wei, Jian; Zhang, Ying-Yu; Luo, Jie; Wang, Ju-Qiong; Zhou, Yu-Xia; Miao, Hong-Hua; Shi, Xiong-Jie; Qu, Yu-Xiu; Xu, Jie; Li, Bo-Liang; Song, Bao-Liang

2017-06-27

Proper intracellular cholesterol trafficking is critical for cellular function. Two lysosome-resident proteins, NPC1 and NPC2, mediate the egress of low-density lipoprotein-derived cholesterol from lysosomes. However, other proteins involved in this process remain largely unknown. Through amphotericin B-based selection, we isolated two cholesterol transport-defective cell lines. Subsequent whole-transcriptome-sequencing analysis revealed two cell lines bearing the same mutation in the vacuolar protein sorting 53 (Vps53) gene. Depletion of VPS53 or other subunits of the Golgi-associated retrograde protein (GARP) complex impaired NPC2 sorting to lysosomes and caused cholesterol accumulation. GARP deficiency blocked the retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR) to the trans-Golgi network. Further, Vps54 mutant mice displayed reduced cellular NPC2 protein levels and increased cholesterol accumulation, underscoring the physiological role of the GARP complex in cholesterol transport. We conclude that the GARP complex contributes to intracellular cholesterol transport by targeting NPC2 to lysosomes in a CI-MPR-dependent manner. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Strolling Toward New Concepts.

PubMed

Ito, Koreaki

2016-09-08

For more than four decades now, I have been studying how genetic information is transformed into protein-based cellular functions. This has included investigations into the mechanisms supporting cellular localization of proteins, disulfide bond formation, quality control of membranes, and translation. I tried to extract new principles and concepts that are universal among living organisms from our observations of Escherichia coli. While I wanted to distill complex phenomena into basic principles, I also tried not to overlook any serendipitous observations. In the first part of this article, I describe personal experiences during my studies of the Sec pathway, which have centered on the SecY translocon. In the second part, I summarize my views of the recent revival of translation studies, which has given rise to the concept that nonuniform polypeptide chain elongation is relevant for the subsequent fates of newly synthesized proteins. Our studies of a class of regulatory nascent polypeptides advance this concept by showing that the dynamic behaviors of the extraribosomal part of the nascent chain affect the ongoing translation process. Vibrant and regulated molecular interactions involving the ribosome, mRNA, and nascent polypeptidyl-tRNA are based, at least partly, on their autonomously interacting properties.

Maintenance of cellular ATP level by caloric restriction correlates chronological survival of budding yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Joon-Seok; Lee, Cheol-Koo, E-mail: cklee2005@korea.ac.kr

Highlights: •CR decreases total ROS and mitochondrial superoxide during the chronological aging. •CR does not affect the levels of oxidative damage on protein and DNA. •CR contributes extension of chronological lifespan by maintenance of ATP level -- Abstract: The free radical theory of aging emphasizes cumulative oxidative damage in the genome and intracellular proteins due to reactive oxygen species (ROS), which is a major cause for aging. Caloric restriction (CR) has been known as a representative treatment that prevents aging; however, its mechanism of action remains elusive. Here, we show that CR extends the chronological lifespan (CLS) of budding yeastmore » by maintaining cellular energy levels. CR reduced the generation of total ROS and mitochondrial superoxide; however, CR did not reduce the oxidative damage in proteins and DNA. Subsequently, calorie-restricted yeast had higher mitochondrial membrane potential (MMP), and it sustained consistent ATP levels during the process of chronological aging. Our results suggest that CR extends the survival of the chronologically aged cells by improving the efficiency of energy metabolism for the maintenance of the ATP level rather than reducing the global oxidative damage of proteins and DNA.« less

Highly Dynamic Cellular-Level Response of Symbiotic Coral to a Sudden Increase in Environmental Nitrogen

PubMed Central

Kopp, C.; Pernice, M.; Domart-Coulon, I.; Djediat, C.; Spangenberg, J. E.; Alexander, D. T. L.; Hignette, M.; Meziane, T.; Meibom, A.

2013-01-01

ABSTRACT Metabolic interactions with endosymbiotic photosynthetic dinoflagellate Symbiodinium spp. are fundamental to reef-building corals (Scleractinia) thriving in nutrient-poor tropical seas. Yet, detailed understanding at the single-cell level of nutrient assimilation, translocation, and utilization within this fundamental symbiosis is lacking. Using pulse-chase 15N labeling and quantitative ion microprobe isotopic imaging (NanoSIMS; nanoscale secondary-ion mass spectrometry), we visualized these dynamic processes in tissues of the symbiotic coral Pocillopora damicornis at the subcellular level. Assimilation of ammonium, nitrate, and aspartic acid resulted in rapid incorporation of nitrogen into uric acid crystals (after ~45 min), forming temporary N storage sites within the dinoflagellate endosymbionts. Subsequent intracellular remobilization of this metabolite was accompanied by translocation of nitrogenous compounds to the coral host, starting at ~6 h. Within the coral tissue, nitrogen is utilized in specific cellular compartments in all four epithelia, including mucus chambers, Golgi bodies, and vesicles in calicoblastic cells. Our study shows how nitrogen-limited symbiotic corals take advantage of sudden changes in nitrogen availability; this opens new perspectives for functional studies of nutrient storage and remobilization in microbial symbioses in changing reef environments. PMID:23674611

Cell death and renewal during prey capture and digestion in the carnivorous sponge Asbestopluma hypogea (Porifera: Poecilosclerida).

PubMed

Martinand-Mari, Camille; Vacelet, Jean; Nickel, Michael; Wörheide, Gert; Mangeat, Paul; Baghdiguian, Stephen

2012-11-15

The sponge Asbestopluma hypogea is unusual among sponges due to its peculiar carnivorous feeding habit. During various stages of its nutrition cycle, the sponge is subjected to spectacular morphological modifications. Starved animals are characterized by many elongated filaments, which are crucial for the capture of prey. After capture, and during the digestion process, these filaments actively regress before being regenerated during a subsequent period of starvation. Here, we demonstrate that these morphological events rely on a highly dynamic cellular turnover, implying a coordinated sequence of programmed cell death (apoptosis and autophagy), cell proliferation and cell migration. A candidate niche for cell renewal by stem cell proliferation and differentiation was identified at the base of the sponge peduncle, characterized by higher levels of BrdU/EdU incorporation. Therefore, BrdU/EdU-positive cells of the peduncle base are candidate motile cells responsible for the regeneration of the prey-capturing main sponge body, i.e. the dynamic filaments. Altogether, our results demonstrate that dynamics of cell renewal in sponge appear to be regulated by cellular mechanisms as multiple and complex as those already identified in bilaterian metazoans.

Preparation of BFV Gag antiserum and preliminary study on cellular distribution of BFV.

PubMed

Wang, Jian; Guo, Hong-yan; Jia, Rui; Xu, Xuan; Tan, Juan; Geng, Yun-qi; Qiao, Wen-tao

2010-04-01

Viruses (e.g. Human immunodeficiency virus, Human simplex virus and Prototype foamy virus) are obligate intracellular parasites and therefore depend on the cellular machinery for cellular trafficking. Bovine foamy virus (BFV) is a member of the Spumaretrovirinae subfamily of Retroviruses, however, details of its cellular trafficking remain unknown. In this study, we cloned the BFV gag gene into prokaryotic expression vector pET28a and purified the denaturalized Gag protein. The protein was used to immunize BALB/c mouse to produce antiserum, which could specifically recognize the BFV Gag protein in BFV-infected cells through western blot assay. Additionally, these results demonstrated that both the optimal and suboptimal cleavage of Gag protein occur in BFV-infected cells. Subsequently, the Gag antiserum was used to investigate subcellular localization of BFV. In immunofluorescence microscopy assays, colocalization microtubules (MTs) and assembling viral particles were clearly observed, which implied that BFV may transport along cellular MTs in host cells. Furthermore, MTs-depolymerizing assay indicated MTs were required for the efficient replication of BFV. In conclusion, our study suggests that BFV has evolved the mechanism to hijack the cellular cytoskeleton for its replication.

Rapid directed evolution of stabilized proteins with cellular high-throughput encapsulation solubilization and screening (CHESS).

PubMed

Yong, K J; Scott, D J

2015-03-01

Directed evolution is a powerful method for engineering proteins towards user-defined goals and has been used to generate novel proteins for industrial processes, biological research and drug discovery. Typical directed evolution techniques include cellular display, phage display, ribosome display and water-in-oil compartmentalization, all of which physically link individual members of diverse gene libraries to their translated proteins. This allows the screening or selection for a desired protein function and subsequent isolation of the encoding gene from diverse populations. For biotechnological and industrial applications there is a need to engineer proteins that are functional under conditions that are not compatible with these techniques, such as high temperatures and harsh detergents. Cellular High-throughput Encapsulation Solubilization and Screening (CHESS), is a directed evolution method originally developed to engineer detergent-stable G proteins-coupled receptors (GPCRs) for structural biology. With CHESS, library-transformed bacterial cells are encapsulated in detergent-resistant polymers to form capsules, which serve to contain mutant genes and their encoded proteins upon detergent mediated solubilization of cell membranes. Populations of capsules can be screened like single cells to enable rapid isolation of genes encoding detergent-stable protein mutants. To demonstrate the general applicability of CHESS to other proteins, we have characterized the stability and permeability of CHESS microcapsules and employed CHESS to generate thermostable, sodium dodecyl sulfate (SDS) resistant green fluorescent protein (GFP) mutants, the first soluble proteins to be engineered using CHESS. © 2014 Wiley Periodicals, Inc.

Transcription Regulation of HYPK by Heat Shock Factor 1

PubMed Central

Das, Srijit; Bhattacharyya, Nitai Pada

2014-01-01

HYPK (Huntingtin Yeast Partner K) was originally identified by yeast two-hybrid assay as an interactor of Huntingtin, the protein mutated in Huntington's disease. HYPK was characterized earlier as an intrinsically unstructured protein having chaperone-like activity in vitro and in vivo. HYPK has the ability of reducing rate of aggregate formation and subsequent toxicity caused by mutant Huntingtin. Further investigation revealed that HYPK is involved in diverse cellular processes and required for normal functioning of cells. In this study we observed that hyperthermia increases HYPK expression in human and mouse cells in culture. Expression of exogenous Heat Shock Factor 1 (HSF1), upon heat treatment could induce HYPK expression, whereas HSF1 knockdown reduced endogenous as well as heat-induced HYPK expression. Putative HSF1-binding site present in the promoter of human HYPK gene was identified and validated by reporter assay. Chromatin immunoprecipitation revealed in vivo interaction of HSF1 and RNA polymerase II with HYPK promoter sequence. Additionally, acetylation of histone H4, a known epigenetic marker of inducible HSF1 binding, was observed in response to heat shock in HYPK gene promoter. Overexpression of HYPK inhibited cells from lethal heat-induced death whereas knockdown of HYPK made the cells susceptible to lethal heat shock-induced death. Apart from elevated temperature, HYPK was also upregulated by hypoxia and proteasome inhibition, two other forms of cellular stress. We concluded that chaperone-like protein HYPK is induced by cellular stress and under transcriptional regulation of HSF1. PMID:24465598

O-GlcNAc and the Cardiovascular System

PubMed Central

Dassanayaka, Sujith; Jones, Steven P.

2014-01-01

The cardiovascular system is capable of robust changes in response to physiologic and pathologic stimuli through intricate signaling mechanisms. The area of metabolism has witnessed a veritable renaissance in the cardiovascular system. In particular, the post-translational β-O-linkage of N-acetylglucosamine (O-GlcNAc) to cellular proteins represents one such signaling pathway that has been implicated in the pathophysiology of cardiovascular disease. This highly dynamic protein modification may induce functional changes in proteins and regulate key cellular processes including translation, transcription, and cell death. In addition, its potential interplay with phosphorylation provides an additional layer of complexity to post-translational regulation. The hexosamine biosynthetic pathway generally requires glucose to form the nucleotide sugar, UDP-GlcNAc. Accordingly, O-GlcNAcylation may be altered in response to nutrient availability and cellular stress. Recent literature supports O-GlcNAcylation as an autoprotective response in models of acute stress (hypoxia, ischemia, oxidative stress). Models of sustained stress, such as pressure overload hypertrophy, and infarct-induced heart failure, may also require protein O-GlcNAcylation as a partial compensatory mechanism. Yet, in models of Type II diabetes, O-GlcNAcylation has been implicated in the subsequent development of vascular, and even cardiac, dysfunction. This review will address this apparent paradox and discuss the potential mechanisms of O-GlcNAc-mediated cardioprotection and cardiovascular dysfunction. This discussion will also address potential targets for pharmacologic interventions and the unique considerations related to such targets. PMID:24287310

O-GlcNAc and the cardiovascular system.

PubMed

Dassanayaka, Sujith; Jones, Steven P

2014-04-01

The cardiovascular system is capable of robust changes in response to physiologic and pathologic stimuli through intricate signaling mechanisms. The area of metabolism has witnessed a veritable renaissance in the cardiovascular system. In particular, the post-translational β-O-linkage of N-acetylglucosamine (O-GlcNAc) to cellular proteins represents one such signaling pathway that has been implicated in the pathophysiology of cardiovascular disease. This highly dynamic protein modification may induce functional changes in proteins and regulate key cellular processes including translation, transcription, and cell death. In addition, its potential interplay with phosphorylation provides an additional layer of complexity to post-translational regulation. The hexosamine biosynthetic pathway generally requires glucose to form the nucleotide sugar, UDP-GlcNAc. Accordingly, O-GlcNAcylation may be altered in response to nutrient availability and cellular stress. Recent literature supports O-GlcNAcylation as an autoprotective response in models of acute stress (hypoxia, ischemia, oxidative stress). Models of sustained stress, such as pressure overload hypertrophy, and infarct-induced heart failure, may also require protein O-GlcNAcylation as a partial compensatory mechanism. Yet, in models of Type II diabetes, O-GlcNAcylation has been implicated in the subsequent development of vascular, and even cardiac, dysfunction. This review will address this apparent paradox and discuss the potential mechanisms of O-GlcNAc-mediated cardioprotection and cardiovascular dysfunction. This discussion will also address potential targets for pharmacologic interventions and the unique considerations related to such targets. Copyright © 2013 Elsevier Inc. All rights reserved.

Endocytosis and Signaling: Cell Logistics Shape the Eukaryotic Cell Plan

PubMed Central

Sigismund, Sara; Confalonieri, Stefano; Ciliberto, Andrea; Polo, Simona; Scita, Giorgio; Di Fiore, Pier Paolo

2017-01-01

Our understanding of endocytosis has evolved remarkably in little more than a decade. This is the result not only of advances in our knowledge of its molecular and biological workings, but also of a true paradigm shift in our understanding of what really constitutes endocytosis and of its role in homeostasis. Although endocytosis was initially discovered and studied as a relatively simple process to transport molecules across the plasma membrane, it was subsequently found to be inextricably linked with almost all aspects of cellular signaling. This led to the notion that endocytosis is actually the master organizer of cellular signaling, providing the cell with understandable messages that have been resolved in space and time. In essence, endocytosis provides the communications and supply routes (the logistics) of the cell. Although this may seem revolutionary, it is still likely to be only a small part of the entire story. A wealth of new evidence is uncovering the surprisingly pervasive nature of endocytosis in essentially all aspects of cellular regulation. In addition, many newly discovered functions of endocytic proteins are not immediately interpretable within the classical view of endocytosis. A possible framework, to rationalize all this new knowledge, requires us to “upgrade” our vision of endocytosis. By combining the analysis of biochemical, biological, and evolutionary evidence, we propose herein that endocytosis constitutes one of the major enabling conditions that in the history of life permitted the development of a higher level of organization, leading to the actuation of the eukaryotic cell plan. PMID:22298658

Laser light propagation in adipose tissue and laser effects on adipose cell membranes

NASA Astrophysics Data System (ADS)

Solarte, Efraín; Rebolledo, Aldo; Gutierrez, Oscar; Criollo, William; Neira, Rodrigo; Arroyave, José; Ramírez, Hugo

2006-01-01

Recently Neira et al. have presented a new liposuction technique that demonstrated the movement of fat from inside to outside of the cell, using a low-level laser device during a liposuction procedure with Ultrawet solution. The clinical observations, allowed this new surgical development, started a set of physical, histological and pharmacological studies aimed to determine the mechanisms involved in the observed fat mobilization concomitant to external laser application in liposuction procedures. Scanning and Transmission Electron Microscopy, studies show that the cellular arrangement of normal adipose tissue changes when laser light from a diode laser: 10 mW, 635 nm is applied. Laser exposures longer than 6 minutes cause the total destruction of the adipocyte panicles. Detailed observation of the adipose cells show that by short irradiation times (less than four minutes) the cell membrane exhibits dark zones, that collapse by longer laser exposures. Optical measurements show that effective penetration length depends on the laser intensity. Moreover, the light scattering is enhanced by diffraction and subsequent interference effects, and the tumescent solution produces a clearing of the tissue optical medium. Finally, isolate adipose cell observation show that fat release from adipocytes is a concomitant effect between the tumescent solution (adrenaline) and laser light, revealing a synergism which conduces to the aperture, and maybe the disruption, of the cell membrane. All these studies were consistent with a laser induced cellular process, which causes fat release from inside the adipocytes into the intercellular space, besides a strong modification of the cellular membranes.

Endocytosis and signaling: cell logistics shape the eukaryotic cell plan.

PubMed

Sigismund, Sara; Confalonieri, Stefano; Ciliberto, Andrea; Polo, Simona; Scita, Giorgio; Di Fiore, Pier Paolo

2012-01-01

Our understanding of endocytosis has evolved remarkably in little more than a decade. This is the result not only of advances in our knowledge of its molecular and biological workings, but also of a true paradigm shift in our understanding of what really constitutes endocytosis and of its role in homeostasis. Although endocytosis was initially discovered and studied as a relatively simple process to transport molecules across the plasma membrane, it was subsequently found to be inextricably linked with almost all aspects of cellular signaling. This led to the notion that endocytosis is actually the master organizer of cellular signaling, providing the cell with understandable messages that have been resolved in space and time. In essence, endocytosis provides the communications and supply routes (the logistics) of the cell. Although this may seem revolutionary, it is still likely to be only a small part of the entire story. A wealth of new evidence is uncovering the surprisingly pervasive nature of endocytosis in essentially all aspects of cellular regulation. In addition, many newly discovered functions of endocytic proteins are not immediately interpretable within the classical view of endocytosis. A possible framework, to rationalize all this new knowledge, requires us to "upgrade" our vision of endocytosis. By combining the analysis of biochemical, biological, and evolutionary evidence, we propose herein that endocytosis constitutes one of the major enabling conditions that in the history of life permitted the development of a higher level of organization, leading to the actuation of the eukaryotic cell plan.

Dermal quercetin smartCrystals®: Formulation development, antioxidant activity and cellular safety.

PubMed

Hatahet, T; Morille, M; Hommoss, A; Dorandeu, C; Müller, R H; Bégu, S

2016-05-01

Flavonoids are natural plant pigments, which possess high antioxidative and antiradical activities. However, their poor water solubility led to a limited bioavailability. To overcome this major hurdle, quercetin nanocrystals were produced implementing smartCrystals® technology. This process combines bead milling and subsequent high-pressure homogenization at relatively low pressure (300bar). To test the possibility to develop a dermal formulation from quercetin smartCrystals®, quercetin nanosuspensions were admixed to Lutrol® F127 and hydroxythylcellulose nonionic gels. The physicochemical properties (morphology, size and charge), saturation solubility, dissolution velocity and the antioxidant properties (DPPH assay) as well as the cellular interaction of the produced quercetin smartCrystals® were studied and compared to crude quercetin powder. Quercetin smartCrystals® showed a strong increase in the saturation solubility and the dissolution velocity (7.6 fold). SmartCrystals® loaded or not into gels proved to be physically stable over a period of three months at 25°C. Interestingly, in vitro DPPH assay confirmed the preservation of quercetin antioxidative properties after nanonization. In parallel, the nanocrystalline form did not display cellular toxicity, even at high concentration (50μg/ml), as assayed on an epithelial cell line (VERO cells). In addition, the nanocrystalline form confirmed a protective activity for VERO cells against hydrogen peroxide induced toxicity in vitro. This new formulation presents a promising approach to deliver quercetin efficiently to skin in well-tolerated formulations. Copyright © 2016 Elsevier B.V. All rights reserved.

Endocrine disrupting chemicals affect the adipogenic differentiation of mesenchymal stem cells in distinct ontogenetic windows

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biemann, Ronald, E-mail: ronald.biemann@medizin.uni-halle.de; Navarrete Santos, Anne; Navarrete Santos, Alexander

Highlights: Black-Right-Pointing-Pointer Endocrine disrupting chemicals affect adipogenesis in mesenchymal stem cells (MSC). Black-Right-Pointing-Pointer The adipogenic impact depends strongly on the window of exposure. Black-Right-Pointing-Pointer Bisphenol A reduces the potential of MSC to differentiate into adipocytes. Black-Right-Pointing-Pointer DEHP and TBT trigger the adipogenic differentiation of mesenchymal stem cells. Black-Right-Pointing-Pointer BPA, DEHP and TBT did not affect adipogenesis in embryonic stem cells. -- Abstract: Endocrine disrupting chemicals (EDC) like bisphenol A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and tributyltin (TBT) are ubiquitously present in the environment and in human tissues. They bind to nuclear hormone receptors and affect cellular and developmental processes. In this study,more » we show that BPA, DEHP and TBT affect the adipogenic differentiation of murine mesenchymal stem cells (MSC, C3H/10T1/2) in a concentration-, stage- and compound-specific manner. C3H/10T1/2 cells and embryonic stem cells (CGR8) were exposed to BPA, DEHP or TBT at different stages of cell determination and differentiation (undifferentiated growth, adipogenic induction and terminal adipogenic differentiation). The final amount of differentiated adipocytes, cellular triglyceride content and mRNA expression of adipogenic marker genes (adiponectin, FABP4, PPAR{gamma}2, LPL) were quantified and compared with corresponding unexposed cells. BPA (10 {mu}M) decreased subsequent adipogenic differentiation of MSC, when cells were exposed during undifferentiated growth. In contrast, DEHP (100 {mu}M) during the hormonal induction period, and TBT (100 nM) in all investigated stages, enhanced adipogenesis. Importantly, exposure of undifferentiated murine embryonic stem cells did not show any effect of the investigated EDC on subsequent adipogenic differentiation.« less

Gliogenesis: historical perspectives, 1839-1985.

PubMed

Webster, Henry deF; Aström, Karl E

2009-01-01

This historical review of gliogenesis begins with Schwann's introduction of the cell doctrine in 1839. Subsequent microscopic studies revealed the cellular structure of many organs and tissues, but the CNS was thought to be different. In 1864, Virchow created the concept that nerve cells are held together by a "Nervenkitte" which he called"glia" (for glue). He and his contemporaries thought that "glia" was an unstructured, connective tissue-like ground substance that separated nerve cells from each other and from blood vessels. Dieters, a pupil of Virchow, discovered that this ground substance contained cells, which he described and illustrated. Improvements in microscopes and discovery of metallic impregnation methods finally showed convincingly that the "glia" was not a binding substance. Instead, it was composed of cells, each separate and distinct from neighboring cells and each with its own characteristic array of processes. Light microscopic studies of developing and mature nervous tissue led to the discovery of different types of glial cells-astroglia, oligodendroglia, microglia, and ependymal cells in the CNS, and Schwann cells in the peripheral nervous system (PNS). Subsequent studies characterized the origins and development of each type of glial cell. A new era began with the introduction of electron microscopy, immunostaining, and in vitro maintenance of both central and peripheral nervous tissue. Other methods and models greatly expanded our understanding of how glia multiply, migrate, and differentiate. In 1985, almost a century and a half of study had produced substantial progress in our understanding of glial cells, including their origins and development. Major advances were associated with the discovery of new methods. These are summarized first. Then the origins and development of astroglia, oligodendroglia, microglia, ependymal cells, and Schwann cells are described and discussed. In general, morphology is emphasized. Findings related to cytodifferentiation, cellular interactions, functions, and regulation of developing glia have also been included.

Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk Is Essential for Toll-like Receptor Activation and Cellular Signaling*

PubMed Central

Abdulkhalek, Samar; Amith, Schammim Ray; Franchuk, Susan L.; Jayanth, Preethi; Guo, Merry; Finlay, Trisha; Gilmour, Alanna; Guzzo, Christina; Gee, Katrina; Beyaert, Rudi; Szewczuk, Myron R.

2011-01-01

The signaling pathways of mammalian Toll-like receptors (TLRs) are well characterized, but the precise mechanism(s) by which TLRs are activated upon ligand binding remains poorly defined. Recently, we reported a novel membrane sialidase-controlling mechanism that depends on ligand binding to its TLR to induce mammalian neuraminidase-1 (Neu1) activity, to influence receptor desialylation, and subsequently to induce TLR receptor activation and the production of nitric oxide and proinflammatory cytokines in dendritic and macrophage cells. The α-2,3-sialyl residue of TLR was identified as the specific target for hydrolysis by Neu1. Here, we report a membrane signaling paradigm initiated by endotoxin lipopolysaccharide (LPS) binding to TLR4 to potentiate G protein-coupled receptor (GPCR) signaling via membrane Gαi subunit proteins and matrix metalloproteinase-9 (MMP9) activation to induce Neu1. Central to this process is that a Neu1-MMP9 complex is bound to TLR4 on the cell surface of naive macrophage cells. Specific inhibition of MMP9 and GPCR Gαi-signaling proteins blocks LPS-induced Neu1 activity and NFκB activation. Silencing MMP9 mRNA using lentivirus MMP9 shRNA transduction or siRNA transfection of macrophage cells and MMP9 knock-out primary macrophage cells significantly reduced Neu1 activity and NFκB activation associated with LPS-treated cells. These findings uncover a molecular organizational signaling platform of a novel Neu1 and MMP9 cross-talk in alliance with TLR4 on the cell surface that is essential for ligand activation of TLRs and subsequent cellular signaling. PMID:21873432

High-Concentrate Diet-Induced Change of Cellular Metabolism Leads to Decreases of Immunity and Imbalance of Cellular Activities in Rumen Epithelium.

PubMed

Lu, Zhongyan; Shen, Hong; Shen, Zanming

2018-01-01

In animals, the immune and cellular processes of tissue largely depend on the status of local metabolism. However, in the rumen epithelium, how the cellular metabolism affects epithelial immunity, and cellular processes, when the diet is switched from energy-rich to energy-excess status, with regard to animal production and health, have not as yet been reported. RNA-seq was applied to compare the biological processes altered by an increase of dietary concentration from 10% to 35% with those altered by an increase of dietary concentration from 35% to 65% (dietary concentrate: the non-grass component in diet, including corn, soya bean meal and additive. High concentrate diet composed of 35% grass, 55% corn, 8% soya bean meal and 2% additive). In addition to the functional analysis of enriched genes in terms of metabolism, the immune system, and cellular process, the highly correlated genes to the enriched metabolism genes were identified, and the function and signaling pathways related to the differentially expressed neighbors were compared among the groups. The variation trends of molar proportions of ruminal SCFAs and those of enriched pathways belonging to metabolism, immune system, and cellular process were altered with the change of diets. With regard to metabolism, lipid metabolism and amino acid metabolism were most affected. According to the correlation analysis, both innate and adaptive immune responses were promoted by the metabolism genes enriched under the 65% concentrate diet. However, the majority of immune responses were suppressed under the 35% concentrate diet. Moreover, the exclusive upregulation of cell growth and dysfunction of cellular transport and catabolism were induced by the metabolism genes enriched under the 65% concentrate diet. On the contrary, a balanced regulation of cellular processes was detected under the 35% concentrate diet. These results indicated that the alterations of cellular metabolism promote the alterations in cellular immunity, repair, and homeostasis in the rumen epithelium, thereby leading to the switch of concentrate effects from positive to negative with regard to animal production and health. © 2018 The Author(s). Published by S. Karger AG, Basel.

A rapid HPLC-APCI-MS method to detect fluoroacetate in plants

USDA-ARS?s Scientific Manuscript database

Many plant species worldwide can cause sudden death of grazing livestock. One diagnostic differential is the presence of monofluoroacetate (MFA) that is metabolised to fluorocitrate that subsequently inhibits the Kreb’s Cycle (the tricarboxylic acid cycle) leading to cellular respiration dysfunction...

Cpd-1 Null Mice Display a Subtle Neurological Phenotype

PubMed Central

Kular, Rupinder K.; Gogliotti, Rocky G.; Opal, Puneet

2010-01-01

Background CPD1 (also known as ANP32-E) belongs to a family of evolutionarily conserved acidic proteins with leucine rich repeats implicated in a variety of cellular processes regulating gene expression, vesicular trafficking, intracellular signaling and apoptosis. Because of its spatiotemporal expression pattern, CPD1 has been proposed to play an important role in brain morphogenesis and synaptic development. Methodology/Principal Findings We have generated CPD1 knock-out mice that we have subsequently characterized. These mice are viable and fertile. However, they display a subtle neurological clasping phenotype and mild motor deficits. Conclusions/Significance CPD1 is not essential for normal development; however, it appears to play a role in the regulation of fine motor functions. The minimal phenotype suggests compensatory biological mechanisms. PMID:20844742

Herpesvirus gB: A Finely Tuned Fusion Machine

PubMed Central

Cooper, Rebecca S.; Heldwein, Ekaterina E.

2015-01-01

Enveloped viruses employ a class of proteins known as fusogens to orchestrate the merger of their surrounding envelope and a target cell membrane. Most fusogens accomplish this task alone, by binding cellular receptors and subsequently catalyzing the membrane fusion process. Surprisingly, in herpesviruses, these functions are distributed among multiple proteins: the conserved fusogen gB, the conserved gH/gL heterodimer of poorly defined function, and various non-conserved receptor-binding proteins. We summarize what is currently known about gB from two closely related herpesviruses, HSV-1 and HSV-2, with emphasis on the structure of the largely uncharted membrane interacting regions of this fusogen. We propose that the unusual mechanism of herpesvirus fusion could be linked to the unique architecture of gB. PMID:26690469

Gravity and neuronal adaptation, in vitro and in vivo-from neuronal cells up to neuromuscular responses: a first model.

PubMed

Kohn, Florian P M; Ritzmann, Ramona

2018-03-01

For decades it has been shown that acute changes in gravity have an effect on neuronal systems of human and animals on different levels, from the molecular level to the whole nervous system. The functional properties and gravity-dependent adaptations of these system levels have been investigated with no or barely any interconnection. This review summarizes the gravity-dependent adaptation processes in human and animal organisms from the in vitro cellular level with its biophysical properties to the in vivo motor responses and underlying sensorimotor functions of human subjects. Subsequently, a first model for short-term adaptation of neuronal transmission is presented and discussed for the first time, which integrates the responses of the different levels of organization to changes in gravity.

The Battle of RNA Synthesis: Virus versus Host.

PubMed

Harwig, Alex; Landick, Robert; Berkhout, Ben

2017-10-21

Transcription control is the foundation of gene regulation. Whereas a cell is fully equipped for this task, viruses often depend on the host to supply tools for their transcription program. Over the course of evolution and adaptation, viruses have found diverse ways to optimally exploit cellular host processes such as transcription to their own benefit. Just as cells are increasingly understood to employ nascent RNAs in transcription regulation, recent discoveries are revealing how viruses use nascent RNAs to benefit their own gene expression. In this review, we first outline the two different transcription programs used by viruses, i.e., transcription (DNA-dependent) and RNA-dependent RNA synthesis. Subsequently, we use the distinct stages (initiation, elongation, termination) to describe the latest insights into nascent RNA-mediated regulation in the context of each relevant stage.

Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers

PubMed Central

Fischer, Audrey; Holden, Matthew A.; Pentelute, Brad L.; Collier, R. John

2011-01-01

Many bacterial toxins form proteinaceous pores that facilitate the translocation of soluble effector proteins across cellular membranes. With anthrax toxin this process may be monitored in real time by electrophysiology, where fluctuations in ionic current through these pores inserted in model membranes are used to infer the translocation of individual protein molecules. However, detecting the minute quantities of translocated proteins has been a challenge. Here, we describe use of the droplet-interface bilayer system to follow the movement of proteins across a model membrane separating two submicroliter aqueous droplets. We report the capture and subsequent direct detection of as few as 100 protein molecules that have translocated through anthrax toxin pores. The droplet-interface bilayer system offers new avenues of approach to the study of protein translocation. PMID:21949363

Modularized TGFbeta-Smad Signaling Pathway

NASA Technical Reports Server (NTRS)

Li, Yongfeng; Wang, M.; Carra, C.; Cucinotta, F. A.

2011-01-01

The Transforming Growth Factor beta (TGFbeta) signaling pathway is a prominent regulatory signaling pathway controlling various important cellular processes. It can be induced by several factors, including ionizing radiation. It is regulated by Smads in a negative feedback loop through promoting increases in the regulatory Smads in the cell nucleus, and subsequent expression of inhibitory Smad, Smad7 to form a ubiquitin ligase with Smurf targeting active TGF receptors for degradation. In this work, we proposed a mathematical model to study the radiation-induced Smad-regulated TGF signaling pathway. By modularization, we are able to analyze each module (subsystem) and recover the nonlinear dynamics of the entire network system. Meanwhile the excitability, a common feature observed in the biological systems, along the TGF signaling pathway is discussed by mathematical analysis and numerical simulation.

Interaction measurement of particles bound to a lipid membrane

NASA Astrophysics Data System (ADS)

Sarfati, Raphael; Dufresne, Eric

2015-03-01

The local shape and dynamics of the plasma membrane play important roles in many cellular processes. Local membrane deformations are often mediated by the adsorption of proteins (notably from the BAR family), and their subsequent self-assembly. The emerging hypothesis is that self-assembly arises from long-range interactions of individual proteins through the membrane's deformation field. We study these interactions in a model system of micron-sized colloidal particles adsorbed onto a lipid bilayer. We use fluorescent microscopy, optical tweezers and particle tracking to measure dissipative and conservative forces as a function of the separation between the particles. We find that particles are driven together with forces of order 100 fN and remain bound in a potential well with a stiffness of order 100 fN/micron.

Mechanistic Insights into Neurotoxicity Induced by Anesthetics in the Developing Brain

PubMed Central

Lei, Xi; Guo, Qihao; Zhang, Jun

2012-01-01

Compelling evidence has shown that exposure to anesthetics used in the clinic can cause neurodegeneration in the mammalian developing brain, but the basis of this is not clear. Neurotoxicity induced by exposure to anesthestics in early life involves neuroapoptosis and impairment of neurodevelopmental processes such as neurogenesis, synaptogenesis and immature glial development. These effects may subsequently contribute to behavior abnormalities in later life. In this paper, we reviewed the possible mechanisms of anesthetic-induced neurotoxicity based on new in vitro and in vivo findings. Also, we discussed ways to protect against anesthetic-induced neurotoxicity and their implications for exploring cellular and molecular mechanisms of neuroprotection. These findings help in improving our understanding of developmental neurotoxicology and in avoiding adverse neurological outcomes in anesthesia practice. PMID:22837663

Multicenter Cell Processing for Cardiovascular Regenerative Medicine Applications - The Cardiovascular Cell Therapy Research Network (CCTRN) Experience

PubMed Central

Gee, Adrian P.; Richman, Sara; Durett, April; McKenna, David; Traverse, Jay; Henry, Timothy; Fisk, Diann; Pepine, Carl; Bloom, Jeannette; Willerson, James; Prater, Karen; Zhao, David; Koç, Jane Reese; Ellis, Steven; Taylor, Doris; Cogle, Christopher; Moyé, Lemuel; Simari, Robert; Skarlatos, Sonia

2013-01-01

Background Aims Multi-center cellular therapy clinical trials require the establishment and implementation of standardized cell processing protocols and associated quality control mechanisms. The aims here were to develop such an infrastructure in support of the Cardiovascular Cell Therapy Research Network (CCTRN) and to report on the results of processing for the first 60 patients. Methods Standardized cell preparations, consisting of autologous bone marrow mononuclear cells, prepared using the Sepax device were manufactured at each of the five processing facilities that supported the clinical treatment centers. Processing staff underwent centralized training that included proficiency evaluation. Quality was subsequently monitored by a central quality control program that included product evaluation by the CCTRN biorepositories. Results Data from the first 60 procedures demonstrate that uniform products, that met all release criteria, could be manufactured at all five sites within 7 hours of receipt of the bone marrow. Uniformity was facilitated by use of the automated systems (the Sepax for processing and the Endosafe device for endotoxin testing), standardized procedures and centralized quality control. Conclusions Complex multicenter cell therapy and regenerative medicine protocols can, where necessary, successfully utilize local processing facilities once an effective infrastructure is in place to provide training, and quality control. PMID:20524773

MicroRNAs and cancer.

PubMed

Cowland, Jack B; Hother, Christoffer; Grønbaek, Kirsten

2007-10-01

MicroRNAs (miRNAs) are a recently discovered group of small RNA molecules involved in the regulation of gene expression. Analogously to mRNAs, the non-protein-encoding pri-miRNAs are synthesized by RNA polymerase II and post-transcriptionally modified by addition of a 5'-cap and a 3'-poly (A) tail. Subsequently, the pri-miRNA undergoes a number of processing steps in the nucleus and cytoplasm, and ends up as a mature approximately 22 nt miRNA, which can exert its function by binding to the 3'-untranslated region of a subset of mRNAs. Binding of the miRNA to the mRNA results in a reduced translation rate and/or increased degradation of the mRNA. In this way a large number of cellular pathways, such as cellular proliferation, differentiation, and apoptosis, are regulated by mi-RNAs. As corruption of these pathways is the hallmark of many cancers, dysregulation of miRNA biogenesis or expression levels may lead to tumorigenesis. The mechanisms that alter the expression of miRNAs are similar to those that change the expression levels of mRNAs of tumor suppressor- and oncogenes, i.e. gross genomic aberrations, epigenetic changes, and minor mutations affecting the expression level, processing, or target-interaction potential of the miRNA. Furthermore, expression profiling of miRNAs has been found to be useful for classification of different tumor types. Taken together, miRNAs can be classified as onco-miRs or tumor suppressor-miRs, and may turn out to be potential targets for cancer therapy.

Heme Oxygenases in Cardiovascular Health and Disease

PubMed Central

Ayer, Anita; Zarjou, Abolfazl; Agarwal, Anupam; Stocker, Roland

2016-01-01

Heme oxygenases are composed of two isozymes, Hmox1 and Hmox2, that catalyze the degradation of heme to carbon monoxide (CO), ferrous iron, and biliverdin, the latter of which is subsequently converted to bilirubin. While initially considered to be waste products, CO and biliverdin/bilirubin have been shown over the last 20 years to modulate key cellular processes, such as inflammation, cell proliferation, and apoptosis, as well as antioxidant defense. This shift in paradigm has led to the importance of heme oxygenases and their products in cell physiology now being well accepted. The identification of the two human cases thus far of heme oxygenase deficiency and the generation of mice deficient in Hmox1 or Hmox2 have reiterated a role for these enzymes in both normal cell function and disease pathogenesis, especially in the context of cardiovascular disease. This review covers the current knowledge on the function of both Hmox1 and Hmox2 at both a cellular and tissue level in the cardiovascular system. Initially, the roles of heme oxygenases in vascular health and the regulation of processes central to vascular diseases are outlined, followed by an evaluation of the role(s) of Hmox1 and Hmox2 in various diseases such as atherosclerosis, intimal hyperplasia, myocardial infarction, and angiogenesis. Finally, the therapeutic potential of heme oxygenases and their products are examined in a cardiovascular disease context, with a focus on how the knowledge we have gained on these enzymes may be capitalized in future clinical studies. PMID:27604527

Is meiosis a fundamental cause of inviability among sexual and asexual plants and animals?

PubMed

Levitis, Daniel A; Zimmerman, Kolea; Pringle, Anne

2017-08-16

Differences in viability between asexually and sexually generated offspring strongly influence the selective advantage and therefore the prevalence of sexual reproduction (sex). However, no general principle predicts when sexual offspring will be more viable than asexual offspring. We hypothesize that when any kind of reproduction is based on a more complex cellular process, it will encompass more potential failure points, and therefore lower offspring viability. Asexual reproduction (asex) can be simpler than sex, when offspring are generated using only mitosis. However, when asex includes meiosis and meiotic restitution, gamete production is more complex than in sex. We test our hypothesis by comparing the viability of asexual and closely related sexual offspring across a wide range of plants and animals, and demonstrate that meiotic asex does result in lower viability than sex; without meiosis, asex is mechanistically simple and provides higher viability than sex. This phylogenetically robust pattern is supported in 42 of 44 comparisons drawn from diverse plants and animals, and is not explained by the other variables included in our model. Other mechanisms may impact viability, such as effects of reproductive mode on heterozygosity and subsequent viability, but we propose the complexity of cellular processes of reproduction, particularly meiosis, as a fundamental cause of early developmental failure and mortality. Meiosis, the leading cause of inviability in humans, emerges as a likely explanation of offspring inviability among diverse eukaryotes. © 2017 The Author(s).

Nitrones as Therapeutics

PubMed Central

Floyd, Robert A.; Kopke, Richard D.; Choi, Chul-Hee; Foster, Steven B.; Doblas, Sabrina; Towner, Rheal A.

2008-01-01

Nitrones have the general chemical formula X-CH=NO-Y. They were first used to trap free radicals in chemical systems and then subsequently in biochemical systems. More recently several nitrones including PBN (α-phenyl-tert-butylnitrone) have been shown to have potent biological activity in many experimental animal models. Many diseases of aging including stroke, cancer development, Parkinson’s disease and Alzheimer’s disease are known to have enhanced levels of free radicals and oxidative stress. Some derivatives of PBN are significantly more potent than PBN and have undergone extensive commercial development in stroke. Recent research has shown that PBN-related nitrones also have anti-cancer activity in several experimental cancer models and have potential as therapeutics in some cancers. Also in recent observations nitrones have been shown to act synergistically in combination with antioxidants in the prevention of acute acoustic noise induced hearing loss. The mechanistic basis of the potent biological activity of PBN-related nitrones is not known. Even though PBN-related nitrones do decrease oxidative stress and oxidative damage, their potent biological anti-inflammatory activity and their ability to alter cellular signaling processes can not readily be explained by conventional notions of free radical trapping biochemistry. This review is focused on our observations and others where the use of selected nitrones as novel therapeutics have been evaluated in experimental models in the context of free radical biochemical and cellular processes considered important in pathologic conditions and age-related diseases. PMID:18793715

LiZIP3 is a cellular zinc transporter that mediates the tightly regulated import of zinc in Leishmania infantum parasites

PubMed Central

Carvalho, Sandra; da Silva, Rosa Barreira; Shawki, Ali; Castro, Helena; Lamy, Márcia; Eide, David; Costa, Vítor; Mackenzie, Bryan; Tomás, Ana M.

2016-01-01

Summary Cellular zinc homeostasis ensures that the intracellular concentration of this element is kept within limits that enable its participation in critical physiological processes without exerting toxic effects. We report here the identification and characterization of the first mediator of zinc homeostasis in Leishmania infantum, LiZIP3, a member of the ZIP family of divalent metal-ion transporters. The zinc transporter activity of LiZIP3 was first disclosed by its capacity to rescue the growth of Saccharomyces cerevisiae strains deficient in zinc acquisition. Subsequent expression of LiZIP3 in Xenopus laevis oocytes was shown to stimulate the uptake of a broad range of metal ions, among which Zn2+ was the preferred LiZIP3 substrate (K0.5 ≈ 0.1 μM). Evidence that LiZIP3 functions as a zinc importer in L. infantum came from the observations that the protein locates to the cell membrane and that its overexpression leads to augmented zinc internalization. Importantly, expression and cell-surface location of LiZIP3 are lost when parasites face high zinc bioavailability. LiZIP3 decline in response to zinc is regulated at the mRNA level in a process involving (a) short-lived protein(s). Collectively, our data reveal that LiZIP3 enables L. infantum to acquire zinc in a highly regulated manner, hence contributing to zinc homeostasis. PMID:25644708

Adipose, Bone and Myeloma: Contributions from the Microenvironment

PubMed Central

McDonald, Michelle; Fairfield, Heather; Falank, Carolyne; Reagan, Michaela R.

2017-01-01

Researchers globally are working towards finding a cure for multiple myeloma (MM), a destructive blood cancer diagnosed yearly in ~750,000 people worldwide [1]. Although MM targets multiple organ systems, it is the devastating skeletal destruction experienced by over 90% of patients that often most severely impacts patient morbidity, pain, and quality of life. Preventing bone disease is therefore a priority in MM treatment, and understanding how and why myeloma cells target the bone marrow (BM) is fundamental to this process. This review focuses on a key area of MM research: the contributions of the bone microenvironment to disease origins, progression, and drug resistance. We describe some of the key cell types in the BM niche: osteoclasts, osteoblasts, osteocytes, adipocytes and mesenchymal stem cells. We then focus on how these key cellular players are, or could be, regulating a range of disease-related processes spanning MM growth, drug resistance, and bone disease (including osteolysis, fracture, and hypercalcemia). We summarize the literature regarding MM-bone cell and MM-adipocyte relationships and subsequent phenotypic changes or adaptations in MM cells, with the aim of providing a deeper understanding of how myeloma cells grow in the skeleton to cause bone destruction. We identify avenues and therapies that intervene in these networks to stop tumor growth and/or induce bone regeneration. Overall, we aim to illustrate how novel therapeutic target molecules, proteins, and cellular mediators may offer new avenues to attack this disease while reviewing currently utilized therapies. PMID:27343063

Impact of Heat Stress on Cellular and Transcriptional Adaptation of Mammary Epithelial Cells in Riverine Buffalo (Bubalus Bubalis)

PubMed Central

Kapila, Neha; Sharma, Ankita; Kishore, Amit; Sodhi, Monika; Tripathi, Pawan K.; Mohanty, Ashok K.

2016-01-01

The present study aims to identify the heat responsive genes and biological pathways in heat stressed buffalo mammary epithelial cells (MECs). The primary mammary epithelial cells of riverine buffalo were exposed to thermal stress at 42°C for one hour. The cells were subsequently allowed to recover at 37°C and harvested at different time intervals (30 min to 48 h) along with control samples (un-stressed). In order to assess the impact of heat stress in buffalo MECs, several in-vitro cellular parameters (lactate dehydrogenase activity, cell proliferation assay, cellular viability, cell death and apoptosis) and transcriptional studies were conducted. The heat stress resulted in overall decrease in cell viability and cell proliferation of MECs while induction of cellular apoptosis and necrosis. The transcriptomic profile of heat stressed MECs was generated using Agilent 44 K bovine oligonucleotide array and at cutoff criteria of ≥3-or ≤3 fold change, a total of 153 genes were observed to be upregulated while 8 genes were down regulated across all time points post heat stress. The genes that were specifically up-regulated or down-regulated were identified as heat responsive genes. The upregulated genes in heat stressed MECs belonged to heat shock family viz., HSPA6, HSPB8, DNAJB2, HSPA1A. Along with HSPs, genes like BOLA, MRPL55, PFKFB3, PSMC2, ENDODD1, ARID5A, and SENP3 were also upregulated. Microarray data revealed that the heat responsive genes belonged to different functional classes viz., chaperons; immune responsive; cell proliferation and metabolism related. Gene ontology analysis revealed enrichment of several biological processes like; cellular process, metabolic process, response to stimulus, biological regulation, immune system processes and signaling. The transcriptome analysis data was further validated by RT-qPCR studies. Several HSP (HSP40, HSP60, HSP70, HSP90, and HSPB1), apoptotic (Bax and Bcl2), immune (IL6, TNFα and NF-kβ) and oxidative stress (GPX1 and DUSP1) related genes showed differential expression profile at different time points post heat stress. The transcriptional data strongly indicated the induction of survival/apoptotic mechanism in heat stressed buffalo MECs. The overrepresented pathways across all time points were; electron transport chain, cytochrome P450, apoptosis, MAPK, FAS and stress induction of HSP regulation, delta Notch signaling, apoptosis modulation by HSP70, EGFR1 signaling, cytokines and inflammatory response, oxidative stress, TNF-alpha and NF- kB signaling pathway. The study thus identified several genes from different functional classes and biological pathways that could be termed as heat responsive in buffalo MEC. The responsiveness of buffalo MECs to heat stress in the present study clearly suggested its suitability as a model to understand the modulation of buffalo mammary gland expression signature in response to environmental heat load. PMID:27682256

Environmental phototoxicity: Solar ultraviolet radiation affects the toxicity of natural and man-made chemicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larson, R.A.; Berenbaum, M.R.

1988-04-01

Ultraviolet radiation appears to be toxic to all forms of unpigmented living cells, including bacteria, protozoa, nematodes, arthropods, fish, birds, and mammals. In addition to the direct absorption of solar energy by cellular constituents, toxicity may occur because of the absorption of sunlight by xenobiotics (or by naturally occurring compounds outside the target cell); these may be converted by light or by subsequent light-promoted reactions that induce cellular damage. This article describes the phototoxicity of photodynamic dyes, light-activated synthetic herbicides, petroleum and its constituents, and naturally occurring chemicals from plants. Detoxification mechanisms are also discussed.

2D spatially controlled polymer micro patterning for cellular behavior studies

NASA Astrophysics Data System (ADS)

Dinca, V.; Palla-Papavlu, A.; Paraico, I.; Lippert, T.; Wokaun, A.; Dinescu, M.

2011-04-01

A simple and effective method to functionalize glass surfaces that enable polymer micropatterning and subsequent spatially controlled adhesion of cells is reported in this paper. The method involves the application of laser induced forward transfer (LIFT) to achieve polymer patterning in a single step onto cell repellent substrates (i.e. polyethyleneglycol (PEG)). This approach was used to produce micron-size polyethyleneimine (PEI)-patterns alternating with cell-repellent areas. The focus of this work is the ability of SH-SY5Y human neuroblastoma cells to orient, migrate, and produce organized cellular arrangements on laser generated PEI patterns.

Analysis of Human Mobility Based on Cellular Data

NASA Astrophysics Data System (ADS)

Arifiansyah, F.; Saptawati, G. A. P.

2017-01-01

Nowadays not only adult but even teenager and children have then own mobile phones. This phenomena indicates that the mobile phone becomes an important part of everyday’s life. Based on these indication, the amount of cellular data also increased rapidly. Cellular data defined as the data that records communication among mobile phone users. Cellular data is easy to obtain because the telecommunications company had made a record of the data for the billing system of the company. Billing data keeps a log of the users cellular data usage each time. We can obtained information from the data about communication between users. Through data visualization process, an interesting pattern can be seen in the raw cellular data, so that users can obtain prior knowledge to perform data analysis. Cellular data processing can be done using data mining to find out human mobility patterns and on the existing data. In this paper, we use frequent pattern mining and finding association rules to observe the relation between attributes in cellular data and then visualize them. We used weka tools for finding the rules in stage of data mining. Generally, the utilization of cellular data can provide supporting information for the decision making process and become a data support to provide solutions and information needed by the decision makers.

Method of forming a continuous polymeric skin on a cellular foam material

DOEpatents

Duchane, David V.; Barthell, Barry L.

1985-01-01

Hydrophobic cellular material is coated with a thin hydrophilic polymer skin which stretches tightly over the outer surface of the foam but which does not fill the cells of the foam, thus resulting in a polymer-coated foam structure having a smoothness which was not possible in the prior art. In particular, when the hydrophobic cellular material is a specially chosen hydrophobic polymer foam and is formed into arbitrarily chosen shapes prior to the coating with hydrophilic polymer, inertial confinement fusion (ICF) targets of arbitrary shapes can be produced by subsequently coating the shapes with metal or with any other suitable material. New articles of manufacture are produced, including improved ICF targets, improved integrated circuits, and improved solar reflectors and solar collectors. In the coating method, the cell size of the hydrophobic cellular material, the viscosity of the polymer solution used to coat, and the surface tensin of the polymer solution used to coat are all very important to the coating.

MACF1 regulates the migration of pyramidal neurons via microtubule dynamics and GSK-3 signaling

PubMed Central

Ka, Minhan; Jung, Eui-Man; Mueller, Ulrich; Kim, Woo-Yang

2014-01-01

Neuronal migration and subsequent differentiation play critical roles for establishing functional neural circuitry in the developing brain. However, the molecular mechanisms that regulate these processes are poorly understood. Here, we show that microtubule actin crosslinking factor 1 (MACF1) determines neuronal positioning by regulating microtubule dynamics and mediating GSK-3 signaling during brain development. First, using MACF1 floxed allele mice and in utero gene manipulation, we find that MACF1 deletion suppresses migration of cortical pyramidal neurons and results in aberrant neuronal positioning in the developing brain. The cell autonomous deficit in migration is associated with abnormal dynamics of leading processes and centrosomes. Furthermore, microtubule stability is severely damaged in neurons lacking MACF1, resulting in abnormal microtubule dynamics. Finally, MACF1 interacts with and mediates GSK-3 signaling in developing neurons. Our findings establish a cellular mechanism underlying neuronal migration and provide insights into the regulation of cytoskeleton dynamics in developing neurons. PMID:25224226

MACF1 regulates the migration of pyramidal neurons via microtubule dynamics and GSK-3 signaling.

PubMed

Ka, Minhan; Jung, Eui-Man; Mueller, Ulrich; Kim, Woo-Yang

2014-11-01

Neuronal migration and subsequent differentiation play critical roles for establishing functional neural circuitry in the developing brain. However, the molecular mechanisms that regulate these processes are poorly understood. Here, we show that microtubule actin crosslinking factor 1 (MACF1) determines neuronal positioning by regulating microtubule dynamics and mediating GSK-3 signaling during brain development. First, using MACF1 floxed allele mice and in utero gene manipulation, we find that MACF1 deletion suppresses migration of cortical pyramidal neurons and results in aberrant neuronal positioning in the developing brain. The cell autonomous deficit in migration is associated with abnormal dynamics of leading processes and centrosomes. Furthermore, microtubule stability is severely damaged in neurons lacking MACF1, resulting in abnormal microtubule dynamics. Finally, MACF1 interacts with and mediates GSK-3 signaling in developing neurons. Our findings establish a cellular mechanism underlying neuronal migration and provide insights into the regulation of cytoskeleton dynamics in developing neurons. Copyright © 2014 Elsevier Inc. All rights reserved.

The molecular network governing nodule organogenesis and infection in the model legume Lotus japonicus.

PubMed

Madsen, Lene H; Tirichine, Leïla; Jurkiewicz, Anna; Sullivan, John T; Heckmann, Anne B; Bek, Anita S; Ronson, Clive W; James, Euan K; Stougaard, Jens

2010-04-12

Bacterial infection of interior tissues of legume root nodules is controlled at the epidermal cell layer and is closely coordinated with progressing organ development. Using spontaneous nodulating Lotus japonicus plant mutants to uncouple nodule organogenesis from infection, we have determined the role of 16 genes in these two developmental processes. We show that host-encoded mechanisms control three alternative entry processes operating in the epidermis, the root cortex and at the single cell level. Single cell infection did not involve the formation of trans-cellular infection threads and was independent of host Nod-factor receptors and bacterial Nod-factor signals. In contrast, Nod-factor perception was required for epidermal root hair infection threads, whereas primary signal transduction genes preceding the secondary Ca2+ oscillations have an indirect role. We provide support for the origin of rhizobial infection through direct intercellular epidermal invasion and subsequent evolution of crack entry and root hair invasions observed in most extant legumes.

Mechanical properties of porous and cellular materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sieradzki, K.; Green, D.J.; Gibson, L.J.

1991-01-01

This symposium successfully brought scientists together from a wide variety of disciplines to focus on the mechanical behavior of porous and cellular solids composed of metals, ceramics, polymers, or biological materials. For cellular materials, papers ranged from processing techniques through microstructure-mechanical property relationships to design. In an overview talk, Mike Ashby (Cambridge Univ.) showed how porous cellular materials can be more efficient than dense materials in designs that require minimum weight. He indicated that many biological materials have been able to accomplish such efficiency but there exists an opportunity to design even more efficient, manmade materials controlling microstructures at differentmore » scale levels. In the area of processing, James Aubert (Sandia National Laboratories) discussed techiques for manipulating polymersolvent phase equilibria to control the microstructure of microcellular foams. Other papers on processing discussed the production of cellular ceramics by CVD, HIPing and sol- gel techniques. Papers on the mechanical behavior of cellular materials considered various ceramics microcellular polymers, conventional polymer foams and apples. There were also contributions that considered optimum design procedures for cellular materials. Steven Cowin (City Univ. of New York) discussed procedures to match the discrete microstructural aspects of cellular materials with the continuum mechanics approach to their elastic behavior.« less

Toward Multiscale Models of Cyanobacterial Growth: A Modular Approach

PubMed Central

Westermark, Stefanie; Steuer, Ralf

2016-01-01

Oxygenic photosynthesis dominates global primary productivity ever since its evolution more than three billion years ago. While many aspects of phototrophic growth are well understood, it remains a considerable challenge to elucidate the manifold dependencies and interconnections between the diverse cellular processes that together facilitate the synthesis of new cells. Phototrophic growth involves the coordinated action of several layers of cellular functioning, ranging from the photosynthetic light reactions and the electron transport chain, to carbon-concentrating mechanisms and the assimilation of inorganic carbon. It requires the synthesis of new building blocks by cellular metabolism, protection against excessive light, as well as diurnal regulation by a circadian clock and the orchestration of gene expression and cell division. Computational modeling allows us to quantitatively describe these cellular functions and processes relevant for phototrophic growth. As yet, however, computational models are mostly confined to the inner workings of individual cellular processes, rather than describing the manifold interactions between them in the context of a living cell. Using cyanobacteria as model organisms, this contribution seeks to summarize existing computational models that are relevant to describe phototrophic growth and seeks to outline their interactions and dependencies. Our ultimate aim is to understand cellular functioning and growth as the outcome of a coordinated operation of diverse yet interconnected cellular processes. PMID:28083530

DOUGLAS LEA MEMORIAL LECTURE: From targets to genes: a brief history of radiosensitivity

NASA Astrophysics Data System (ADS)

Steel, G. Gordon

1996-02-01

The biological work of Douglas Lea spanned the period from 1934 to his early death in 1947, and during this short period he made important contributions to the theory of radiation action. He interpreted experimental data relating to the effects of radiation on viruses, bacteria, bean roots, etc in terms of the inactivation of discrete targets, which he identified with cellular genes. He thus laid the foundation of much subsequent research. It is now well recognized that mammalian cells differ substantially in radiosensitivity, especially in the low-dose region of the survival curve. The dependence of radiosensitivity on dose rate has been widely studied; this has practical significance for clinical radiotherapy as well as mechanistic implications. Since Lea's time there have been a number of efforts to describe models that can relate cell killing to radiation dose, dose rate, and track structure. So far these have not led to a comprehensive and widely accepted picture. Microdosimetric considerations lead to the concept of differing severity of lesions induced in DNA. Much is known about the sequence of processes that subsequently lead to cell inactivation: this can be divided into phases of induction, processing, and manifestation. Chromosomal events are currently attracting much attention, as they did in Lea's time. Considerable progress has also been made in identifying genes that control the repair of radiation damage. It has been found that mutation is frequently associated with the loss of a large segment of the genome around the damage site and this will have important implications for interactive processes between particle tracks.

Umbilical cord tissue-derived mesenchymal stromal cells maintain immunomodulatory and angiogenic potencies after cryopreservation and subsequent thawing.

PubMed

Bárcia, Rita N; Santos, Jorge M; Teixeira, Mariana; Filipe, Mariana; Pereira, Ana Rita S; Ministro, Augusto; Água-Doce, Ana; Carvalheiro, Manuela; Gaspar, Maria Manuela; Miranda, Joana P; Graça, Luis; Simões, Sandra; Santos, Susana Constantino Rosa; Cruz, Pedro; Cruz, Helder

2017-03-01

The effect of cryopreservation on mesenchymal stromal cell (MSC) therapeutic properties has become highly controversial. However, data thus far have indiscriminately involved the assessment of different types of MSCs with distinct production processes. This study assumed that MSC-based products are affected differently depending on the tissue source and manufacturing process and analyzed the effect of cryopreservation on a specific population of umbilical cord tissue-derived MSCs (UC-MSCs), UCX ® . Cell phenotype was assessed by flow cytometry through the evaluation of the expression of relevant surface markers such as CD14, CD19, CD31, CD34, CD44, CD45, CD90, CD105, CD146, CD200, CD273, CD274 and HLA-DR. Immunomodulatory activity was analyzed in vitro through the ability to inhibit activated T cells and in vivo by the ability to reverse the signs of inflammation in an adjuvant-induced arthritis (AIA) model. Angiogenic potential was evaluated in vitro using a human umbilical vein endothelial cell-based angiogenesis assay, and in vivo using a mouse model for hindlimb ischemia. Phenotype and immunomodulatory and angiogenic potencies of this specific UC-MSC population were not impaired by cryopreservation and subsequent thawing, both in vitro and in vivo. This study suggests that potency impairment related to cryopreservation in a given tissue source can be avoided by the production process. The results have positive implications for the development of advanced-therapy medicinal products. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Ultrasound and Microbubble Guided Drug Delivery: Mechanistic Understanding and Clinical Implications

PubMed Central

Wang, Tzu-Yin; Wilson, Katheryne E.; Machtaler, Steven; Willmann, Jürgen K.

2014-01-01

Ultrasound mediated drug delivery using microbubbles is a safe and noninvasive approach for spatially localized drug administration. This approach can create temporary and reversible openings on cellular membranes and vessel walls (a process called “sonoporation”), allowing for enhanced transport of therapeutic agents across these natural barriers. It is generally believed that the sonoporation process is highly associated with the energetic cavitation activities (volumetric expansion, contraction, fragmentation, and collapse) of the microbubble. However, a thorough understanding of the process was unavailable until recently. Important progress on the mechanistic understanding of sonoporation and the corresponding physiological responses in vitro and in vivo has been made. Specifically, recent research shed light on the cavitation process of microbubbles and fluid motion during insonation of ultrasound, on the spatio-temporal interactions between microbubbles and cells or vessel walls, as well as on the temporal course of the subsequent biological effects. These findings have significant clinical implications on the development of optimal treatment strategies for effective drug delivery. In this article, current progress in the mechanistic understanding of ultrasound and microbubble mediated drug delivery and its implications for clinical translation is discussed. PMID:24372231

Gravitropism in cut flower stalks of snapdragon

NASA Astrophysics Data System (ADS)

Philosoph-Hadas, S.; Friedman, H.; Meir, S.; Berkovitz-SimanTov, R.; Rosenberger, I.; Halevy, A. H.; Kaufman, P. B.; Balk, P.; Woltering, E. J.

The negative gravitropic response of cut flower stalks is a complex multistep process that requires the participation of various cellular components acting in succession or in parallel. The process was particularly characterized in snapdragon (Antirrhinum majus L.) spikes with regard to (1) gravity stimulus perception associated with amyloplast reorientation; (2) stimulus transduction mediated through differential changes in the level, action and related genes of auxin and ethylene and their possible interaction; (3) stimulus response associated with differential growth leading to stalk curvature; (4) involvement of cytosolic calcium and actin cytoskeleton. Results show that the gravity-induced amyloplast reorientation, differential over-expression of two early auxin responsive genes and asymmetrical distribution of free IAA are early events in the bending process. These precede the asymmetrical ethylene production and differential stem growth, which was derived from initial shrinkage of the upper stem side and a subsequent elongation of the lower stem side. Results obtained with various calcium- and cytoskeleton-related agents indicate that cytosolic calcium and actin filaments may play essential roles in gravitropism-related processes of cut flower stalks. Therefore, modulators of these two physiological mediators may serve as means for controlling any undesired gravitropic bending.

Cellular and laminar expression of Dab-1 during the postnatal critical period in cat visual cortex and the effects of dark rearing.

PubMed

Kiser, Paul J; Liu, Zijing; Wilt, Steven D; Mower, George D

2011-04-06

This study describes postnatal critical period changes in cellular and laminar expression of Dab-1, a gene shown to play a role in controlling neuronal positioning during embryonic brain development, in cat visual cortex and the effects of dark rearing (DR). At 1week, there is dense cellular staining which is uniform across cortical layers and very light neuropil staining. At the peak of the critical period (5weeks), dense cell staining is largely restricted to large pyramidal cells of deep layer III and layer V, there is faint cell body staining throughout all cortical layers, neuropil staining is markedly increased and uniform in layers III to VI. This dramatic change in laminar and cellular labeling is independent of visual input, since immunostaining is similar in 5-week DR cats. By 10weeks, the mature laminar and cellular staining pattern is established and the major subsequent change is a further reduction in the density of cellular staining in all cortical layers. Neuropil staining is pronounced and uniform across cortical layers. These developmental changes are altered by DR. Quantification by cell counts indicated that age and DR interact such that differences in cellular expression are opposite in direction between 5- and 20-week-old cats. This bidirectional regulation of cellular expression is the same in all cortical laminae. The bidirectional regulation of cellular expression matches the effects of age and DR on physiological plasticity during the critical period as assessed by ocular dominance shifts in response to monocular deprivation. Copyright © 2011 Elsevier B.V. All rights reserved.

Alginic acid cell entrapment: a novel method for measuring in vivo macrophage cholesterol homeostasis

PubMed Central

Sontag, Timothy J.; Chellan, Bijoy; Bhanvadia, Clarissa V.; Getz, Godfrey S.; Reardon, Catherine A.

2015-01-01

Macrophage conversion to atherosclerotic foam cells is partly due to the balance of uptake and efflux of cholesterol. Cholesterol efflux from cells by HDL and its apoproteins for subsequent hepatic elimination is known as reverse cholesterol transport. Numerous methods have been developed to measure in vivo macrophage cholesterol efflux. Most methods do not allow for macrophage recovery for analysis of changes in cellular cholesterol status. We describe a novel method for measuring cellular cholesterol balance using the in vivo entrapment of macrophages in alginate, which retains incorporated cells while being permeable to lipoproteins. Recipient mice were injected subcutaneously with CaCl2 forming a bubble into which a macrophage/alginate suspension was injected, entrapping the macrophages. Cells were recovered after 24 h. Cellular free and esterified cholesterol mass were determined enzymatically and normalized to cellular protein. Both normal and cholesterol loaded macrophages undergo measureable changes in cell cholesterol when injected into WT and apoA-I-, LDL-receptor-, or apoE-deficient mice. Cellular cholesterol balance is dependent on initial cellular cholesterol status, macrophage cholesterol transporter expression, and apolipoprotein deficiency. Alginate entrapment allows for the in vivo measurement of macrophage cholesterol homeostasis and is a novel platform for investigating the role of genetics and therapeutic interventions in atherogenesis. PMID:25465389

Eicosanoids up-regulate production of reactive oxygen species by NADPH-dependent oxidase in Spodoptera exigua phagocytic hemocytes

USDA-ARS?s Scientific Manuscript database

Eicosanoids mediate cellular immune responses in insects, including phagocytosis of invading microbes. Phagocytosis entails two major steps, the internalization of microbes and the subsequent killing of them via formation of reactive oxygen species (ROS). Here, we posed the hypothesis that eicosanoi...

Activation of Pax7-Positive Cells in a Non-Contractile Tissue Contributes to Regeneration of Myogenic Tissues in the Electric Fish S. macrurus

PubMed Central

Weber, Christopher M.; Martindale, Mark Q.; Tapscott, Stephen J.; Unguez, Graciela A.

2012-01-01

The ability to regenerate tissues is shared across many metazoan taxa, yet the type and extent to which multiple cellular mechanisms come into play can differ across species. For example, urodele amphibians can completely regenerate all lost tissues, including skeletal muscles after limb amputation. This remarkable ability of urodeles to restore entire limbs has been largely linked to a dedifferentiation-dependent mechanism of regeneration. However, whether cell dedifferentiation is the fundamental factor that triggers a robust regeneration capacity, and whether the loss or inhibition of this process explains the limited regeneration potential in other vertebrates is not known. Here, we studied the cellular mechanisms underlying the repetitive regeneration of myogenic tissues in the electric fish S. macrurus. Our in vivo microinjection studies of high molecular weight cell lineage tracers into single identified adult myogenic cells (muscle or noncontractile muscle-derived electrocytes) revealed no fragmentation or cellularization proximal to the amputation plane. In contrast, ultrastructural and immunolabeling studies verified the presence of myogenic stem cells that express the satellite cell marker Pax7 in mature muscle fibers and electrocytes of S. macrurus. These data provide the first example of Pax-7 positive muscle stem cells localized within a non-contractile electrogenic tissue. Moreover, upon amputation, Pax-7 positive cells underwent a robust replication and were detected exclusively in regions that give rise to myogenic cells and dorsal spinal cord components revealing a regeneration process in S. macrurus that is dependent on the activation of myogenic stem cells for the renewal of both skeletal muscle and the muscle-derived electric organ. These data are consistent with the emergent concept in vertebrate regeneration that different tissues provide a distinct progenitor cell population to the regeneration blastema, and these progenitor cells subsequently restore the original tissue. PMID:22685526

Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression.

PubMed

Gurevich, David B; Severn, Charlotte E; Twomey, Catherine; Greenhough, Alexander; Cash, Jenna; Toye, Ashley M; Mellor, Harry; Martin, Paul

2018-06-04

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti-angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor-α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

HAMLET - A protein-lipid complex with broad tumoricidal activity.

PubMed

Ho, James C S; Nadeem, Aftab; Svanborg, Catharina

2017-01-15

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a tumoricidal protein-lipid complex with broad effects against cancer cells of different origin. The therapeutic potential is emphasized by a high degree of specificity for tumor tissue. Here we review early studies of HAMLET, in collaboration with the Orrenius laboratory, and some key features of the subsequent development of the HAMLET project. The early studies focused on the apoptotic response that accompanies death in HAMLET treated tumor cells and the role of mitochondria in this process. In subsequent studies, we have identified a sequence of interactions that starts with the membrane integration of HAMLET and the activation of ion fluxes followed by HAMLET internalization, progressive inhibition of MAPK kinases and GTPases and sorting of HAMLET to different cellular compartments, including the nuclei. Therapeutic efficacy of HAMLET has been demonstrated in animal models of glioblastoma, bladder cancer and intestinal cancer. In clinical studies, HAMLET has been shown to target skin papillomas and bladder cancers. The findings identify HAMLET as a new drug candidate with promising selectivity for cancer cells and a strong therapeutic potential. Copyright © 2016 Elsevier Inc. All rights reserved.

Mammalian synthetic biology for studying the cell

PubMed Central

Mathur, Melina; Xiang, Joy S.

2017-01-01

Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. PMID:27932576

Landauer in the Age of Synthetic Biology: Energy Consumption and Information Processing in Biochemical Networks

NASA Astrophysics Data System (ADS)

Mehta, Pankaj; Lang, Alex H.; Schwab, David J.

2016-03-01

A central goal of synthetic biology is to design sophisticated synthetic cellular circuits that can perform complex computations and information processing tasks in response to specific inputs. The tremendous advances in our ability to understand and manipulate cellular information processing networks raises several fundamental physics questions: How do the molecular components of cellular circuits exploit energy consumption to improve information processing? Can one utilize ideas from thermodynamics to improve the design of synthetic cellular circuits and modules? Here, we summarize recent theoretical work addressing these questions. Energy consumption in cellular circuits serves five basic purposes: (1) increasing specificity, (2) manipulating dynamics, (3) reducing variability, (4) amplifying signal, and (5) erasing memory. We demonstrate these ideas using several simple examples and discuss the implications of these theoretical ideas for the emerging field of synthetic biology. We conclude by discussing how it may be possible to overcome these limitations using "post-translational" synthetic biology that exploits reversible protein modification.

Effect of mechanical disruption on the effectiveness of three reactors used for dilute acid pretreatment of corn stover Part 2: morphological and structural substrate analysis

PubMed Central

2014-01-01

Background Lignocellulosic biomass is a renewable, naturally mass-produced form of stored solar energy. Thermochemical pretreatment processes have been developed to address the challenge of biomass recalcitrance, however the optimization, cost reduction, and scalability of these processes remain as obstacles to the adoption of biofuel production processes at the industrial scale. In this study, we demonstrate that the type of reactor in which pretreatment is carried out can profoundly alter the micro- and nanostructure of the pretreated materials and dramatically affect the subsequent efficiency, and thus cost, of enzymatic conversion of cellulose. Results Multi-scale microscopy and quantitative image analysis was used to investigate the impact of different biomass pretreatment reactor configurations on plant cell wall structure. We identify correlations between enzymatic digestibility and geometric descriptors derived from the image data. Corn stover feedstock was pretreated under the same nominal conditions for dilute acid pretreatment (2.0 wt% H2SO4, 160°C, 5 min) using three representative types of reactors: ZipperClave® (ZC), steam gun (SG), and horizontal screw (HS) reactors. After 96 h of enzymatic digestion, biomass treated in the SG and HS reactors achieved much higher cellulose conversions, 88% and 95%, respectively, compared to the conversion obtained using the ZC reactor (68%). Imaging at the micro- and nanoscales revealed that the superior performance of the SG and HS reactors could be explained by reduced particle size, cellular dislocation, increased surface roughness, delamination, and nanofibrillation generated within the biomass particles during pretreatment. Conclusions Increased cellular dislocation, surface roughness, delamination, and nanofibrillation revealed by direct observation of the micro- and nanoscale change in accessibility explains the superior performance of reactors that augment pretreatment with physical energy. PMID:24690534

Transient gibberellin application promotes Arabidopsis thaliana hypocotyl cell elongation without maintaining transverse orientation of microtubules on the outer tangential wall of epidermal cells.

PubMed

Sauret-Güeto, Susanna; Calder, Grant; Harberd, Nicholas P

2012-02-01

The phytohormone gibberellin (GA) promotes plant growth by stimulating cellular expansion. Whilst it is known that GA acts by opposing the growth-repressing effects of DELLA proteins, it is not known how these events promote cellular expansion. Here we present a time-lapse analysis of the effects of a single pulse of GA on the growth of Arabidopsis hypocotyls. Our analyses permit kinetic resolution of the transient growth effects of GA on expanding cells. We show that pulsed application of GA to the relatively slowly growing cells of the unexpanded light-grown Arabidopsis hypocotyl results in a transient burst of anisotropic cellular growth. This burst, and the subsequent restoration of initial cellular elongation rates, occurred respectively following the degradation and subsequent reappearance of a GFP-tagged DELLA (GFP-RGA). In addition, we used a GFP-tagged α-tubulin 6 (GFP-TUA6) to visualise the behaviour of microtubules (MTs) on the outer tangential wall (OTW) of epidermal cells. In contrast to some current hypotheses concerning the effect of GA on MTs, we show that the GA-induced boost of hypocotyl cell elongation rate is not dependent upon the maintenance of transverse orientation of the OTW MTs. This confirms that transverse alignment of outer face MTs is not necessary to maintain rapid elongation rates of light-grown hypocotyls. Together with future studies on MT dynamics in other faces of epidermal cells and in cells deeper within the hypocotyl, our observations advance understanding of the mechanisms by which GA promotes plant cell and organ growth. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Optimization of a Biomimetic Apatite Nanoparticle Delivery System for Non-viral Gene Transfection---a Simulated Body Fluid Approach

NASA Astrophysics Data System (ADS)

Das, Debobrato

Current methods for gene delivery utilize nanocarriers such as liposomes and viral vectors that may produce in vivo toxicity, immunogenicity, or mutagenesis. Moreover, these common high-cost systems have a low efficacy of gene-vehicle transport across the cell plasma membrane followed by inadequate release and weak intracellular stability of the genetic sequence. Thus, this study aims to maximize gene transfection while minimizing cytotoxicity by utilizing supersaturated blood-plasma ions derived from simulated body fluids (SBF). With favorable electrostatic interactions to create biocompatible calcium-phosphate nanoparticles (NPs) derived from biomimetic apatite (BA), results suggest that the SBF system, though naturally sensitive to reaction conditions, after optimization can serve as a tunable and versatile platform for the delivery of various types of nucleic acids. From a systematic exploration of the effects of nucleation pH, incubation temperature, and time on transfection efficiency, the study proposes distinct characteristic trends in SBF BA-NP morphology, cellular uptake, cell viability, and gene modulation. Specifically, with aggressive nucleation and growth of BA-NPs in solution (observed via scanning electron microscopy), the ensuing microenvironment imposes a more toxic cellular interaction (indicated by alamarBlue and BCA assays), limiting particle uptake (fluorescence experiments) and subsequent gene knockdown (quantitative loss of function assays). Controlled precipitation of BA-NPs function to increase particle accessibility by surrounding cells, and subsequently enhance uptake and transfection efficiency. By closely examining such trends, an optimal fabrication condition of pH 6.5-37C can be observed where particle growth is more tamed and less chaotic, providing improved, favorable cellular interactions that increase cell uptake and consequently maximize gene transfection, without compromising cellular viability.

Protecting the retinal neurons from glaucoma: lowering ocular pressure is not enough.

PubMed

Pascale, Alessia; Drago, Filippo; Govoni, Stefano

2012-07-01

The retina is theater of a number of biochemical reactions allowing, within its layers, the conversion of light impulses into electrical signals. The axons of the last neuronal elements, the ganglion cells, form the optic nerve and transfer the signals to the brain. Therefore, an appropriate cellular communication, not only within the different retinal cells, but also between the retina itself and the other brain structures, is fundamental. One of the most diffuse pathologies affecting retinal function and communication, which thus reverberates in the whole visual system, is glaucoma. This insidious disease is characterized by a progressive optic nerve degeneration and sight loss which may finally lead to irreversible blindness. Nevertheless, the progressive nature of this pathology offers an opportunity for therapeutic intervention. To better understand the cellular processes implicated in the development of glaucoma useful to envision a targeted pharmacological strategy, this manuscript first examines the complex cellular and functional organization of the retina and subsequently identifies the targets sensitive to neurodegeneration. Within this context, high ocular pressure represents a key risk factor. However, recent literature findings highlight the concept that lowering ocular pressure is not enough to prevent/slow down glaucomatous damage, suggesting the importance of combining the hypotensive treatment with other pharmacological approaches, such as the use of neuroprotectants. Therefore, this important and more novel aspect is extensively considered in this review, also emphasizing the idea that the neuroprotective strategy should be extended to the entire visual system and not restricted to the retina. Copyright © 2012 Elsevier Ltd. All rights reserved.

Mammalian synthetic biology for studying the cell.

PubMed

Mathur, Melina; Xiang, Joy S; Smolke, Christina D

2017-01-02

Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. © 2017 Mathur et al.

Structure and Function of Viral Deubiquitinating Enzymes.

PubMed

Bailey-Elkin, Ben A; Knaap, Robert C M; Kikkert, Marjolein; Mark, Brian L

2017-11-10

Post-translational modification of cellular proteins by ubiquitin regulates numerous cellular processes, including innate and adaptive immune responses. Ubiquitin-mediated control over these processes can be reversed by cellular deubiquitinating enzymes (DUBs), which remove ubiquitin from cellular targets and depolymerize polyubiquitin chains. The importance of protein ubiquitination to host immunity has been underscored by the discovery of viruses that encode proteases with deubiquitinating activity, many of which have been demonstrated to actively corrupt cellular ubiquitin-dependent processes to suppress innate antiviral responses and promote viral replication. DUBs have now been identified in diverse viral lineages, and their characterization is providing valuable insights into virus biology and the role of the ubiquitin system in host antiviral mechanisms. Here, we provide an overview of the structural biology of these fascinating viral enzymes and their role innate immune evasion and viral replication. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cell proliferation inhibition in reduced gravity

NASA Technical Reports Server (NTRS)

Moos, P. J.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

1994-01-01

Extended durations of spaceflight have been shown to be deleterious on an organismic level; however, mechanisms underlying cellular sensitivity to the gravitational environment remain to be elucidated. The majority of the gravitational studies to date indicates that cell regulatory pathways may be influenced by their gravitational environment. Still, few cell biology experiments have been performed in space flight and even fewer experiments have been repeated on subsequent flights. With flight opportunities on STS-50, 54, and 57, Sf9 cells were flown in the BioServe Fluids Processing Apparatus and cell proliferation was measured with and without exposure to a cell regulatory sialoglycopeptide (CeReS) inhibitor. Results from these flights indicate that the Sf9 cells grew comparable to ground controls, that the CeReS inhibitor bound to its specific receptor, and that its signal transduction cascade was not gravity sensitive.

Natural Compounds and Neuroprotection: Mechanisms of Action and Novel Delivery Systems.

PubMed

Bagli, Eleni; Goussia, Anna; Moschos, Marilita M; Agnantis, Niki; Kitsos, Georgios

Neurodegeneration characterizes pathologic conditions, ranging from Alzheimer's disease to glaucoma, with devastating social and economic effects. It is a complex process implicating a series of molecular and cellular events, such as oxidative stress, mitochondrial dysfunction, protein misfolding, excitotoxicity and inflammation. Natural compounds, because of their broad spectrum of pharmacological and biological activities, could be possible candidates for the management of such multifactorial morbidities. However, their therapeutic potential against neurodegenerative diseases has been hampered by their poor bioavailability and subsequent insufficient delivery to the brain. This article provides an overview of the molecular mechanisms through which natural compounds exert their neuroprotective effects, as well as the development of novel natural compound-loaded delivery systems that could improve their neuroavailability. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Methods for the Detection of Autophagy in Mammalian Cells

PubMed Central

Zhang, Ziyan; Singh, Rajat; Aschner, Michael

2016-01-01

Macroautophagy (hereafter referred to as autophagy) is a degradation pathway that delivers cytoplasmic materials to lysosomes via double-membraned vesicles designated autophagosomes. Cytoplasmic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes, where the cargo is degraded. Autophagy is a crucial mechanism involved in many aspects of cell function, including cellular metabolism and energy balance; and alterations in autophagy have been linked to various human pathological processes. Thus, methods that accurately measure autophagic activity are necessary. In this unit, we introduce several approaches to analyze autophagy in mammalian cells, including immunoblotting analysis of LC3 and p62, detection of autophagosome formation by fluorescence microscopy, and monitoring autophagosome maturation by tandem mRFP-GFP fluorescence microscopy. Overall, we recommend a combined use of multiple methods to accurately assess the autophagic activity in any given biological setting. PMID:27479363

Modularized Smad-regulated TGFβ signaling pathway.

PubMed

Li, Yongfeng; Wang, Minli; Carra, Claudio; Cucinotta, Francis A

2012-12-01

The transforming Growth Factor β (TGFβ) signaling pathway is a prominent regulatory signaling pathway controlling various important cellular processes. TGFβ signaling can be induced by several factors including ionizing radiation. The pathway is regulated in a negative feedback loop through promoting the nuclear import of the regulatory Smads and a subsequent expression of inhibitory Smad7, that forms ubiquitin ligase with Smurf2, targeting active TGFβ receptors for degradation. In this work, we proposed a mathematical model to study the Smad-regulated TGFβ signaling pathway. By modularization, we are able to analyze mathematically each component subsystem and recover the nonlinear dynamics of the entire network system. Meanwhile the excitability, a common feature observed in the biological systems, in the TGFβ signaling pathway is discussed and supported as well by numerical simulation, indicating the robustness of the model. Published by Elsevier Inc.

Polyamine biosynthesis during germination of yeast ascospores.

PubMed Central

Brawley, J V; Ferro, A J

1979-01-01

The role of the diamine putrescine during germination and outgrowth of ascospores of Saccharomyces cerevisiae was examined. Ornithine decarboxylase activity increased and declined rapidly during germination and outgrowth; peak activity was attained after the cells had proceeded through the G1 interval of the cell cycle, whereas minimal activity was present at the completion of the first cell division. alpha-Methylornithine inhibited both ornithine decarboxylase activity and the in vivo accumulation of putrescine. In the presence of alpha-methylornithireak dormancy and proceed through one cell division. Subsequent cellular growth, however, was retarded but not completely inhibited. The supplementation of Methylglyoxal bis(guanylhydrazone) to sporulation medium greatly inhibited this sexual process. These data suggest that the synthesis of putrescine is not required for the breaking of spore dormancy, but that polyamine biosynthesis may be essential for meiosis and sporulation. PMID:387744

Loss of the E3 ubiquitin ligase LRSAM1 sensitizes peripheral axons to degeneration in a mouse model of Charcot-Marie-Tooth disease.

PubMed

Bogdanik, Laurent P; Sleigh, James N; Tian, Cong; Samuels, Mark E; Bedard, Karen; Seburn, Kevin L; Burgess, Robert W

2013-05-01

Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous condition characterized by peripheral axon degeneration with subsequent motor and sensory deficits. Several CMT gene products function in endosomal sorting and trafficking to the lysosome, suggesting that defects in this cellular pathway might present a common pathogenic mechanism for these conditions. LRSAM1 is an E3 ubiquitin ligase that is implicated in this process, and mutations in LRSAM1 have recently been shown to cause CMT. We have generated mouse mutations in Lrsam1 to create an animal model of this form of CMT (CMT2P). Mouse Lrsam1 is abundantly expressed in the motor and sensory neurons of the peripheral nervous system. Both homozygous and heterozygous mice have largely normal neuromuscular performance and only a very mild neuropathy phenotype with age. However, Lrsam1 mutant mice are more sensitive to challenge with acrylamide, a neurotoxic agent that causes axon degeneration, indicating that the axons in the mutant mice are indeed compromised. In transfected cells, LRSAM1 primarily localizes in a perinuclear compartment immediately beyond the Golgi and shows little colocalization with components of the endosome to lysosome trafficking pathway, suggesting that other cellular mechanisms also merit consideration.

Determining Protease Activity In Vivo by Fluorescence Cross-Correlation Analysis

PubMed Central

Kohl, Tobias; Haustein, Elke; Schwille, Petra

2005-01-01

To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ubiquitous, but so far there is no standard technique permitting online analysis of these processes in vivo. Featuring single-molecule sensitivity combined with intrinsic averaging, fluorescence correlation spectroscopy is a minimally invasive technique ideally suited to monitor proteins. Moreover, endogenous fluorescence-based assays can be established by genetically encoding fusions of autofluorescent proteins and cellular proteins, thus avoiding the disadvantages of in vitro protein labeling and subsequent delivery to cells. Here, we present an in vivo protease assay as a model system: Green and red autofluorescent proteins were connected by Caspase-3- sensitive and insensitive protein linkers to create double-labeled protease substrates. Then, dual-color fluorescence cross-correlation spectroscopy was employed to study the protease reaction in situ. Allowing assessment of multiple dynamic parameters simultaneously, this method provided internal calibration and improved experimental resolution for quantifying protein stability. This approach, which is easily extended to reversible protein-protein interactions, seems very promising for elucidating intracellular protein functions. PMID:16055538

Evidence of parasexual activity in "asexual amoebae" Cochliopodium spp. (Amoebozoa): extensive cellular and nuclear fusion.

PubMed

Tekle, Yonas I; Anderson, O Roger; Lecky, Ariel F

2014-09-01

The majority of microbial eukaryotes have long been considered asexual, though new evidence indicates sex, or sexual-like (parasexual) behaviors that deviate from the usual union of two gametes, among other variant aspects. Over a dozen amoebozoans are implicated to have sexual stages. However, the exact mechanism by which sex occurs in these lineages remains elusive. This is mainly due to the diverse quality and cryptic nature of their life cycle. In this study we present evidence of some previously unreported aspects of the life cycle of an amoeba, Cochliopodium, that undergoes unusual intraspecific interactions using light microscopy and immunocytochemistry. Similar to other amoebozoans, Cochliopodium, is considered asexual with no published reports of sex or parasexuality. We also investigated environmental conditions that govern the observed intraspecific interactions. Both light microscopic and immunocytochemistry evidence demonstrates Cochliopodium undergoes cellular fusion (plasmogamy) and nuclear fusion (karyogamy). Large plasmodia eventually undergo karyogamy and contain large fused, polyploid, nuclei. These are observed to fragment, subsequently, by karyotomy (nuclear fission) and cytoplasmic fission to yield uninucleated amoebae. This process could lead to a non-meiotic, parasexual exchange of chromosomes in Cochliopodium. These findings strongly suggest that Cochliopodium is involved in parasexual activity and should no longer be considered strictly asexual. Copyright © 2014 Elsevier GmbH. All rights reserved.

LAMP-3 (Lysosome-Associated Membrane Protein 3) Promotes the Intracellular Proliferation of Salmonella typhimurium.

PubMed

Lee, Eun-Ju; Park, Kwan-Sik; Jeon, In-Sook; Choi, Jae-Woon; Lee, Sang-Jeon; Choy, Hyun E; Song, Ki-Duk; Lee, Hak-Kyo; Choi, Joong-Kook

2016-07-01

Lysosomes are cellular organelles containing diverse classes of catabolic enzymes that are implicated in diverse cellular processes including phagocytosis, autophagy, lipid transport, and aging. Lysosome-associated membrane proteins (LAMP-1 and LAMP-2) are major glycoproteins important for maintaining lysosomal integrity, pH, and catabolism. LAMP-1 and LAMP-2 are constitutively expressed in Salmonella-infected cells and are recruited to Salmonella-containing vacuoles (SCVs) as well as Salmonella-induced filaments (Sifs) that promote the survival and proliferation of the Salmonella. LAMP-3, also known as DC-LAMP/CD208, is a member of the LAMP family of proteins, but its role during Salmonella infection remains unclear. DNA microarray analysis identified LAMP-3 as one of the genes responding to LPS stimulation in THP-1 macrophage cells. Subsequent analyses reveal that LPS and Salmonella induced the expression of LAMP-3 at both the transcriptional and translational levels. Confocal Super resolution N-SIM imaging revealed that LAMP-3, like LAMP-2, shifts its localization from the cell surface to alongside Salmonella. Knockdown of LAMP-3 by specific siRNAs decreased the number of Salmonella recovered from the infected cells. Therefore, we conclude that LAMP-3 is induced by Salmonella infection and recruited to the Salmonella pathogen for intracellular proliferation.

LAMP-3 (Lysosome-Associated Membrane Protein 3) Promotes the Intracellular Proliferation of Salmonella typhimurium

PubMed Central

Lee, Eun-Ju; Park, Kwan-Sik; Jeon, In-Sook; Choi, Jae-Woon; Lee, Sang-Jeon; Choy, Hyun E.; Song, Ki-Duk; Lee, Hak-Kyo; Choi, Joong-Kook

2016-01-01

Lysosomes are cellular organelles containing diverse classes of catabolic enzymes that are implicated in diverse cellular processes including phagocytosis, autophagy, lipid transport, and aging. Lysosome-associated membrane proteins (LAMP-1 and LAMP-2) are major glycoproteins important for maintaining lysosomal integrity, pH, and catabolism. LAMP-1 and LAMP-2 are constitutively expressed in Salmonella-infected cells and are recruited to Salmonella-containing vacuoles (SCVs) as well as Salmonella-induced filaments (Sifs) that promote the survival and proliferation of the Salmonella. LAMP-3, also known as DC-LAMP/CD208, is a member of the LAMP family of proteins, but its role during Salmonella infection remains unclear. DNA microarray analysis identified LAMP-3 as one of the genes responding to LPS stimulation in THP-1 macrophage cells. Subsequent analyses reveal that LPS and Salmonella induced the expression of LAMP-3 at both the transcriptional and translational levels. Confocal Super resolution N-SIM imaging revealed that LAMP-3, like LAMP-2, shifts its localization from the cell surface to alongside Salmonella. Knockdown of LAMP-3 by specific siRNAs decreased the number of Salmonella recovered from the infected cells. Therefore, we conclude that LAMP-3 is induced by Salmonella infection and recruited to the Salmonella pathogen for intracellular proliferation. PMID:27329040

Mechanical Coupling of Smooth Muscle Cells Using Microengineered Substrates and Local Stimulation

NASA Astrophysics Data System (ADS)

Copeland, Craig; Hunter, David; Tung, Leslie; Chen, Christopher; Reich, Daniel

2013-03-01

Mechanical stresses directly affect many cellular processes, including signal transduction, growth, differentiation, and survival. Cells can themselves generate such stresses by activating myosin to contract the actin cytoskeleton, which in turn can regulate both cell-substrate and cell-cell interactions. We are studying mechanical forces at cell-cell and cell-substrate interactions using arrays of selectively patterned flexible PDMS microposts combined with the ability to apply local chemical stimulation. Micropipette ``spritzing'', a laminar flow technique, uses glass micropipettes mounted on a microscope stage to deliver drugs to controlled regions within a cellular construct while cell traction forces are recorded via the micropost array. The pipettes are controlled by micromanipulators allowing for rapid and precise movement across the array and the ability to treat multiple constructs within a sample. This technique allows for observing the propagation of a chemically induced mechanical stimulus through cell-cell and cell-substrate interactions. We have used this system to administer the acto-myosin inhibitors Blebbistatin and Y-27632 to single cells and observed the subsequent decrease in cell traction forces. Experiments using trypsin-EDTA have shown this system to be capable of single cell manipulation through removal of one cell within a pair configuration while leaving the other cell unaffected. This project is supported in part by NIH grant HL090747

Yeast Reporter Assay to Identify Cellular Components of Ricin Toxin A Chain Trafficking.

PubMed

Becker, Björn; Schnöder, Tina; Schmitt, Manfred J

2016-12-06

RTA, the catalytic A-subunit of the ribosome inactivating A/B toxin ricin, inhibits eukaryotic protein biosynthesis by depurination of 28S rRNA. Although cell surface binding of ricin holotoxin is mainly mediated through its B-subunit (RTB), sole application of RTA is also toxic, albeit to a significantly lower extent, suggesting alternative pathways for toxin uptake and transport. Since ricin toxin trafficking in mammalian cells is still not fully understood, we developed a GFP-based reporter assay in yeast that allows rapid identification of cellular components required for RTA uptake and subsequent transport through a target cell. We hereby show that Ypt6p, Sft2p and GARP-complex components play an important role in RTA transport, while neither the retromer complex nor COPIB vesicles are part of the transport machinery. Analyses of yeast knock-out mutants with chromosomal deletion in genes whose products regulate ADP-ribosylation factor GTPases (Arf-GTPases) and/or retrograde Golgi-to-ER (endoplasmic reticulum) transport identified Sso1p, Snc1p, Rer1p, Sec22p, Erv46p, Gea1p and Glo3p as novel components in RTA transport, suggesting the developed reporter assay as a powerful tool to dissect the multistep processes of host cell intoxication in yeast.

Uptake, sequestration and tolerance of cadmium at cellular levels in the hyperaccumulator plant species Sedum alfredii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Shengke; Xie, Ruohan; Wang, Haixin

Sedum alfredii is one of a few plant species known to hyperaccumulate cadmium (Cd). Uptake, localization, and tolerance of Cd at cellular levels in shoots were compared in hyperaccumulating (HE) and non-hyperaccumulating (NHE) ecotypes of Sedum alfredii. X-ray fluorescence images of Cd in stems and leaves showed only a slight Cd signal restricted within vascular bundles in the NHEs, while enhanced localization of Cd, with significant tissue- and age-dependent variations, was detected in HEs. In contrast to the vascular-enriched Cd in young stems, parenchyma cells in leaf mesophyll, stem pith and cortex tissues served as terminal storage sites for Cdmore » sequestration in HEs. Kinetics of Cd transport into individual leaf protoplasts of the two ecotypes showed little difference in Cd accumulation. However, far more efficient storage of Cd in vacuoles was apparent in HEs. Subsequent analysis of cell viability and hydrogen peroxide levels suggested that HE protoplasts exhibited higher resistance to Cd than those of NHE protoplasts. These results suggest that efficient sequestration into vacuoles, as opposed to rapid transport into parenchyma cells, is a pivotal process in Cd accumulation and homeostasis in shoots of HE S. alfredii. This is in addition to its efficient root-to-shoot translocation of Cd.« less

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke.

PubMed

Talley Watts, Lora; Zheng, Wei; Garling, R Justin; Frohlich, Victoria C; Lechleiter, James Donald

2015-06-23

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Virus reactivation: a panoramic view in human infections

PubMed Central

Traylen, Christopher M; Patel, Hersh R; Fondaw, Wylder; Mahatme, Sheran; Williams, John F; Walker, Lia R; Dyson, Ossie F; Arce, Sergio; Akula, Shaw M

2011-01-01

Viruses are obligate intracellular parasites, relying to a major extent on the host cell for replication. An active replication of the viral genome results in a lytic infection characterized by the release of new progeny virus particles, often upon the lysis of the host cell. Another mode of virus infection is the latent phase, where the virus is ‘quiescent’ (a state in which the virus is not replicating). A combination of these stages, where virus replication involves stages of both silent and productive infection without rapidly killing or even producing excessive damage to the host cells, falls under the umbrella of a persistent infection. Reactivation is the process by which a latent virus switches to a lytic phase of replication. Reactivation may be provoked by a combination of external and/or internal cellular stimuli. Understanding this mechanism is essential in developing future therapeutic agents against viral infection and subsequent disease. This article examines the published literature and current knowledge regarding the viral and cellular proteins that may play a role in viral reactivation. The focus of the article is on those viruses known to cause latent infections, which include herpes simplex virus, varicella zoster virus, Epstein–Barr virus, human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, Kaposi’s sarcoma-associated herpesvirus, JC virus, BK virus, parvovirus and adenovirus. PMID:21799704

NeuroCa: integrated framework for systematic analysis of spatiotemporal neuronal activity patterns from large-scale optical recording data

PubMed Central

Jang, Min Jee; Nam, Yoonkey

2015-01-01

Abstract. Optical recording facilitates monitoring the activity of a large neural network at the cellular scale, but the analysis and interpretation of the collected data remain challenging. Here, we present a MATLAB-based toolbox, named NeuroCa, for the automated processing and quantitative analysis of large-scale calcium imaging data. Our tool includes several computational algorithms to extract the calcium spike trains of individual neurons from the calcium imaging data in an automatic fashion. Two algorithms were developed to decompose the imaging data into the activity of individual cells and subsequently detect calcium spikes from each neuronal signal. Applying our method to dense networks in dissociated cultures, we were able to obtain the calcium spike trains of ∼1000 neurons in a few minutes. Further analyses using these data permitted the quantification of neuronal responses to chemical stimuli as well as functional mapping of spatiotemporal patterns in neuronal firing within the spontaneous, synchronous activity of a large network. These results demonstrate that our method not only automates time-consuming, labor-intensive tasks in the analysis of neural data obtained using optical recording techniques but also provides a systematic way to visualize and quantify the collective dynamics of a network in terms of its cellular elements. PMID:26229973

An Immature Myeloid/Myeloid-Suppressor Cell Response Associated with Necrotizing Inflammation Mediates Lethal Pulmonary Tularemia

PubMed Central

Periasamy, Sivakumar; Avram, Dorina; McCabe, Amanda; MacNamara, Katherine C.; Sellati, Timothy J.; Harton, Jonathan A.

2016-01-01

Inhalation of Francisella tularensis (Ft) causes acute and fatal pneumonia. The lung cytokine milieu favors exponential Ft replication, but the mechanisms underlying acute pathogenesis and death remain unknown. Evaluation of the sequential and systemic host immune response in pulmonary tularemia reveals that in contrast to overwhelming bacterial burden or cytokine production, an overt innate cellular response to Ft drives tissue pathology and host mortality. Lethal infection with Ft elicits medullary and extra-medullary myelopoiesis supporting recruitment of large numbers of immature myeloid cells and MDSC to the lungs. These cells fail to mature and die, leading to subsequent necrotic lung damage, loss of pulmonary function, and host death that is partially dependent upon immature Ly6G+ cells. Acceleration of this process may account for the rapid lethality seen with Ft SchuS4. In contrast, during sub-lethal infection with Ft LVS the pulmonary cellular response is characterized by a predominance of mature neutrophils and monocytes required for protection, suggesting a required threshold for lethal bacterial infection. Further, eliciting a mature phagocyte response provides transient, but dramatic, innate protection against Ft SchuS4. This study reveals that the nature of the myeloid cell response may be the primary determinant of host mortality versus survival following Francisella infection. PMID:27015566

Effects of short-term hypersalinity exposure on the susceptibility to wasting disease in the subtropical seagrass Thalassia testudinum.

PubMed

Trevathan, Stacey M; Kahn, Amanda; Ross, Cliff

2011-09-01

Seagrass meadows are a vital component of coastal ecosystems and have experienced declines in abundance due to a series of environmental stressors including elevated salinity and incidence of disease. This study evaluated the impacts of short-term hypersalinity stress on the early stages of infection in Thalassia testudinum Banks ex König by assessing changes in cellular physiology and metabolism. Seagrass short shoots were exposed to ambient (30 psu) and elevated (45 psu) salinities for 7 days and subsequently infected for one week by the causative pathogen of wasting disease, Labyrinthula sp. The occurrence of wasting disease was significantly lower in the hypersalinity treatments. Additionally, while exposure to elevated salinity caused a reduction in chlorophyll a and b content, T. testudinum's health, in terms of photochemical efficiency, was not significantly compromised by hypersalinity or infection. In contrast, plant respiratory demand was significantly enhanced as a function of infection. Elevated salinity caused T. testudinum to significantly increase its in vivo H(2)O(2) concentrations to levels that exceeded those which inhibited Labyrinthula growth in a liquid in vitro assay. The results suggest that while short-term exposure to hypersalinity alters selected cellular processes this does not necessarily lead to an immediate increase in wasting disease susceptibility. Published by Elsevier Masson SAS.

Comparative Genomics and Reverse Genetics Analysis Reveal Indispensable Functions of the Serine Acetyltransferase Gene Family in Arabidopsis[W][OA

PubMed Central

Watanabe, Mutsumi; Mochida, Keiichi; Kato, Tomohiko; Tabata, Satoshi; Yoshimoto, Naoko; Noji, Masaaki; Saito, Kazuki

2008-01-01

Ser acetyltransferase (SERAT), which catalyzes O-acetyl-Ser (OAS) formation, plays a key role in sulfur assimilation and Cys synthesis. Despite several studies on SERATs from various plant species, the in vivo function of multiple SERAT genes in plant cells remains unaddressed. Comparative genomics studies with the five genes of the SERAT gene family in Arabidopsis thaliana indicated that all three Arabidopsis SERAT subfamilies are conserved across five plant species with available genome sequences. Single and multiple knockout mutants of all Arabidopsis SERAT gene family members were analyzed. All five quadruple mutants with a single gene survived, with three mutants showing dwarfism. However, the quintuple mutant lacking all SERAT genes was embryo-lethal. Thus, all five isoforms show functional redundancy in vivo. The developmental and compartment-specific roles of each SERAT isoform were also demonstrated. Mitochondrial SERAT2;2 plays a predominant role in cellular OAS formation, while plastidic SERAT2;1 contributes less to OAS formation and subsequent Cys synthesis. Three cytosolic isoforms, SERAT1;1, SERAT3;1, and SERAT3;2, may play a major role during seed development. Thus, the evolutionally conserved SERAT gene family is essential in cellular processes, and the substrates and products of SERAT must be exchangeable between the cytosol and organelles. PMID:18776059

A study of adsorption behavior of human serum albumin and ovalbumin on hydroxyapatite/chitosan composite.

PubMed

Xu, Chao; He, Deliang; Zeng, Liping; Luo, Shenglian

2009-10-15

In situ adsorption of human serum albumin (HSA) and ovalbumin (OVA) was real-time monitored by piezoelectric quartz crystal impedance (PQCI) technique to fully understand the initial cellular response on hydroxyapatite/chitosan (HAP/CS) composite. The PQCI parameters, such as resonant frequency (f), static capacitance (C(s)), and motional resistance (R(m)) were measured for investigating the kinetic adsorption behaviors of both proteins. The change in frequency shifts (Deltaf) depends on the amount of the adsorbed protein, and the change in motional resistance (DeltaR(m)) results from the microporosity variation of HAP/CS coating. The results show that the amount of the absorbed HSA is much greater than that of OVA on HAP/CS coating because of the unique construction of HSA as well as a flexible protein. Furthermore, Deltaf and DeltaR(m) data were fitted according to the kinetic exponential decay equations. It can be seen that there is only one adsorption process for OVA, but the absorption process for HSA is followed by a rearrangement process, and the former process is faster than the rearrangement process. Subsequently, the composite binding with proteins were demonstrated by the Fourier transform infrared (FTIR), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS).

Selenium and selenocysteine: roles in cancer, health and development

PubMed Central

Hatfield, Dolph L.; Tsuji, Petra A.; Carlson, Bradley A.; Gladyshev, Vadim N.

2014-01-01

The many biological and biomedical effects of selenium are relatively unknown outside the selenium field. This fascinating element, initially described as a toxin, was subsequently shown to be essential for health and development. By the mid 1990s, selenium emerged as one of the most promising cancer chemopreventive agents, but subsequent human clinical trials yielded contradictory results. However, basic research on selenium continued to move at a rapid pace elucidating its many roles in health, development, and cancer prevention and promotion. Dietary selenium acts principally through selenoproteins, most of which are oxidoreductases involved in diverse cellular functions. PMID:24485058

Fgf3 and Fgf10a work in concert to promote maturation of the epibranchial placodes in zebrafish.

PubMed

McCarroll, Matthew N; Nechiporuk, Alex V

2013-01-01

Essential cellular components of the paired sensory organs of the vertebrate head are derived from transient thickenings of embryonic ectoderm known as cranial placodes. The epibranchial (EB) placodes give rise to sensory neurons of the EB ganglia that are responsible for relaying visceral sensations form the periphery to the central nervous system. Development of EB placodes and subsequent formation of EB ganglia is a multistep process regulated by various extrinsic factors, including fibroblast growth factors (Fgfs). We discovered that two Fgf ligands, Fgf3 and Fgf10a, cooperate to promote EB placode development. Whereas EB placodes are induced in the absence of Fgf3 and Fgf10a, they fail to express placode specific markers Pax2a and Sox3. Expression analysis and mosaic rescue experiments demonstrate that Fgf3 signal is derived from the endoderm, whereas Fgf10a is emitted from the lateral line system and the otic placode. Further analyses revealed that Fgf3 and Fgf10a activities are not required for cell proliferation or survival, but are required for placodal cells to undergo neurogenesis. Based on these data, we conclude that a combined loss of these Fgf factors results in a failure of the EB placode precursors to initiate a transcriptional program needed for maturation and subsequent neurogenesis. These findings highlight the importance and complexity of reiterated Fgf signaling during cranial placode formation and subsequent sensory organ development.

Fgf3 and Fgf10a Work in Concert to Promote Maturation of the Epibranchial Placodes in Zebrafish

PubMed Central

McCarroll, Matthew N.; Nechiporuk, Alex V.

2013-01-01

Essential cellular components of the paired sensory organs of the vertebrate head are derived from transient thickenings of embryonic ectoderm known as cranial placodes. The epibranchial (EB) placodes give rise to sensory neurons of the EB ganglia that are responsible for relaying visceral sensations form the periphery to the central nervous system. Development of EB placodes and subsequent formation of EB ganglia is a multistep process regulated by various extrinsic factors, including fibroblast growth factors (Fgfs). We discovered that two Fgf ligands, Fgf3 and Fgf10a, cooperate to promote EB placode development. Whereas EB placodes are induced in the absence of Fgf3 and Fgf10a, they fail to express placode specific markers Pax2a and Sox3. Expression analysis and mosaic rescue experiments demonstrate that Fgf3 signal is derived from the endoderm, whereas Fgf10a is emitted from the lateral line system and the otic placode. Further analyses revealed that Fgf3 and Fgf10a activities are not required for cell proliferation or survival, but are required for placodal cells to undergo neurogenesis. Based on these data, we conclude that a combined loss of these Fgf factors results in a failure of the EB placode precursors to initiate a transcriptional program needed for maturation and subsequent neurogenesis. These findings highlight the importance and complexity of reiterated Fgf signaling during cranial placode formation and subsequent sensory organ development. PMID:24358375

Environmentally Induced Epigenetic Transgenerational Inheritance of Altered Sertoli Cell Transcriptome and Epigenome: Molecular Etiology of Male Infertility

PubMed Central

Guerrero-Bosagna, Carlos; Savenkova, Marina; Haque, Md. Muksitul; Nilsson, Eric; Skinner, Michael K.

2013-01-01

Environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of adult onset disease, including testis disease and male infertility. The current study was designed to determine the impact of an altered sperm epigenome on the subsequent development of an adult somatic cell (Sertoli cell) that influences the onset of a specific disease (male infertility). A gestating female rat (F0 generation) was exposed to the agriculture fungicide vinclozolin during gonadal sex determination and then the subsequent F3 generation progeny used for the isolation of Sertoli cells and assessment of testis disease. As previously observed, enhanced spermatogenic cell apoptosis was observed. The Sertoli cells provide the physical and nutritional support for the spermatogenic cells. Over 400 genes were differentially expressed in the F3 generation control versus vinclozolin lineage Sertoli cells. A number of specific cellular pathways were identified to be transgenerationally altered. One of the key metabolic processes affected was pyruvate/lactate production that is directly linked to spermatogenic cell viability. The Sertoli cell epigenome was also altered with over 100 promoter differential DNA methylation regions (DMR) modified. The genomic features and overlap with the sperm DMR were investigated. Observations demonstrate that the transgenerational sperm epigenetic alterations subsequently alters the development of a specific somatic cell (Sertoli cell) epigenome and transcriptome that correlates with adult onset disease (male infertility). The environmentally induced epigenetic transgenerational inheritance of testis disease appears to be a component of the molecular etiology of male infertility. PMID:23555832

Computational challenges in modeling gene regulatory events.

PubMed

Pataskar, Abhijeet; Tiwari, Vijay K

2016-10-19

Cellular transcriptional programs driven by genetic and epigenetic mechanisms could be better understood by integrating "omics" data and subsequently modeling the gene-regulatory events. Toward this end, computational biology should keep pace with evolving experimental procedures and data availability. This article gives an exemplified account of the current computational challenges in molecular biology.

Melt-processed polymeric cellular dosage forms for immediate drug release.

PubMed

Blaesi, Aron H; Saka, Nannaji

2015-12-28

The present immediate-release solid dosage forms, such as the oral tablets and capsules, comprise granular matrices. While effective in releasing the drug rapidly, they are fraught with difficulties inherent in processing particulate matter. By contrast, liquid-based processes would be far more predictable; but the standard cast microstructures are unsuited for immediate-release because they resist fluid percolation and penetration. In this article, we introduce cellular dosage forms that can be readily prepared from polymeric melts by incorporating the nucleation, growth, and coalescence of microscopic gas bubbles in a molding process. We show that the cell topology and formulation of such cellular structures can be engineered to reduce the length-scale of the mass-transfer step, which determines the time of drug release, from as large as the dosage form itself to as small as the thickness of the cell wall. This allows the cellular dosage forms to achieve drug release rates over an order of magnitude faster compared with those of cast matrices, spanning the entire spectrum of immediate-release and beyond. The melt-processed polymeric cellular dosage forms enable predictive design of immediate-release solid dosage forms by tailoring microstructures, and could be manufactured efficiently in a single step.

A Simple Microscopy Assay to Teach the Processes of Phagocytosis and Exocytosis

ERIC Educational Resources Information Center

Gray, Ross; Gray, Andrew; Fite, Jessica L.; Jordan, Renee; Stark, Sarah; Naylor, Kari

2012-01-01

Phagocytosis and exocytosis are two cellular processes involving membrane dynamics. While it is easy to understand the purpose of these processes, it can be extremely difficult for students to comprehend the actual mechanisms. As membrane dynamics play a significant role in many cellular processes ranging from cell signaling to cell division to…

Biotechnologies as a Context for Enhancing Junior High-School Students' Ability to Ask Meaningful Questions about Abstract Biological Processes.

ERIC Educational Resources Information Center

Olsher, G.; Dreyfus, A.

1999-01-01

Suggests a new approach to teaching about biochemical cellular processes by stimulating student interest in those biochemical processes that allowed for the outcomes of modern biotechnologies. Discusses the development of students' ability to ask meaningful questions about intra-cellular processes, and the resulting meaningful learning of relevant…

Cellular context-dependent consequences of Apc mutations on gene regulation and cellular behavior.

PubMed

Hashimoto, Kyoichi; Yamada, Yosuke; Semi, Katsunori; Yagi, Masaki; Tanaka, Akito; Itakura, Fumiaki; Aoki, Hitomi; Kunisada, Takahiro; Woltjen, Knut; Haga, Hironori; Sakai, Yoshiharu; Yamamoto, Takuya; Yamada, Yasuhiro

2017-01-24

The spectrum of genetic mutations differs among cancers in different organs, implying a cellular context-dependent effect for genetic aberrations. However, the extent to which the cellular context affects the consequences of oncogenic mutations remains to be fully elucidated. We reprogrammed colon tumor cells in an Apc Min/+ (adenomatous polyposis coli) mouse model, in which the loss of the Apc gene plays a critical role in tumor development and subsequently, established reprogrammed tumor cells (RTCs) that exhibit pluripotent stem cell (PSC)-like signatures of gene expression. We show that the majority of the genes in RTCs that were affected by Apc mutations did not overlap with the genes affected in the intestine. RTCs lacked pluripotency but exhibited an increased expression of Cdx2 and a differentiation propensity that was biased toward the trophectoderm cell lineage. Genetic rescue of the mutated Apc allele conferred pluripotency on RTCs and enabled their differentiation into various cell types in vivo. The redisruption of Apc in RTC-derived differentiated cells resulted in neoplastic growth that was exclusive to the intestine, but the majority of the intestinal lesions remained as pretumoral microadenomas. These results highlight the significant influence of cellular context on gene regulation, cellular plasticity, and cellular behavior in response to the loss of the Apc function. Our results also imply that the transition from microadenomas to macroscopic tumors is reprogrammable, which underscores the importance of epigenetic regulation on tumor promotion.

Cellular context-dependent consequences of Apc mutations on gene regulation and cellular behavior

PubMed Central

Hashimoto, Kyoichi; Yamada, Yosuke; Semi, Katsunori; Yagi, Masaki; Tanaka, Akito; Itakura, Fumiaki; Aoki, Hitomi; Kunisada, Takahiro; Woltjen, Knut; Haga, Hironori; Sakai, Yoshiharu; Yamamoto, Takuya; Yamada, Yasuhiro

2017-01-01

The spectrum of genetic mutations differs among cancers in different organs, implying a cellular context-dependent effect for genetic aberrations. However, the extent to which the cellular context affects the consequences of oncogenic mutations remains to be fully elucidated. We reprogrammed colon tumor cells in an ApcMin/+ (adenomatous polyposis coli) mouse model, in which the loss of the Apc gene plays a critical role in tumor development and subsequently, established reprogrammed tumor cells (RTCs) that exhibit pluripotent stem cell (PSC)-like signatures of gene expression. We show that the majority of the genes in RTCs that were affected by Apc mutations did not overlap with the genes affected in the intestine. RTCs lacked pluripotency but exhibited an increased expression of Cdx2 and a differentiation propensity that was biased toward the trophectoderm cell lineage. Genetic rescue of the mutated Apc allele conferred pluripotency on RTCs and enabled their differentiation into various cell types in vivo. The redisruption of Apc in RTC-derived differentiated cells resulted in neoplastic growth that was exclusive to the intestine, but the majority of the intestinal lesions remained as pretumoral microadenomas. These results highlight the significant influence of cellular context on gene regulation, cellular plasticity, and cellular behavior in response to the loss of the Apc function. Our results also imply that the transition from microadenomas to macroscopic tumors is reprogrammable, which underscores the importance of epigenetic regulation on tumor promotion. PMID:28057861

Mitochondrial correlates of signaling processes involved with the cellular response to eimeria infection in broiler chickens

USDA-ARS?s Scientific Manuscript database

Host cellular responses to coccidiosis infection are consistent with elements of apoptosis, autophagy, and necrosis. These processes are enhanced in the cell through cell-directed signaling or repressed through parasite-derived inhibitors of these processes favoring the survival of the parasite. Acr...

Methods and Devices for Micro-Isolation, Extraction, and/or Analysis of Microscale Components

NASA Technical Reports Server (NTRS)

Wade, Lawrence A. (Inventor); Kartalov, Emil P. (Inventor); Taylor, Clive (Inventor); Shibata, Darryl (Inventor)

2014-01-01

Provided herein are devices and methods for the micro-isolation of biological cellular material. A micro-isolation apparatus described can comprise a photomask that protects regions of interest against DNA-destroying illumination. The micro-isolation apparatus can further comprise photosensitive material defining access wells following illumination and subsequent developing of the photosensitive material. The micro-isolation apparatus can further comprise a chambered microfluidic device comprising channels providing access to wells defined in photosensitive material. The micro-isolation apparatus can comprise a chambered microfluidic device without access wells defined in photosensitive material where valves control the flow of gases or liquids through the channels of the microfluidic device. Also included are methods for selectively isolating cellular material using the apparatuses described herein, as are methods for biochemical analysis of individual regions of interest of cellular material using the devices described herein. Further included are methods of making masking arrays useful for the methods described herein.

Coated foams, preparation, uses and articles

DOEpatents

Duchane, D.V.; Barthell, B.L.

1982-10-21

Hydrophobic cellular material is coated with a thin hydrophilic polymer skin which stretches tightly over the foam but which does not fill the cells of the foam, thus resulting in a polymer-coated foam structure having a smoothness which was not possible in the prior art. In particular, when the hydrophobic cellular material is a specially chosen hydrophobic polymer foam and is formed into arbitrarily chosen shapes prior to the coating with hydrophilic polymer, inertial confinement fusion (ICF) targets of arbitrary shapes can be produced by subsequently coating the shapes with metal or with any other suitable material. New articles of manufacture are produced, including improved ICF targets, improved integrated circuits, and improved solar reflectors and solar collectors. In the coating method, the cell size of the hydrophobic cellular material, the viscosity of the polymer solution used to coat, and the surface tension of the polymer solution used to coat are all very important to the coating.

Selective Cytotoxicity of Rhodium Metalloinsertors in Mismatch Repair-Deficient Cells†

PubMed Central

Ernst, Russell J.; Komor, Alexis C.; Barton, Jacqueline K.

2011-01-01

Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. Rhodium metalloinsertors bind to DNA mismatches with high affinity and specificity and represent a promising strategy to target mismatches in cells. Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands in cells deficient in MMR versus those that are MMR-proficient. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents. PMID:22103240

Autophagosome and phagosome.

PubMed

Deretic, Vojo

2008-01-01

Autophagy and phagocytosis are evolutionarily ancient processes functioning in capture and digestion of material found in the cellular interior and exterior, respectively. In their most primordial form, both processes are involved in cellular metabolism and feeding, supplying cells with externally obtained particulate nutrients or using portions of cell's own cytoplasm to generate essential nutrients and energy at times of starvation. Although autophagy and phagocytosis are commonly treated as completely separate biological phenomena, they are topologically similar and can be, at least morphologically, viewed as different manifestations of a spectrum of related processes. Autophagy is the process of sequestering portions of cellular interior (cytosol and intracellular organelles) into a membranous organelle (autophagosome), whereas phagocystosis is its topological equivalent engaged in sequestering cellular exterior. Both autophagosomes and phagosomes mature into acidified, degradative organelles, termed autolysosomes and phagolysosomes, respectively. The basic role of autophagy as a nutritional process, and that of phagocytosis where applicable, has survived in present-day organisms ranging from yeast to man. It has in addition evolved into a variety of specialized processes in metazoans, with a major role in cellular/cytoplasmic homeostasis. In humans, autophagy has been implicated in many health and disease states, including cancer, neurodegeneration, aging and immunity, while phagocytosis plays a role in immunity and tissue homeostasis. Autophagy and phagocytosis cooperate in the latter two processes. In this chapter, we briefly review the regulatory and execution stages of both autophagy and phagocytosis.

Light Weight Biomorphous Cellular Ceramics from Cellulose Templates

NASA Technical Reports Server (NTRS)

Singh, Mrityunjay; Yee, Bo-Moon; Gray, Hugh R. (Technical Monitor)

2003-01-01

Bimorphous ceramics are a new class of materials that can be fabricated from the cellulose templates derived from natural biopolymers. These biopolymers are abundantly available in nature and are produced by the photosynthesis process. The wood cellulose derived carbon templates have three- dimensional interconnectivity. A wide variety of non-oxide and oxide based ceramics have been fabricated by template conversion using infiltration and reaction-based processes. The cellular anatomy of the cellulose templates plays a key role in determining the processing parameters (pyrolysis, infiltration conditions, etc.) and resulting ceramic materials. The processing approach, microstructure, and mechanical properties of the biomorphous cellular ceramics (silicon carbide and oxide based) have been discussed.

Model-based design of experiments for cellular processes.

PubMed

Chakrabarty, Ankush; Buzzard, Gregery T; Rundell, Ann E

2013-01-01

Model-based design of experiments (MBDOE) assists in the planning of highly effective and efficient experiments. Although the foundations of this field are well-established, the application of these techniques to understand cellular processes is a fertile and rapidly advancing area as the community seeks to understand ever more complex cellular processes and systems. This review discusses the MBDOE paradigm along with applications and challenges within the context of cellular processes and systems. It also provides a brief tutorial on Fisher information matrix (FIM)-based and Bayesian experiment design methods along with an overview of existing software packages and computational advances that support MBDOE application and adoption within the Systems Biology community. As cell-based products and biologics progress into the commercial sector, it is anticipated that MBDOE will become an essential practice for design, quality control, and production. Copyright © 2013 Wiley Periodicals, Inc.

SIRTUIN 1 AND SIRTUIN 3: PHYSIOLOGICAL MODULATORS OF METABOLISM

PubMed Central

Nogueiras, Ruben; Habegger, Kirk M.; Chaudhary, Nilika; Finan, Brian; Banks, Alexander S.; Dietrich, Marcelo O.; Horvath, Tamas L.; Sinclair, David A.; Pfluger, Paul T.; Tschöop, Matthias H.

2013-01-01

The sirtuins are a family of highly conserved NAD+-dependent deacetylases that act as cellular sensors to detect energy availability and modulate metabolic processes. Two sirtuins that are central to the control of metabolic processes are mammalian sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3), which are localized to the nucleus and mitochondria, respectively. Both are activated by high NAD+ levels, a condition caused by low cellular energy status. By deacetylating a variety of proteins that induce catabolic processes while inhibiting anabolic processes, SIRT1 and SIRT3 coordinately increase cellular energy stores and ultimately maintain cellular energy homeostasis. Defects in the pathways controlled by SIRT1 and SIRT3 are known to result in various metabolic disorders. Consequently, activation of sirtuins by genetic or pharmacological means can elicit multiple metabolic benefits that protect mice from diet-induced obesity, type 2 diabetes, and nonalcoholic fatty liver disease. PMID:22811431

RNA-Binding Protein FXR1 Regulates p21 and TERC RNA to Bypass p53-Mediated Cellular Senescence in OSCC

PubMed Central

Majumder, Mrinmoyee; House, Reniqua; Palanisamy, Nallasivam; Qie, Shuo; Day, Terrence A.; Neskey, David; Diehl, J. Alan

2016-01-01

RNA-binding proteins (RBP) regulate numerous aspects of co- and post-transcriptional gene expression in cancer cells. Here, we demonstrate that RBP, fragile X-related protein 1 (FXR1), plays an essential role in cellular senescence by utilizing mRNA turnover pathway. We report that overexpressed FXR1 in head and neck squamous cell carcinoma targets (G-quadruplex (G4) RNA structure within) both mRNA encoding p21 (Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A, Cip1) and the non-coding RNA Telomerase RNA Component (TERC), and regulates their turnover to avoid senescence. Silencing of FXR1 in cancer cells triggers the activation of Cyclin-Dependent Kinase Inhibitors, p53, increases DNA damage, and ultimately, cellular senescence. Overexpressed FXR1 binds and destabilizes p21 mRNA, subsequently reduces p21 protein expression in oral cancer cells. In addition, FXR1 also binds and stabilizes TERC RNA and suppresses the cellular senescence possibly through telomerase activity. Finally, we report that FXR1-regulated senescence is irreversible and FXR1-depleted cells fail to form colonies to re-enter cellular proliferation. Collectively, FXR1 displays a novel mechanism of controlling the expression of p21 through p53-dependent manner to bypass cellular senescence in oral cancer cells. PMID:27606879

Cementocyte cell death occurs in rat cellular cementum during orthodontic tooth movement.

PubMed

Matsuzawa, Humihiro; Toriya, Naoko; Nakao, Yuya; Konno-Nagasaka, Moe; Arakawa, Toshiya; Okayama, Miki; Mizoguchi, Itaru

2017-05-01

To clarify the mechanism of root resorption during orthodontic treatment, we examined cementocyte cell death and root resorption in the cellular cementum on the pressure side during experimental tooth movement. Using 8-week-old male Wistar rats, the right first molar was pushed mesiobuccally with a force of 40 g by a Ni-Ti alloy wire while the contralateral first molar was used as a control. Localization and number of cleaved caspase-3-positive and single-stranded DNA (ssDNA) - positive cells were evaluated using dual-label immunohistochemistry with anticleaved caspase-3 and anti-ssDNA antibodies. In addition, tartrate-resistant acid phosphatase (TRAP)-positive cells in the cellular cementum were evaluated using TRAP histochemical staining. Caspase-3- and ssDNA-positive cells appeared at 12 hours, but were restricted to the compressed periodontal ligament (PDL) and not the cellular cementum. Cleaved caspase-3-positive cementocytes were observed in the cellular cementum adjacent to the compressed PDL on day 1. From days 2 to 4, the number of caspase-3- and ssDNA-positive cementocytes increased. TRAP-positive cells appeared on the cellular cementum at the periphery of the hyalinized tissue on day 7, and resorption progressed into the broad surface of the cementum by day 14. Cementocytes adjacent to the hyalinized tissue underwent apoptotic cell death during orthodontic tooth movement, which might have been associated with subsequent root resorption.

A Novel Pattern of Yolk Processing in Developing Snake Eggs (Colubridae: Lampropeltini) and its Functional and Evolutionary Implications.

PubMed

Powers, Kathryn G; Blackburn, Daniel G

2017-07-01

Early amniotic vertebrates evolved large-yolked eggs that permitted production of well-developed, terrestrial hatchlings. This reproductive pattern required new mechanisms for cellularizing the yolk and mobilizing it for embryonic use. In birds, cells that line the yolk sac cavity phagocytose and digest the yolk material, a pattern that is commonly assumed to be universal among oviparous amniotes. However, recent evidence challenges the assumption that all squamate reptiles conform to the avian developmental pattern. In this paper, scanning electron microscopy and histology were used to study mechanisms of yolk processing in two colubrid snakes, the kingsnake Lampropeltis getula and the milksnake L. triangulum. Endodermal cells from the yolk sac splanchnopleure proliferate massively as they invade the yolk sac cavity, forming elaborate chains of interlinked cells. These cells grow in size as they phagocytose yolk material. Subsequently, vitelline capillaries invade the masses of yolk-laden cells and become coated with the endodermal cells, forming an elaborate meshwork of cell-coated strands. The close association of cells, yolk, and blood vessels allows yolk material to be cellularized, digested, and transported for embryonic use. The overall pattern is like that of the corn snake Pantherophis guttatus, but contrasts markedly with that of birds. Given recent evidence that this developmental pattern may also occur in certain lizards, we postulate that it is ancestral for squamates. Studies of lizards, crocodilians, and turtles are needed to clarify the evolutionary history of this pattern and its implications for the evolution of the amniotic (terrestrial) vertebrate egg. © 2017 Wiley Periodicals, Inc.

Adipose, Bone, and Myeloma: Contributions from the Microenvironment.

PubMed

McDonald, Michelle M; Fairfield, Heather; Falank, Carolyne; Reagan, Michaela R

2017-05-01

Researchers globally are working towards finding a cure for multiple myeloma (MM), a destructive blood cancer diagnosed yearly in ~750,000 people worldwide (Podar et al. in Expert Opin Emerg Drugs 14:99-127, 2009). Although MM targets multiple organ systems, it is the devastating skeletal destruction experienced by over 90 % of patients that often most severely impacts patient morbidity, pain, and quality of life. Preventing bone disease is therefore a priority in MM treatment, and understanding how and why myeloma cells target the bone marrow (BM) is fundamental to this process. This review focuses on a key area of MM research: the contributions of the bone microenvironment to disease origins, progression, and drug resistance. We describe some of the key cell types in the BM niche: osteoclasts, osteoblasts, osteocytes, adipocytes, and mesenchymal stem cells. We then focus on how these key cellular players are, or could be, regulating a range of disease-related processes spanning MM growth, drug resistance, and bone disease (including osteolysis, fracture, and hypercalcemia). We summarize the literature regarding MM-bone cell and MM-adipocyte relationships and subsequent phenotypic changes or adaptations in MM cells, with the aim of providing a deeper understanding of how myeloma cells grow in the skeleton to cause bone destruction. We identify avenues and therapies that intervene in these networks to stop tumor growth and/or induce bone regeneration. Overall, we aim to illustrate how novel therapeutic target molecules, proteins, and cellular mediators may offer new avenues to attack this disease while reviewing currently utilized therapies.

Metabolic Conversion of Ceramides in HeLa Cells - A Cholesteryl Phosphocholine Delivery Approach

PubMed Central

Kjellberg, Matti A.; Lönnfors, Max; Slotte, J. Peter; Mattjus, Peter

2015-01-01

Ceramides can be delivered to cultured cells without solvents in the form of complexes with cholesteryl phosphocholine. We have analysed the delivery of three different radiolabeled D-erythro-ceramides (C6-Cer, C10-Cer and C16-Cer) to HeLa cells, and followed their metabolism as well as the cell viability. We found that all three ceramides were successfully taken up by HeLa cells when complexed to CholPC in an equimolar ratio, and show that the ceramides show different rates of cellular uptake and metabolic fate. The C6-Cer had the highest incorporation rate, followed by C10-Cer and C16-Cer, respectively. The subsequent effect on cell viability strongly correlated with the rate of incorporation, where C6-Cer had the strongest apoptotic effects. Low-dose (1 μM) treatment with C6-Cer favoured conversion of the precursor to sphingomyelin, whereas higher concentrations (25–100 μM) yielded increased conversion to C6-glucosylceramide. Similar results were obtained for C10-Cer. In the lower-dose C16-Cer experiments, most of the precursor was degraded, whereas at high-dose concentrations the precursor remained un-metabolized. Using this method, we demonstrate that ceramides with different chain lengths clearly exhibit varying rates of cellular uptake. The cellular fate of the externally delivered ceramides are clearly connected to their rate of incorporation and their subsequent effects on cell viability may be in part determined by their chain length. PMID:26599810

A multi-stage process including transient polyploidization and EMT precedes the emergence of chemoresistent ovarian carcinoma cells with a dedifferentiated and pro-inflammatory secretory phenotype.

PubMed

Rohnalter, Verena; Roth, Katrin; Finkernagel, Florian; Adhikary, Till; Obert, Julia; Dorzweiler, Kristina; Bensberg, Maike; Müller-Brüsselbach, Sabine; Müller, Rolf

2015-11-24

DNA-damaging drugs induce a plethora of molecular and cellular alterations in tumor cells, but their interrelationship is largely obscure. Here, we show that carboplatin treatment of human ovarian carcinoma SKOV3 cells triggers an ordered sequence of events, which precedes the emergence of mitotic chemoresistant cells. The initial phase of cell death after initiation of carboplatin treatment is followed around day 14 by the emergence of a mixed cell population consisting of cycling, cell cycle-arrested and senescent cells. At this stage, giant cells make up >80% of the cell population, p21 (CDKN1A) in strongly induced, and cell numbers remain nearly static. Subsequently, cell death decreases, p21 expression drops to a low level and cell divisions increase, including regular mitoses of giant cells and depolyploidization by multi-daughter divisions. These events are accompanied by the upregulation of stemness markers and a pro-inflammatory secretory phenotype, peaking after approximately 14 days of treatment. At the same time the cells initiate epithelial to mesenchymal transition, which over the subsequent weeks continuously increases, concomitantly with the emergence of highly proliferative, migratory, dedifferentiated, pro-inflammatory and chemoresistant cells (SKOV3-R). These cells are anchorage-independent and grow in a 3D collagen matrix, while cells on day 14 do not survive under these conditions, indicating that SKOV3-R cells were generated thereafter by the multi-stage process described above. This process was essentially recapitulated with the ovarian carcinoma cell line IGROV-1. Our observations suggest that transitory cells characterized by polyploidy, features of stemness and a pro-inflammatory secretory phenotype contribute to the acquisition of chemoresistance.

DNA repair efficiency in germ cells and early mouse embryos and consequences for radiation-induced transgenerational genomic damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marchetti, Francesco; Wyrobek, Andrew J.

Exposure to ionizing radiation and other environmental agents can affect the genomic integrity of germ cells and induce adverse health effects in the progeny. Efficient DNA repair during gametogenesis and the early embryonic cycles after fertilization is critical for preventing transmission of DNA damage to the progeny and relies on maternal factors stored in the egg before fertilization. The ability of the maternal repair machinery to repair DNA damage in both parental genomes in the fertilizing egg is especially crucial for the fertilizing male genome that has not experienced a DNA repair-competent cellular environment for several weeks prior to fertilization.more » During the DNA repair-deficient period of spermatogenesis, DNA lesions may accumulate in sperm and be carried into the egg where, if not properly repaired, could result in the formation of heritable chromosomal aberrations or mutations and associated birth defects. Studies with female mice deficient in specific DNA repair genes have shown that: (i) cell cycle checkpoints are activated in the fertilized egg by DNA damage carried by the sperm; and (ii) the maternal genotype plays a major role in determining the efficiency of repairing genomic lesions in the fertilizing sperm and directly affect the risk for abnormal reproductive outcomes. There is also growing evidence that implicates DNA damage carried by the fertilizing gamete as a mediator of postfertilization processes that contribute to genomic instability in subsequent generations. Transgenerational genomic instability most likely involves epigenetic mechanisms or error-prone DNA repair processes in the early embryo. Maternal and embryonic DNA repair processes during the early phases of mammalian embryonic development can have far reaching consequences for the genomic integrity and health of subsequent generations.« less

A multi-stage process including transient polyploidization and EMT precedes the emergence of chemoresistent ovarian carcinoma cells with a dedifferentiated and pro-inflammatory secretory phenotype

PubMed Central

Rohnalter, Verena; Roth, Katrin; Finkernagel, Florian; Adhikary, Till; Obert, Julia; Dorzweiler, Kristina; Bensberg, Maike; Müller-Brüsselbach, Sabine; Müller, Rolf

2015-01-01

DNA-damaging drugs induce a plethora of molecular and cellular alterations in tumor cells, but their interrelationship is largely obscure. Here, we show that carboplatin treatment of human ovarian carcinoma SKOV3 cells triggers an ordered sequence of events, which precedes the emergence of mitotic chemoresistant cells. The initial phase of cell death after initiation of carboplatin treatment is followed around day 14 by the emergence of a mixed cell population consisting of cycling, cell cycle-arrested and senescent cells. At this stage, giant cells make up >80% of the cell population, p21 (CDKN1A) in strongly induced, and cell numbers remain nearly static. Subsequently, cell death decreases, p21 expression drops to a low level and cell divisions increase, including regular mitoses of giant cells and depolyploidization by multi-daughter divisions. These events are accompanied by the upregulation of stemness markers and a pro-inflammatory secretory phenotype, peaking after approximately 14 days of treatment. At the same time the cells initiate epithelial to mesenchymal transition, which over the subsequent weeks continuously increases, concomitantly with the emergence of highly proliferative, migratory, dedifferentiated, pro-inflammatory and chemoresistant cells (SKOV3-R). These cells are anchorage-independent and grow in a 3D collagen matrix, while cells on day 14 do not survive under these conditions, indicating that SKOV3-R cells were generated thereafter by the multi-stage process described above. This process was essentially recapitulated with the ovarian carcinoma cell line IGROV-1. Our observations suggest that transitory cells characterized by polyploidy, features of stemness and a pro-inflammatory secretory phenotype contribute to the acquisition of chemoresistance. PMID:26503466

AGCVIII Kinases: at the crossroads of cellular signaling

USDA-ARS?s Scientific Manuscript database

AGCVIII kinases regulate diverse developmental and cellular processes in plants. As putative mediators of secondary messengers, AGCVIII kinases potentially integrate developmental and environmental cues into specific cellular responses through substrate phosphorylation. Here we discuss the functiona...

Differential growth of wrinkled biofilms

NASA Astrophysics Data System (ADS)

Espeso, D. R.; Carpio, A.; Einarsson, B.

2015-02-01

Biofilms are antibiotic-resistant bacterial aggregates that grow on moist surfaces and can trigger hospital-acquired infections. They provide a classical example in biology where the dynamics of cellular communities may be observed and studied. Gene expression regulates cell division and differentiation, which affect the biofilm architecture. Mechanical and chemical processes shape the resulting structure. We gain insight into the interplay between cellular and mechanical processes during biofilm development on air-agar interfaces by means of a hybrid model. Cellular behavior is governed by stochastic rules informed by a cascade of concentration fields for nutrients, waste, and autoinducers. Cellular differentiation and death alter the structure and the mechanical properties of the biofilm, which is deformed according to Föppl-Von Kármán equations informed by cellular processes and the interaction with the substratum. Stiffness gradients due to growth and swelling produce wrinkle branching. We are able to reproduce wrinkled structures often formed by biofilms on air-agar interfaces, as well as spatial distributions of differentiated cells commonly observed with B. subtilis.

Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries.

PubMed

Stoner, Gary David; Wang, Li-Shu; Casto, Bruce Cordell

2008-09-01

Reactive oxygen species (ROS) are a major cause of cellular injury in an increasing number of diseases, including cancer. Most ROS are created in the cell through normal cellular metabolism. They can be produced by environmental insults such as ultraviolet light and toxic chemicals, as well as by the inflammatory process. Interception of ROS or limiting their cellular effects is a major role of antioxidants. Due to their content of phenolic and flavonoid compounds, berries exhibit high antioxidant potential, exceeding that of many other foodstuffs. Through their ability to scavenge ROS and reduce oxidative DNA damage, stimulate antioxidant enzymes, inhibit carcinogen-induced DNA adduct formation and enhance DNA repair, berry compounds have been shown to inhibit mutagenesis and cancer initiation. Berry constituents also influence cellular processes associated with cancer progression including signaling pathways associated with cell proliferation, differentiation, apoptosis and angiogenesis. This review article summarizes laboratory and human studies, demonstrating the protective effects of berries and berry constituents on oxidative and other cellular processes leading to cancer development.

Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries

PubMed Central

Stoner, Gary David; Wang, Li-Shu; Casto, Bruce Cordell

2008-01-01

Reactive oxygen species (ROS) are a major cause of cellular injury in an increasing number of diseases, including cancer. Most ROS are created in the cell through normal cellular metabolism. They can be produced by environmental insults such as ultraviolet light and toxic chemicals, as well as by the inflammatory process. Interception of ROS or limiting their cellular effects is a major role of antioxidants. Due to their content of phenolic and flavonoid compounds, berries exhibit high antioxidant potential, exceeding that of many other foodstuffs. Through their ability to scavenge ROS and reduce oxidative DNA damage, stimulate antioxidant enzymes, inhibit carcinogen-induced DNA adduct formation and enhance DNA repair, berry compounds have been shown to inhibit mutagenesis and cancer initiation. Berry constituents also influence cellular processes associated with cancer progression including signaling pathways associated with cell proliferation, differentiation, apoptosis and angiogenesis. This review article summarizes laboratory and human studies, demonstrating the protective effects of berries and berry constituents on oxidative and other cellular processes leading to cancer development. PMID:18544560

Computational challenges in modeling gene regulatory events

PubMed Central

Pataskar, Abhijeet; Tiwari, Vijay K.

2016-01-01

ABSTRACT Cellular transcriptional programs driven by genetic and epigenetic mechanisms could be better understood by integrating “omics” data and subsequently modeling the gene-regulatory events. Toward this end, computational biology should keep pace with evolving experimental procedures and data availability. This article gives an exemplified account of the current computational challenges in molecular biology. PMID:27390891

The Cellular Mechanisms of Memory Are Modified by Experience

ERIC Educational Resources Information Center

Wiltgen, Brian J.; Wood, Alynda N.; Levy, Brynne

2011-01-01

The N-methyl-D-aspartate receptor (NMDAR) is thought to be essential for synaptic plasticity and learning. However, recent work indicates that the role of this receptor depends on the prior history of the research subject. For example, animals trained on a hippocampus-dependent learning task are subsequently able to acquire new information in the…

The Two Faces of p53 in Embryonic Stem Cells | Center for Cancer Research

Cancer.gov

Mutations in the genome of a cell can have dire effects. This type of damage can be even more devastating if it occurs in stem cells, since any changes will be passed on to all subsequent generations. Consequently, there are many checkpoints to alert cellular machinery of changes in the DNA.

Early cellular evolution.

NASA Technical Reports Server (NTRS)

Margulis, L.

1972-01-01

Study of the evolutionary developments that occurred subsequent to the origin of ancestral cells. Microbial physiology and ecology are potential sharp tools for shaping concepts of microbial evolution. Some popular unjustified assumptions are discussed. It is considered that certain principles derived mainly from the advances of molecular biology can be used to order the natural groups (genera) of extant prokaryotes and their patterns phylogenetically.

Are microRNAs true sensors of ageing and cellular senescence?

PubMed

Williams, Justin; Smith, Flint; Kumar, Subodh; Vijayan, Murali; Reddy, P Hemachandra

2017-05-01

All living beings are programmed to death due to aging and age-related processes. Aging is a normal process of every living species. While all cells are inevitably progressing towards death, many disease processes accelerate the aging process, leading to senescence. Pathologies such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, cardiovascular disease, cancer, and skin diseases have been associated with deregulated aging. Healthy aging can delay onset of all age-related diseases. Genetics and epigenetics are reported to play large roles in accelerating and/or delaying the onset of age-related diseases. Cellular mechanisms of aging and age-related diseases are not completely understood. However, recent molecular biology discoveries have revealed that microRNAs (miRNAs) are potential sensors of aging and cellular senescence. Due to miRNAs capability to bind to the 3' untranslated region (UTR) of mRNA of specific genes, miRNAs can prevent the translation of specific genes. The purpose of our article is to highlight recent advancements in miRNAs and their involvement in cellular changes in aging and senescence. Our article discusses the current understanding of cellular senescence, its interplay with miRNAs regulation, and how they both contribute to disease processes. Copyright © 2016 Elsevier B.V. All rights reserved.

Mass spectrometric profiling of lipids in intestinal tissue from rats fed cereals processed for medical conditions.

PubMed

Dowlatshahi Pour, Masoumeh; Jennische, Eva; Lange, Stefan; Ewing, Andrew G; Malmberg, Per

2016-06-11

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used for lipid profiling of intestine tissue sections from rats fed specially processed cereals and rats fed ordinary feed as a control. This cereal is known to increase the activity of antisecretory factor in plasma and the exact mechanism for the activation process at the cellular level is unclear. ToF-SIMS has been used to track food induced changes in lipid content in intestinal tissue sections to gain insight into the possible mechanisms involved. Data from 20 intestine sections belonging to four different rats from each group of control and specially processed cereals-fed rats were obtained using the stage scan macroraster with a lateral resolution of 5 μm. Data were subsequently subjected to orthogonal partial least squares discriminant analysis. The data clearly show that changes of certain lipids are induced by the specially processed cereal feed. Scores plots show a well-defined separation between the two groups. The corresponding loading plots reveal that the groups separate mainly due to changes of vitamin E, phosphocholine, and phosphosphingolipid fragments, and that for the c18:2 fatty acid. The observed changes in lipids might give insight into the working mechanisms of antisecretory factor in the body, and this has been successfully used to understand the working mechanism of specially processed cereal-induced antisecretory factor activation in intestine.

Slight temperature changes affect protein affinity and cellular uptake/toxicity of nanoparticles

NASA Astrophysics Data System (ADS)

Mahmoudi, Morteza; Shokrgozar, Mohammad A.; Behzadi, Shahed

2013-03-01

It is known that what the cell actually ``sees'' at the nanoscale is an outer shell formed of `protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular/organ temperature.It is known that what the cell actually ``sees'' at the nanoscale is an outer shell formed of `protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular/organ temperature. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr32551b

Understanding the cancer cell phenotype beyond the limitations of current omics analyses.

PubMed

Moreno-Sánchez, Rafael; Saavedra, Emma; Gallardo-Pérez, Juan Carlos; Rumjanek, Franklin D; Rodríguez-Enríquez, Sara

2016-01-01

Efforts to understand the mechanistic principles driving cancer metabolism and proliferation have been lately governed by genomic, transcriptomic and proteomic studies. This paper analyzes the caveats of these approaches. As molecular biology's central dogma proposes a unidirectional flux of information from genes to mRNA to proteins, it has frequently been assumed that monitoring the changes in the gene sequences and in mRNA and protein contents is sufficient to explain complex cellular processes. Such a stance commonly disregards that post-translational modifications can alter the protein function/activity and also that regulatory mechanisms enter into action, to coordinate the protein activities of pathways/cellular processes, in order to keep the cellular homeostasis. Hence, the actual protein activities (as enzymes/transporters/receptors) and their regulatory mechanisms ultimately dictate the final outcomes of a pathway/cellular process. In this regard, it is here documented that the mRNA levels of many metabolic enzymes and transcriptional factors have no correlation with the respective protein contents and activities. The validity of current clinical mRNA-based tests and proposed metabolite biomarkers for cancer detection/prognosis is also discussed. Therefore, it is proposed that, to achieve a thorough understanding of the modifications undergone by proliferating cancer cells, it is mandatory to experimentally analyze the cellular processes at the functional level. This could be achieved (a) locally, by examining the actual protein activities in the cell and their kinetic properties (or at least kinetically characterize the most controlling steps of the pathway/cellular process); (b) systemically, by analyzing the main fluxes of the pathway/cellular process, and how they are modulated by metabolites, all which should contribute to comprehending the regulatory mechanisms that have been altered in cancer cells. By adopting a more holistic approach it may become possible to improve the design of therapeutic strategies that would target cancer cells more specifically. © 2015 FEBS.

Analysis of high-throughput screening reveals the effect of surface topographies on cellular morphology.

PubMed

Hulsman, Marc; Hulshof, Frits; Unadkat, Hemant; Papenburg, Bernke J; Stamatialis, Dimitrios F; Truckenmüller, Roman; van Blitterswijk, Clemens; de Boer, Jan; Reinders, Marcel J T

2015-03-01

Surface topographies of materials considerably impact cellular behavior as they have been shown to affect cell growth, provide cell guidance, and even induce cell differentiation. Consequently, for successful application in tissue engineering, the contact interface of biomaterials needs to be optimized to induce the required cell behavior. However, a rational design of biomaterial surfaces is severely hampered because knowledge is lacking on the underlying biological mechanisms. Therefore, we previously developed a high-throughput screening device (TopoChip) that measures cell responses to large libraries of parameterized topographical material surfaces. Here, we introduce a computational analysis of high-throughput materiome data to capture the relationship between the surface topographies of materials and cellular morphology. We apply robust statistical techniques to find surface topographies that best promote a certain specified cellular response. By augmenting surface screening with data-driven modeling, we determine which properties of the surface topographies influence the morphological properties of the cells. With this information, we build models that predict the cellular response to surface topographies that have not yet been measured. We analyze cellular morphology on 2176 surfaces, and find that the surface topography significantly affects various cellular properties, including the roundness and size of the nucleus, as well as the perimeter and orientation of the cells. Our learned models capture and accurately predict these relationships and reveal a spectrum of topographies that induce various levels of cellular morphologies. Taken together, this novel approach of high-throughput screening of materials and subsequent analysis opens up possibilities for a rational design of biomaterial surfaces. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Premature aging/senescence in cancer cells facing therapy: good or bad?

PubMed

Gonzalez, Llilians Calvo; Ghadaouia, Sabrina; Martinez, Aurélie; Rodier, Francis

2016-02-01

Normal and cancer cells facing their demise following exposure to radio-chemotherapy can actively participate in choosing their subsequent fate. These programmed cell fate decisions include true cell death (apoptosis-necroptosis) and therapy-induced cellular senescence (TIS), a permanent "proliferative arrest" commonly portrayed as premature cellular aging. Despite a permanent loss of proliferative potential, senescent cells remain viable and are highly bioactive at the microenvironment level, resulting in a prolonged impact on tissue architecture and functions. Cellular senescence is primarily documented as a tumor suppression mechanism that prevents cellular transformation. In the context of normal tissues, cellular senescence also plays important roles in tissue repair, but contributes to age-associated tissue dysfunction when senescent cells accumulate. Theoretically, in multi-step cancer progression models, cancer cells have already bypassed cellular senescence during their immortalization step (see hallmarks of cancer). It is then perhaps surprising to find that cancer cells often retain the ability to undergo TIS, or premature aging. This occurs because cellular senescence results from multiple signalling pathways, some retained in cancer cells, aiming to prevent cell cycle progression in damaged cells. Since senescent cancer cells persist after therapy and secrete an array of cytokines and growth factors that can modulate the tumor microenvironment, these cells may have beneficial and detrimental effects regarding immune modulation and survival of remaining proliferation-competent cancer cells. Similarly, while normal cells undergoing senescence are believed to remain indefinitely growth arrested, whether this is true for senescent cancer cells remains unclear, raising the possibility that these cells may represent a reservoir for cancer recurrence after treatment. This review discusses our current knowledge on cancer cell senescence and highlight questions that must be addressed to fully understand the beneficial and detrimental impacts of cellular senescence during cancer therapy.

Emergence of tissue mechanics from cellular processes: shaping a fly wing

NASA Astrophysics Data System (ADS)

Merkel, Matthias; Etournay, Raphael; Popovic, Marko; Nandi, Amitabha; Brandl, Holger; Salbreux, Guillaume; Eaton, Suzanne; Jülicher, Frank

Nowadays, biologistsare able to image biological tissueswith up to 10,000 cells in vivowhere the behavior of each individual cell can be followed in detail.However, how precisely large-scale tissue deformation and stresses emerge from cellular behavior remains elusive. Here, we study this question in the developing wing of the fruit fly. To this end, we first establish a geometrical framework that exactly decomposes tissue deformation into contributions by different kinds of cellular processes. These processes comprise cell shape changes, cell neighbor exchanges, cell divisions, and cell extrusions. As the key idea, we introduce a tiling of the cellular network into triangles. This approach also reveals that tissue deformation can also be created by correlated cellular motion. Based on quantifications using these concepts, we developed a novel continuum mechanical model for the fly wing. In particular, our model includes active anisotropic stresses and a delay in the response of cell rearrangements to material stresses. A different approach to study the emergence of tissue mechanics from cellular behavior are cell-based models. We characterize the properties of a cell-based model for 3D tissues that is a hybrid between single particle models and the so-called vertex models.

Exosomes from iPSCs Delivering siRNA Attenuate Intracellular Adhesion Molecule-1 Expression and Neutrophils Adhesion in Pulmonary Microvascular Endothelial Cells.

PubMed

Ju, Zhihai; Ma, Jinhui; Wang, Chen; Yu, Jie; Qiao, Yeru; Hei, Feilong

2017-04-01

The pro-inflammatory activation of pulmonary microvascular endothelial cells resulting in continuous expression of cellular adhesion molecules, and subsequently recruiting primed neutrophils to form a firm neutrophils-endothelium (PMN-EC) adhesion, has been examined and found to play a vital role in acute lung injury (ALI). RNA interference (RNAi) is a cellular process through harnessing a natural pathway silencing target gene based on recognition and subsequent degradation of specific mRNA sequences. It opens a promising approach for precision medicine. However, this application was hampered by many obstacles, such as immunogenicity, instability, toxicity problems, and difficulty in across the biological membrane. In this study, we reprogrammed urine exfoliated renal epithelial cells into human induced pluripotent stem cells (huiPSCs) and purified the exosomes (Exo) from huiPSCs as RNAi delivery system. Through choosing the episomal system to deliver transcription factors, we obtained a non-integrating huiPSCs. Experiments in both vitro and vivo demonstrated that these huiPSCs possess the pluripotent properties. The exosomes of huiPSCs isolated by differential centrifugation were visualized by transmission electron microscopy (TEM) showing a typical exosomal appearance with an average diameter of 122 nm. Immunoblotting confirmed the presence of the typical exosomal markers, including CD63, TSG 101, and Alix. Co-cultured PKH26-labeled exosomes with human primary pulmonary microvascular endothelial cells (HMVECs) confirmed that they could be internalized by recipient cells at a time-dependent manner. Then, electroporation was used to introduce siRNA against intercellular adhesion molecule-1 (ICAM-1) into exosomes to form an Exo/siRNA compound. The Exo/siRNA compound efficiently delivered the target siRNA into HMVECs causing selective gene silencing, inhibiting the ICAM-1 protein expression, and PMN-EC adhesion induced by lipopolysaccharide (LPS). These data suggest that huiPSCs exosomes could be used as a natural gene delivery vector to transport therapeutic siRNAs for alleviating inflammatory responses in recipient cells.

Detecting the Extent of Cellular Decomposition after Sub-Eutectoid Annealing in Rolled UMo Foils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kautz, Elizabeth J.; Jana, Saumyadeep; Devaraj, Arun

2017-07-31

This report presents an automated image processing approach to quantifying microstructure image data, specifically the extent of eutectoid (cellular) decomposition in rolled U-10Mo foils. An image processing approach is used here to be able to quantitatively describe microstructure image data in order to relate microstructure to processing parameters (time, temperature, deformation).

Protein arginine methylation: Cellular functions and methods of analysis.

PubMed

Pahlich, Steffen; Zakaryan, Rouzanna P; Gehring, Heinz

2006-12-01

During the last few years, new members of the growing family of protein arginine methyltransferases (PRMTs) have been identified and the role of arginine methylation in manifold cellular processes like signaling, RNA processing, transcription, and subcellular transport has been extensively investigated. In this review, we describe recent methods and findings that have yielded new insights into the cellular functions of arginine-methylated proteins, and we evaluate the currently used procedures for the detection and analysis of arginine methylation.

Nitric Oxide and Mitochondrial Function in Neurological Diseases.

PubMed

Ghasemi, Mehdi; Mayasi, Yunis; Hannoun, Anas; Eslami, Seyed Majid; Carandang, Raphael

2018-04-15

Mitochondria are key cellular organelles that play crucial roles in the energy production and regulation of cellular metabolism. Accumulating evidence suggests that mitochondrial activity can be modulated by nitric oxide (NO). As a key neurotransmitter in biologic systems, NO mediates the majority of its function through activation of the cyclic guanylyl cyclase (cGC) signaling pathway and S-nitrosylation of a variety of proteins involved in cellular functioning including those involved in mitochondrial biology. Moreover, excess NO or the formation of reactive NO species (RNS), e.g., peroxynitrite (ONOO - ), impairs mitochondrial functioning and this, in conjunction with nuclear events, eventually affects neuronal cell metabolism and survival, contributing to the pathogenesis of several neurodegenerative diseases. In this review we highlight the possible mechanisms underlying the noxious effects of excess NO and RNS on mitochondrial function including (i) negative effects on electron transport chain (ETC); (ii) ONOO - -mediated alteration in mitochondrial permeability transition; (iii) enhanced mitochondrial fragmentation and autophagy through S-nitrosylation of key proteins involved in this process such as dynamin-related protein 1 (DRP-1) and Parkin/PINK1 (protein phosphatase and tensin homolog-induced kinase 1) complex; (iv) alterations in the mitochondrial metabolic pathways including Krebs cycle, glycolysis, fatty acid metabolism, and urea cycle; and finally (v) mitochondrial ONOO - -induced nuclear toxicity and subsequent release of apoptosis-inducing factor (AIF) from mitochondria, causing neuronal cell death. These proposed mechanisms highlight the multidimensional nature of NO and its signaling in the mitochondrial function. Understanding the mechanisms by which NO mediates mitochondrial (dys)function can provide new insights into the treatment of neurodegenerative diseases. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Ameliorative effects of selenium on arsenic-induced cytotoxicity in PC12 cells via modulating autophagy/apoptosis.

PubMed

Rahman, Md Mostafizur; Uson-Lopez, Rachael A; Sikder, Md Tajuddin; Tan, Gongxun; Hosokawa, Toshiyuki; Saito, Takeshi; Kurasaki, Masaaki

2018-04-01

Arsenic is well known toxicant responsible for human diseases including cancers. On the other hand, selenium is an essential trace element with significant chemopreventive effects, anticancer potentials and antioxidant properties. Although previous studies have reported antagonism/synergism between arsenic and selenium in biological systems, the biomolecular mechanism/s is still inconclusive. Therefore, to elucidate the molecular phenomena in cellular level, we hypothesized that co-exposure of selenium with arsenic may have suppressive effects on arsenic-induced cytotoxicity. We found that selenium in co-exposure with arsenic increases cell viability, and suppresses oxidative stress induced by arsenic in PC12 cells. Consequently, DNA fragmentation due to arsenic exposure was also reduced by arsenic and selenium co-exposure. Furthermore, western blot analyses revealed that simultaneous exposure of both metals significantly inhibited autophagy which further suppressed apoptosis through positively regulation of key proteins; p-mTOR, p-Akt, p-Foxo1A, p62, and expression of ubiquitin, Bax, Bcl2, NFкB, and caspases 3 and 9, although those are negatively regulated by arsenic. In addition, reverse transcriptase PCR analysis confirmed the involvement of caspase cascade in cell death process induced by arsenic and subsequent inhibition by co-exposure of selenium with arsenic. The cellular accumulation study of arsenic in presence/absence of selenium via inductively coupled plasma mass spectrometry confirmed that selenium effectively retarded the uptake of arsenic in PC12 cells. Finally, these findings imply that selenium is capable to modulate arsenic-induced intrinsic apoptosis pathway via enhancement of mTOR/Akt autophagy signaling pathway through employing antioxidant potentials and through inhibiting the cellular accumulation of arsenic in PC12 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

PubMed Central

Nicholls, David G.; Darley-Usmar, Victor M.; Wu, Min; Jensen, Per Bo; Rogers, George W.; Ferrick, David A.

2010-01-01

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity. Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment. Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR. In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell. This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults. PMID:21189469

Investigation of a calcium-responsive contrast agent in cellular model systems: feasibility for use as a smart molecular probe in functional MRI.

PubMed

Angelovski, Goran; Gottschalk, Sven; Milošević, Milena; Engelmann, Jörn; Hagberg, Gisela E; Kadjane, Pascal; Andjus, Pavle; Logothetis, Nikos K

2014-05-21

Responsive or smart contrast agents (SCAs) represent a promising direction for development of novel functional MRI (fMRI) methods for the eventual noninvasive assessment of brain function. In particular, SCAs that respond to Ca(2+) may allow tracking neuronal activity independent of brain vasculature, thus avoiding the characteristic limitations of current fMRI techniques. Here we report an in vitro proof-of-principle study with a Ca(2+)-sensitive, Gd(3+)-based SCA in an attempt to validate its potential use as a functional in vivo marker. First, we quantified its relaxometric response in a complex 3D cell culture model. Subsequently, we examined potential changes in the functionality of primary glial cells following administration of this SCA. Monitoring intracellular Ca(2+) showed that, despite a reduction in the Ca(2+) level, transport of Ca(2+) through the plasma membrane remained unaffected, while stimulation with ATP induced Ca(2+)-transients suggested normal cellular signaling in the presence of low millimolar SCA concentrations. SCAs merely lowered the intracellular Ca(2+) level. Finally, we estimated the longitudinal relaxation times (T1) for an idealized in vivo fMRI experiment with SCA, for extracellular Ca(2+) concentration level changes expected during intense neuronal activity which takes place upon repetitive stimulation. The values we obtained indicate changes in T1 of around 1-6%, sufficient to be robustly detectable using modern MRI methods in high field scanners. Our results encourage further attempts to develop even more potent SCAs and appropriate fMRI protocols. This would result in novel methods that allow monitoring of essential physiological processes at the cellular and molecular level.

Cellular Assays for Studying the Fe-S Cluster Containing Base Excision Repair Glycosylase MUTYH and Homologs.

PubMed

Majumdar, Chandrima; Nuñez, Nicole N; Raetz, Alan G; Khuu, Cindy; David, Sheila S

2018-01-01

Many DNA repair enzymes, including the human adenine glycosylase MUTYH, require iron-sulfur (Fe-S) cluster cofactors for DNA damage recognition and subsequent repair. MUTYH prokaryotic and eukaryotic homologs are a family of adenine (A) glycosylases that cleave A when mispaired with the oxidatively damaged guanine lesion, 8-oxo-7,8-dihydroguanine (OG). Faulty OG:A repair has been linked to the inheritance of missense mutations in the MUTYH gene. These inherited mutations can result in the onset of a familial colorectal cancer disorder known as MUTYH-associated polyposis (MAP). While in vitro studies can be exceptional at unraveling how MutY interacts with its OG:A substrate, cell-based assays are needed to provide a cellular context to these studies. In addition, strategic comparison of in vitro and in vivo studies can provide exquisite insight into the search, selection, excision process, and the coordination with protein partners, required to mediate full repair of the lesion. A commonly used assay is the rifampicin resistance assay that provides an indirect evaluation of the intrinsic mutation rate in Escherichia coli (E. coli or Ec), read out as antibiotic-resistant cell growth. Our laboratory has also developed a bacterial plasmid-based assay that allows for direct evaluation of repair of a defined OG:A mispair. This assay provides a means to assess the impact of catalytic defects in affinity and excision on overall repair. Finally, a mammalian GFP-based reporter assay has been developed that more accurately models features of mammalian cells. Taken together, these assays provide a cellular context to the repair activity of MUTYH and its homologs that illuminates the role these enzymes play in preventing mutations and disease. © 2018 Elsevier Inc. All rights reserved.

Identification and optogenetic manipulation of memory engrams in the hippocampus

PubMed Central

Ramirez, Steve; Tonegawa, Susumu; Liu, Xu

2014-01-01

With the accumulation of our knowledge about how memories are formed, consolidated, retrieved, and updated, neuroscience is now reaching a point where discrete memories can be identified and manipulated at rapid timescales. Here, we start with historical studies that lead to the modern memory engram theory. Then, we will review recent advances in memory engram research that combine transgenic and optogenetic approaches to reveal the underlying neuronal substrates sufficient for activating mnemonic processes. We will focus on three concepts: (1) isolating memory engrams at the level of single cells to tag them for subsequent manipulation; (2) testing the sufficiency of these engrams for memory recall by artificially activating them; and (3) presenting new stimuli during the artificial activation of these engrams to induce an association between the two to form a false memory. We propose that hippocampal cells that show activity-dependent changes during learning construct a cellular basis for contextual memory engrams. PMID:24478647

Increased Arf/p53 activity in stem cells, aging and cancer.

PubMed

Carrasco-Garcia, Estefania; Moreno, Manuel; Moreno-Cugnon, Leire; Matheu, Ander

2017-04-01

Arf/p53 pathway protects the cells against DNA damage induced by acute stress. This characteristic is the responsible for its tumor suppressor activity. Moreover, it regulates the chronic type of stress associated with aging. This is the basis of its anti-aging activity. Indeed, increased gene dosage of Arf/p53 displays elongated longevity and delayed aging. At a cellular level, it has been recently shown that increased dosage of Arf/p53 delays age-associated stem cell exhaustion and the subsequent decline in tissue homeostasis and regeneration. However, p53 can also promote aging if constitutively activated. In this context, p53 reduces tissue regeneration, which correlates with premature exhaustion of stem cells. We discuss here the current evidence linking the Arf/p53 pathway to the processes of aging and cancer through stem cell regulation. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Preparation of sumoylated substrates for biochemical analysis.

PubMed

Knipscheer, Puck; Klug, Helene; Sixma, Titia K; Pichler, Andrea

2009-01-01

Covalent modification of proteins with SUMO (small ubiquitin related modifier) affects many cellular processes like transcription, nuclear transport, DNA repair and cell cycle progression. Although hundreds of SUMO targets have been identified, for several of them the function remains obscure. In the majority of cases sumoylation is investigated via "loss of modification" analysis by mutating the relevant target lysine. However, in other cases this approach is not successful since mapping of the modification site is problematic or mutation does not cause an obvious phenotype. These latter cases ask for different approaches to investigate the target modification. One possibility is to choose the opposite approach, a "gain in modification" analysis by producing both SUMO modified and unmodified protein in vitro and comparing them in functional assays. Here, we describe the purification of the ubiquitin conjugating enzyme E2-25K, its in vitro sumoylation with recombinant enzymes and the subsequent separation and purification of the modified and the unmodified forms.

Non-Neoplastic and Neoplastic Pleural Endpoints Following Fiber Exposure

PubMed Central

Broaddus, V. Courtney; Everitt, Jeffrey I.; Black, Brad; Kane, Agnes B.

2011-01-01

Exposure to asbestos fibers is associated with non-neoplastic pleural diseases including plaques, fibrosis, and benign effusions, as well as with diffuse malignant pleural mesothelioma. Translocation and retention of fibers are fundamental processes in understanding the interactions between the dose and dimensions of fibers retained at this anatomic site and the subsequent pathological reactions. The initial interaction of fibers with target cells in the pleura has been studied in cellular models in vitro and in experimental studies in vivo. The proposed biological mechanisms responsible for non-neoplastic and neoplastic pleural diseases and the physical and chemical properties of asbestos fibers relevant to these mechanisms are critically reviewed. Understanding mechanisms of asbestos fiber toxicity may help us anticipate the problems from future exposures both to asbestos and to novel fibrous materials such as nanotubes. Gaps in our understanding have been outlined as guides for future research. PMID:21534088

Keratinocytes mediate innocuous and noxious touch via ATP-P2X4 signaling

PubMed Central

Moehring, Francie; Cowie, Ashley M; Menzel, Anthony D; Weyer, Andy D; Grzybowski, Michael; Arzua, Thiago; Geurts, Aron M; Palygin, Oleg

2018-01-01

The first point of our body’s contact with tactile stimuli (innocuous and noxious) is the epidermis, the outermost layer of skin that is largely composed of keratinocytes. Here, we sought to define the role that keratinocytes play in touch sensation in vivo and ex vivo. We show that optogenetic inhibition of keratinocytes decreases behavioral and cellular mechanosensitivity. These processes are inherently mediated by ATP signaling, as demonstrated by complementary cutaneous ATP release and degradation experiments. Specific deletion of P2X4 receptors in sensory neurons markedly decreases behavioral and primary afferent mechanical sensitivity, thus positioning keratinocyte-released ATP to sensory neuron P2X4 signaling as a critical component of baseline mammalian tactile sensation. These experiments lay a vital foundation for subsequent studies into the dysfunctional signaling that occurs in cutaneous pain and itch disorders, and ultimately, the development of novel topical therapeutics for these conditions. PMID:29336303

Evaluation of breast tissue with confocal strip-mosaicking microscopy: a test approach emulating pathology-like examination

PubMed Central

Abeytunge, Sanjee; Larson, Bjorg; Peterson, Gary; Morrow, Monica; Rajadhyaksha, Milind

2017-01-01

Abstract. Confocal microscopy is an emerging technology for rapid imaging of freshly excised tissue without the need for frozen- or fixed-section processing. Initial studies have described imaging of breast tissue using fluorescence confocal microscopy with small regions of interest, typically 750×750  μm2. We present exploration with a microscope, termed confocal strip-mosaicking microscope (CSM microscope), which images an area of 2×2  cm2 of tissue with cellular-level resolution in 10 min of excision. Using the CSM microscope, we imaged 34 fresh, human, large breast tissue specimens from 18 patients, blindly analyzed by a board-certified pathologist and subsequently correlated with the corresponding standard fixed histopathology. Invasive tumors and benign tissue were clearly identified in CSM strip-mosaic images. Thirty specimens were concordant for image-to-histopathology correlation while four were discordant. PMID:28327961

Astrocytic glycogen metabolism in the healthy and diseased brain.

PubMed

Bak, Lasse K; Walls, Anne B; Schousboe, Arne; Waagepetersen, Helle S

2018-05-11

The brain contains a fairly low amount of glycogen, mostly located in astrocytes, a fact that has prompted the suggestion that glycogen does not have a significant physiological role in the brain. However, glycogen metabolism in astrocytes is essential for several key physiological processes and is adversely affected in disease. For instance, diminished ability to break down glycogen impinges on learning, and epilepsy, Alzheimer's disease, and type 2 diabetes are all associated with abnormal astrocyte glycogen metabolism. Glycogen metabolism supports astrocytic K + and neurotransmitter glutamate uptake and subsequent glutamine synthesis-three fundamental steps in excitatory signaling at most brain synapses. Thus, there is abundant evidence for a key role of glycogen in brain function. Here, we summarize the physiological brain functions that depend on glycogen, discuss glycogen metabolism in disease, and investigate how glycogen breakdown is regulated at the cellular and molecular levels. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Cell Death Pathways and Phthalocyanine as an Efficient Agent for Photodynamic Cancer Therapy

PubMed Central

Mfouo-Tynga, Ivan; Abrahamse, Heidi

2015-01-01

The mechanisms of cell death can be predetermined (programmed) or not and categorized into apoptotic, autophagic and necrotic pathways. The process of Hayflick limits completes the execution of death-related mechanisms. Reactive oxygen species (ROS) are associated with oxidative stress and subsequent cytodamage by oxidizing and degrading cell components. ROS are also involved in immune responses, where they stabilize and activate both hypoxia-inducible factors and phagocytic effectors. ROS production and presence enhance cytodamage and photodynamic-induced cell death. Photodynamic cancer therapy (PDT) uses non-toxic chemotherapeutic agents, photosensitizer (PS), to initiate a light-dependent and ROS-related cell death. Phthalocyanines (PCs) are third generation and stable PSs with improved photochemical abilities. They are effective inducers of cell death in various neoplastic models. The metallated PCs localize in critical cellular organelles and are better inducers of cell death than other previous generation PSs as they favor mainly apoptotic cell death events. PMID:25955645

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist.

PubMed

Sanchez, Elisa; Huse, Morgan

2018-04-25

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

Root hydrotropism is controlled via a cortex-specific growth mechanism.

PubMed

Dietrich, Daniela; Pang, Lei; Kobayashi, Akie; Fozard, John A; Boudolf, Véronique; Bhosale, Rahul; Antoni, Regina; Nguyen, Tuan; Hiratsuka, Sotaro; Fujii, Nobuharu; Miyazawa, Yutaka; Bae, Tae-Woong; Wells, Darren M; Owen, Markus R; Band, Leah R; Dyson, Rosemary J; Jensen, Oliver E; King, John R; Tracy, Saoirse R; Sturrock, Craig J; Mooney, Sacha J; Roberts, Jeremy A; Bhalerao, Rishikesh P; Dinneny, José R; Rodriguez, Pedro L; Nagatani, Akira; Hosokawa, Yoichiroh; Baskin, Tobias I; Pridmore, Tony P; De Veylder, Lieven; Takahashi, Hideyuki; Bennett, Malcolm J

2017-05-08

Plants can acclimate by using tropisms to link the direction of growth to environmental conditions. Hydrotropism allows roots to forage for water, a process known to depend on abscisic acid (ABA) but whose molecular and cellular basis remains unclear. Here we show that hydrotropism still occurs in roots after laser ablation removed the meristem and root cap. Additionally, targeted expression studies reveal that hydrotropism depends on the ABA signalling kinase SnRK2.2 and the hydrotropism-specific MIZ1, both acting specifically in elongation zone cortical cells. Conversely, hydrotropism, but not gravitropism, is inhibited by preventing differential cell-length increases in the cortex, but not in other cell types. We conclude that root tropic responses to gravity and water are driven by distinct tissue-based mechanisms. In addition, unlike its role in root gravitropism, the elongation zone performs a dual function during a hydrotropic response, both sensing a water potential gradient and subsequently undergoing differential growth.

Pseudomonas fluorescens HK44: Lessons Learned from a Model Whole-Cell Bioreporter with a Broad Application History

PubMed Central

Trögl, Josef; Chauhan, Archana; Ripp, Steven; Layton, Alice C.; Kuncová, Gabriela; Sayler, Gary S.

2012-01-01

Initially described in 1990, Pseudomonas fluorescens HK44 served as the first whole-cell bioreporter genetically endowed with a bioluminescent (luxCDABE) phenotype directly linked to a catabolic (naphthalene degradative) pathway. HK44 was the first genetically engineered microorganism to be released in the field to monitor bioremediation potential. Subsequent to that release, strain HK44 had been introduced into other solids (soils, sands), liquid (water, wastewater), and volatile environments. In these matrices, it has functioned as one of the best characterized chemically-responsive environmental bioreporters and as a model organism for understanding bacterial colonization and transport, cell immobilization strategies, and the kinetics of cellular bioluminescent emission. This review summarizes the characteristics of P. fluorescens HK44 and the extensive range of its applications with special focus on the monitoring of bioremediation processes and biosensing of environmental pollution. PMID:22438725

Mitochondrial Dysfunction in Parkinson's Disease: Pathogenesis and Neuroprotection

PubMed Central

Mounsey, Ross B.; Teismann, Peter

2011-01-01

Mitochondria are vitally important organelles involved in an array of functions. The most notable is their prominent role in energy metabolism, where they generate over 90% of our cellular energy in the form of ATP through oxidative phosphorylation. Mitochondria are involved in various other processes including the regulation of calcium homeostasis and stress response. Mitochondrial complex I impairment and subsequent oxidative stress have been identified as modulators of cell death in experimental models of Parkinson's disease (PD). Identification of specific genes which are involved in the rare familial forms of PD has further augmented the understanding and elevated the role mitochondrial dysfunction is thought to have in disease pathogenesis. This paper provides a review of the role mitochondria may play in idiopathic PD through the study of experimental models and how genetic mutations influence mitochondrial activity. Recent attempts at providing neuroprotection by targeting mitochondria are described and their progress assessed. PMID:21234411

Stress-response transcription factors Msn2 and Msn4 couple TORC2-Ypk1 signaling and mitochondrial respiration to ATG8 gene expression and autophagy.

PubMed

Vlahakis, Ariadne; Lopez Muniozguren, Nerea; Powers, Ted

2017-01-01

Macroautophagy/autophagy is a starvation and stress-induced catabolic process critical for cellular homeostasis and adaptation. Several Atg proteins are involved in the formation of the autophagosome and subsequent degradation of cytoplasmic components, a process termed autophagy flux. Additionally, the expression of several Atg proteins, in particular Atg8, is modulated transcriptionally, yet the regulatory mechanisms involved remain poorly understood. Here we demonstrate that the AGC kinase Ypk1, target of the rapamycin-insensitive TORC2 signaling pathway, controls ATG8 expression by repressing the heterodimeric Zinc-finger transcription factors Msn2 and Msn4. We find that Msn2 and Msn4 promote ATG8 expression downstream of the histone deacetylase complex (HDAC) subunit Ume6, a previously identified negative regulator of ATG8 expression. Moreover, we demonstrate that TORC2-Ypk1 signaling is functionally linked to distinct mitochondrial respiratory complexes. Surprisingly, we find that autophagy flux during amino acid starvation is also dependent upon Msn2-Msn4 activity, revealing a broad role for these transcription factors in the autophagy response.

Genetic Dominance & Cellular Processes

ERIC Educational Resources Information Center

Seager, Robert D.

2014-01-01

In learning genetics, many students misunderstand and misinterpret what "dominance" means. Understanding is easier if students realize that dominance is not a mechanism, but rather a consequence of underlying cellular processes. For example, metabolic pathways are often little affected by changes in enzyme concentration. This means that…

Connecting Photosynthesis and Cellular Respiration: Preservice Teachers' Conceptions

ERIC Educational Resources Information Center

Brown, Mary H.; Schwartz, Renee S.

2009-01-01

The biological processes of photosynthesis and plant cellular respiration include multiple biochemical steps, occur simultaneously within plant cells, and share common molecular components. Yet, learners often compartmentalize functions and specialization of cell organelles relevant to these two processes, without considering the interconnections…

Quantitative phase-digital holographic microscopy: a new imaging modality to identify original cellular biomarkers of diseases

NASA Astrophysics Data System (ADS)

Marquet, P.; Rothenfusser, K.; Rappaz, B.; Depeursinge, C.; Jourdain, P.; Magistretti, P. J.

2016-03-01

Quantitative phase microscopy (QPM) has recently emerged as a powerful label-free technique in the field of living cell imaging allowing to non-invasively measure with a nanometric axial sensitivity cell structure and dynamics. Since the phase retardation of a light wave when transmitted through the observed cells, namely the quantitative phase signal (QPS), is sensitive to both cellular thickness and intracellular refractive index related to the cellular content, its accurate analysis allows to derive various cell parameters and monitor specific cell processes, which are very likely to identify new cell biomarkers. Specifically, quantitative phase-digital holographic microscopy (QP-DHM), thanks to its numerical flexibility facilitating parallelization and automation processes, represents an appealing imaging modality to both identify original cellular biomarkers of diseases as well to explore the underlying pathophysiological processes.

Fluorescence microscopy: A tool to study autophagy

NASA Astrophysics Data System (ADS)

Rai, Shashank; Manjithaya, Ravi

2015-08-01

Autophagy is a cellular recycling process through which a cell degrades old and damaged cellular components such as organelles and proteins and the degradation products are reused to provide energy and building blocks. Dysfunctional autophagy is reported in several pathological situations. Hence, autophagy plays an important role in both cellular homeostasis and diseased conditions. Autophagy can be studied through various techniques including fluorescence based microscopy. With the advancements of newer technologies in fluorescence microscopy, several novel processes of autophagy have been discovered which makes it an essential tool for autophagy research. Moreover, ability to tag fluorescent proteins with sub cellular targets has enabled us to evaluate autophagy processes in real time under fluorescent microscope. In this article, we demonstrate different aspects of autophagy in two different model organisms i.e. yeast and mammalian cells, with the help of fluorescence microscopy.

Determinants of PDT-induced apoptosis

NASA Astrophysics Data System (ADS)

Kessel, David; Luo, Yu; Kim, Hyeong-Reh C.

2000-03-01

Photodynamic therapy can initiate cell death by apoptosis or necrosis. Using agents with known patterns of sub-cellular localization, we examined the correlation between sites of photodamage and the mode of cell death, using murine leukemia cells in vitro. Mitochondrial or mitochondrial/lysosomal photodamage caused the rapid release of cytochrome c. This effect was not temperature sensitive, and could be demonstrated immediately after irradiation of photosensitized cells at 10 degrees C. Subsequent warming to 37 degrees C led to a rapid apoptotic response, consistent with the known ability of cytochrome c to trigger the activation of caspase-3. In contrast, lysosomal or lysosomal/membrane photodamage resulted in the release of cathepsins and other proteolytic enzymes. A subsequent incubation at 37 degrees C resulted in mitochondrial degradation, leading to loss of cytochrome c within 30 min. The apoptotic response was both delayed and incomplete, with many dead cells not exhibiting an apoptotic morphology. The latter outcome was traced to photodamage to procaspase-3, an effect not observed with sensitizers that caused mainly mitochondrial photodamage. Studies in a cell-free system demonstrated that agents with lysosomal and/or membrane targets could bring about photoinactivation of caspase-3. These result are consistent with the proposal that photodynamic therapy can both activate and inactivate components of the apoptotic process.

Cellular water distribution, transport, and its investigation methods for plant-based food material.

PubMed

Khan, Md Imran H; Karim, M A

2017-09-01

Heterogeneous and hygroscopic characteristics of plant-based food material make it complex in structure, and therefore water distribution in its different cellular environments is very complex. There are three different cellular environments, namely the intercellular environment, the intracellular environment, and the cell wall environment inside the food structure. According to the bonding strength, intracellular water is defined as loosely bound water, cell wall water is categorized as strongly bound water, and intercellular water is known as free water (FW). During food drying, optimization of the heat and mass transfer process is crucial for the energy efficiency of the process and the quality of the product. For optimizing heat and mass transfer during food processing, understanding these three types of waters (strongly bound, loosely bound, and free water) in plant-based food material is essential. However, there are few studies that investigate cellular level water distribution and transport. As there is no direct method for determining the cellular level water distributions, various indirect methods have been applied to investigate the cellular level water distribution, and there is, as yet, no consensus on the appropriate method for measuring cellular level water in plant-based food material. Therefore, the main aim of this paper is to present a comprehensive review on the available methods to investigate the cellular level water, the characteristics of water at different cellular levels and its transport mechanism during drying. The effect of bound water transport on quality of food product is also discussed. This review article presents a comparative study of different methods that can be applied to investigate cellular water such as nuclear magnetic resonance (NMR), bioelectric impedance analysis (BIA), differential scanning calorimetry (DSC), and dilatometry. The article closes with a discussion of current challenges to investigating cellular water. Copyright © 2017 Elsevier Ltd. All rights reserved.

Integration of Proteomic, Transcriptional, and Interactome Data Reveals Hidden Signaling Components

PubMed Central

Huang, Shao-shan Carol; Fraenkel, Ernest

2009-01-01

Cellular signaling and regulatory networks underlie fundamental biological processes such as growth, differentiation, and response to the environment. Although there are now various high-throughput methods for studying these processes, knowledge of them remains fragmentary. Typically, the vast majority of hits identified by transcriptional, proteomic, and genetic assays lie outside of the expected pathways. These unexpected components of the cellular response are often the most interesting, because they can provide new insights into biological processes and potentially reveal new therapeutic approaches. However, they are also the most difficult to interpret. We present a technique, based on the Steiner tree problem, that uses previously reported protein-protein and protein-DNA interactions to determine how these hits are organized into functionally coherent pathways, revealing many components of the cellular response that are not readily apparent in the original data. Applied simultaneously to phosphoproteomic and transcriptional data for the yeast pheromone response, it identifies changes in diverse cellular processes that extend far beyond the expected pathways. PMID:19638617

The cellular transducer in bone: What is it?

PubMed

Taylor, David; Hazenberg, Jan; Lee, T Clive

2006-01-01

Bone is able to detect its strain environment and respond accordingly. In particular it is able to adapt to over-use and under-use by bone deposition or resorption. How can bone sense strain? Various physical mechanisms have been proposed for the so-called cellular transducer, but there is no conclusive proof for any one of them. This paper examines the theories and evidence, with particular reference to a new theory proposed by the authors, involving damage to cellular processes by microcracks. Experiments on bone samples ex-vivo showed that cracks cannot fracture osteocytes, but that cellular processes which span the crack can be broken. A theoretical model was developed for predicting the number of broken processes as a function of crack size and applied stress. This showed that signals emitted by fractured processes could be used to detect cracks which needed repairing and to provide information on the overall level of damage which could be used to initiate repair and adaptation responses.

Cellular Imaging With MRI.

PubMed

Makela, Ashley V; Murrell, Donna H; Parkins, Katie M; Kara, Jenna; Gaudet, Jeffrey M; Foster, Paula J

2016-10-01

Cellular magnetic resonance imaging (MRI) is an evolving field of imaging with strong translational and research potential. The ability to detect, track, and quantify cells in vivo and over time allows for studying cellular events related to disease processes and may be used as a biomarker for decisions about treatments and for monitoring responses to treatments. In this review, we discuss methods for labeling cells, various applications for cellular MRI, the existing limitations, strategies to address these shortcomings, and clinical cellular MRI.

Nanobodies and recombinant binders in cell biology

PubMed Central

Helma, Jonas; Cardoso, M. Cristina; Muyldermans, Serge

2015-01-01

Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes. PMID:26056137

Kinetic theory approach to modeling of cellular repair mechanisms under genome stress.

PubMed

Qi, Jinpeng; Ding, Yongsheng; Zhu, Ying; Wu, Yizhi

2011-01-01

Under acute perturbations from outer environment, a normal cell can trigger cellular self-defense mechanism in response to genome stress. To investigate the kinetics of cellular self-repair process at single cell level further, a model of DNA damage generating and repair is proposed under acute Ion Radiation (IR) by using mathematical framework of kinetic theory of active particles (KTAP). Firstly, we focus on illustrating the profile of Cellular Repair System (CRS) instituted by two sub-populations, each of which is made up of the active particles with different discrete states. Then, we implement the mathematical framework of cellular self-repair mechanism, and illustrate the dynamic processes of Double Strand Breaks (DSBs) and Repair Protein (RP) generating, DSB-protein complexes (DSBCs) synthesizing, and toxins accumulating. Finally, we roughly analyze the capability of cellular self-repair mechanism, cellular activity of transferring DNA damage, and genome stability, especially the different fates of a certain cell before and after the time thresholds of IR perturbations that a cell can tolerate maximally under different IR perturbation circumstances.

Evolution of the new vertebrate head by co-option of an ancient chordate skeletal tissue.

PubMed

Jandzik, David; Garnett, Aaron T; Square, Tyler A; Cattell, Maria V; Yu, Jr-Kai; Medeiros, Daniel M

2015-02-26

A defining feature of vertebrates (craniates) is a pronounced head that is supported and protected by a robust cellular endoskeleton. In the first vertebrates, this skeleton probably consisted of collagenous cellular cartilage, which forms the embryonic skeleton of all vertebrates and the adult skeleton of modern jawless and cartilaginous fish. In the head, most cellular cartilage is derived from a migratory cell population called the neural crest, which arises from the edges of the central nervous system. Because collagenous cellular cartilage and neural crest cells have not been described in invertebrates, the appearance of cellular cartilage derived from neural crest cells is considered a turning point in vertebrate evolution. Here we show that a tissue with many of the defining features of vertebrate cellular cartilage transiently forms in the larvae of the invertebrate chordate Branchiostoma floridae (Florida amphioxus). We also present evidence that during evolution, a key regulator of vertebrate cartilage development, SoxE, gained new cis-regulatory sequences that subsequently directed its novel expression in neural crest cells. Together, these results suggest that the origin of the vertebrate head skeleton did not depend on the evolution of a new skeletal tissue, as is commonly thought, but on the spread of this tissue throughout the head. We further propose that the evolution of cis-regulatory elements near an ancient regulator of cartilage differentiation was a major factor in the evolution of the vertebrate head skeleton.

3D Printing Variable Stiffness Foams Using Viscous Thread Instability

NASA Astrophysics Data System (ADS)

Lipton, Jeffrey I.; Lipson, Hod

2016-08-01

Additive manufacturing of cellular structures has numerous applications ranging from fabrication of biological scaffolds and medical implants, to mechanical weight reduction and control over mechanical properties. Various additive manufacturing processes have been used to produce open regular cellular structures limited only by the resolution of the printer. These efforts have focused on printing explicitly designed cells or explicitly planning offsets between strands. Here we describe a technique for producing cellular structures implicitly by inducing viscous thread instability when extruding material. This process allows us to produce complex cellular structures at a scale that is finer than the native resolution of the printer. We demonstrate tunable effective elastic modulus and density that span two orders of magnitude. Fine grained cellular structures allow for fabrication of foams for use in a wide range of fields ranging from bioengineering, to robotics to food printing.

Cell Signaling and Neurotoxicity: 3H-Arachidonic acid release (Phospholipase A2) in cerebellar granule neurons

EPA Science Inventory

Cell signaling is a complex process which controls basic cellular activities and coordinates actions to maintain normal cellular homeostasis. Alterations in signaling processes have been associated with neurological diseases such as Alzheimer's and cerebellar ataxia, as well as, ...

Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications.

PubMed

Ai, Xiangzhao; Lyu, Linna; Mu, Jing; Hu, Ming; Wang, Zhimin; Xing, Bengang

2017-11-10

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical properties, which allow for the absorption of near-infrared (NIR) light and can subsequently convert it into multiplexed emissions that span over a broad range of regions from the UV to the visible to the NIR. This article presents detailed experimental procedures for high-temperature co-precipitation synthesis of core-shell UCNs that incorporate different lanthanide ions into nanocrystals for efficiently converting deep-tissue penetrable NIR excitation (808 nm) into a strong blue emission at 480 nm. By controlling the surface modification with biocompatible polymer (polyacrylic acid, PAA), the as-prepared UCNs acquires great solubility in buffer solutions. The hydrophilic nanocrystals are further functionalized with specific ligands (dibenzyl cyclooctyne, DBCO) for localization on the cell membrane. Upon NIR light (808 nm) irradiation, the upconverted blue emission can effectively activate the light-gated channel protein on the cell membrane and specifically regulate the cation (e.g., Ca 2+ ) influx in the cytoplasm. This protocol provides a feasible methodology for the synthesis of core-shell lanthanide-doped UCNs and subsequent biocompatible surface modification for further cellular applications.

Generic framework for mining cellular automata models on protein-folding simulations.

PubMed

Diaz, N; Tischer, I

2016-05-13

Cellular automata model identification is an important way of building simplified simulation models. In this study, we describe a generic architectural framework to ease the development process of new metaheuristic-based algorithms for cellular automata model identification in protein-folding trajectories. Our framework was developed by a methodology based on design patterns that allow an improved experience for new algorithms development. The usefulness of the proposed framework is demonstrated by the implementation of four algorithms, able to obtain extremely precise cellular automata models of the protein-folding process with a protein contact map representation. Dynamic rules obtained by the proposed approach are discussed, and future use for the new tool is outlined.

Pretreatment of high solid microbial sludges

DOEpatents

Rivard, Christopher J.; Nagle, Nicholas J.

1998-01-01

A process and apparatus for pretreating microbial sludges in order to enhance secondary anaerobic digestion. The pretreatment process involves disrupting the cellular integrity of municipal sewage sludge through a combination of thermal, explosive decompression and shear forces. The sludge is pressurized and pumped to a pretreatment reactor where it is mixed with steam to heat and soften the sludge. The pressure of the sludge is suddenly reduced and explosive decompression forces are imparted which partially disrupt the cellular integrity of the sludge. Shear forces are then applied to the sludge to further disrupt the cellular integrity of the sludge. Disrupting cellular integrity releases both soluble and insoluble organic constituents and thereby renders municipal sewage sludge more amenable to secondary anaerobic digestion.

Magnetization reversal process in (Sm, Dy, Gd) (Co, Fe, Cu, Zr)z magnets with different cellular structures

NASA Astrophysics Data System (ADS)

Liu, Lei; Liu, Zhuang; Zhang, Xin; Feng, Yanping; Wang, Chunxiao; Sun, Yingli; Lee, Don; Yan, Aru; Wu, Qiong

2017-05-01

Magnetization reversal mechanism is found to vary with cellular structures by a comparative study of the magnetization processes of three (Sm, Dy, Gd) (Co, Fe, Cu, Zr)z magnets with different cellular structures. Analysis of domain walls, initial magnetization curves and recoil loops indicates that the morphology of cellular structure has a significant effect on the magnetization process, besides the obvious connection to the difference of domain energy density between cell boundary phase (CBP) and main phase. The magnetization of Sample 2 (with a moderate cell size and uniformly continuous CBPs) behaves as a strong coherence domain-wall pinning effect to the domain wall and lead to a highest coercivity in the magnet. The magnetization of Sample 1 (with thin and discontinuous CBPs) shows an inconsistent pinning effect to the domain wall while that of Sample 3 (with thick and aggregate CBPs) exhibits a two-phase separation magnetization. Both the two cases lead to lower coercivities. A simplified model is given as well to describe the relationships among cellular structure and magnetization behavior.

Programmed death-1 controls T cell survival by regulating oxidative metabolism1

PubMed Central

Tkachev, Victor; Goodell, Stefanie; Opipari, Anthony W.; Hao, Ling-Yang; Franchi, Luigi; Glick, Gary D.; Ferrara, James L.M.; Byersdorfer, Craig A.

2015-01-01

The co-inhibitory receptor programmed death-1 (PD-1) maintains immune homeostasis by negatively regulating T cell function and survival. Blockade of PD-1 increases the severity of graft-versus-host disease (GVHD), but the interplay between PD-1 inhibition and T cell metabolism is not well studied. We found that both murine and human alloreactive T cells concomitantly up-regulated PD-1 expression and increased levels of reactive oxygen species (ROS) following allogeneic bone marrow transplantation. This PD-1HiROSHi phenotype was specific to alloreactive T cells and was not observed in syngeneic T cells during homeostatic proliferation. Blockade of PD-1 signaling decreased both mitochondrial H2O2 and total cellular ROS levels and PD-1 driven increases in ROS were dependent upon the oxidation of fatty acids, as treatment with etomoxir nullified changes in ROS levels following PD-1 blockade. Downstream of PD-1, elevated ROS levels impaired T cell survival in a process reversed by anti-oxidants. Furthermore, PD-1 driven changes in ROS were fundamental to establishing a cell’s susceptibility to subsequent metabolic inhibition, as blockade of PD-1 decreased the efficacy of later F1F0-ATP synthase modulation. These data indicate that PD-1 facilitates apoptosis in alloreactive T cells by increasing reactive oxygen species in a process dependent upon the oxidation of fat. In addition, blockade of PD-1 undermines the potential for subsequent metabolic inhibition, an important consideration given the increasing use of anti-PD-1 therapies in the clinic. PMID:25972478

Endotoxin, Toll-like Receptor-4, and Atherosclerotic Heart Disease

PubMed Central

Horseman, Michael A.; Surani, Salim; Bowman, John D.

2017-01-01

Background: Endotoxin is a lipopolysaccharide (LPS) constituent of the outer membrane of most gram negative bacteria. Ubiquitous in the environment, it has been implicated as a cause or con-tributing factor in several disparate disorders from sepsis to heatstroke and Type II diabetes mellitus. Starting at birth, the innate immune system develops cellular defense mechanisms against environmen-tal microbes that are in part modulated through a series of receptors known as toll-like receptors. Endo-toxin, often referred to as LPS, binds to toll-like receptor 4 (TLR4)/ myeloid differentiation protein 2 (MD2) complexes on various tissues including cells of the innate immune system, smooth muscle and endothelial cells of blood vessels including coronary arteries, and adipose tissue. Entry of LPS into the systemic circulation ultimately leads to intracellular transcription of several inflammatory mediators. The subsequent inflammation has been implicated in the development and progression atherosclerosis and subsequent coronary artery disease and heart failure. Objective: The potential roles of endotoxin and TLR4 are reviewed regarding their role in the pathogen-esis of atherosclerotic heart disease. Conclusion: Atherosclerosis is initiated by inflammation in arterial endothelial and subendothelial cells, and inflammatory processes are implicated in its progression to clinical heart disease. Endotoxin and TLR4 play a central role in the inflammatory process, and represent potential targets for therapeutic intervention. Therapy with HMG-CoA inhibitors may reduce the expression of TLR4 on monocytes. Other therapeutic interventions targeting TLR4 expression or function may prove beneficial in athero-sclerotic disease prevention and treatment.

Biological Behavior of Osteoblast Cell and Apatite Forming Ability of the Surface Modified Ti Alloys.

PubMed

Zhao, Jingming; Hwang, K H; Choi, W S; Shin, S J; Lee, J K

2016-02-01

Titanium as one kind of biomaterials comes in direct contact with the body, making evaluation of biocompatibility an important aspect to biomaterials development. Surface chemistry of titanium plays an important role in osseointegration. Different surface modification alters the surface chemistry and result in different biological response. In this study, three kinds of mixed acid solutions were used to treat Ti specimens to induce Ca-P formation. Following a strong mixed acid activation process, Ca-P coating successfully formed on the Ti surfaces in simulated body fluid. Strong mixed acid increased the roughness of the metal surface, because the porous and rough surface allows better adhesion between Ca-P coatings and substrates. After modification of titanium surface by mixed acidic solution and subsequently H2O2/HCL treatment evaluation of biocompatibility was conducted from hydroxyapatite formation by biomimetic process and cell viability on modified titanium surface. Nano-scale modification of titanium surfaces can alter cellular and tissue responses, which may benefit osseointegration and dental implant therapy. Results from this study indicated that surface treatment methods affect the surface morphology, type of TiO2 layer formed and subsequent apatite deposition and biological responses. The thermo scientific alamarblue cell viability assay reagent is used to quantitatively measure the viability of mammalian cell lines, bacteria and fungi by incorporating a rapid, sensitive and reliable fluorometric/colorimetric growth indicator, without any toxic and side effect to cell line. In addition, mixed acid treatment uses a lower temperature and shorter time period than widely used alkali treatment.

The AICD interacting protein DAB1 is up-regulated in Alzheimer frontal cortex brain samples and causes deregulation of proteins involved in gene expression changes.

PubMed

Müller, T; Loosse, C; Schrötter, A; Schnabel, A; Helling, S; Egensperger, R; Marcus, K

2011-08-01

AICD is the intracellular subdomain of the amyloid precursor protein thought to play a pivotal role as a potential transcription factor that might be of relevance for the pathophysiology of Alzheimer's disease. For its signal transduction potential AICD requires interacting proteins like FE65 and TIP60. However, many other proteins were described being able to bind to AICD. Here, we studied mRNA levels of AICD interacting proteins and found one of them (DAB1) strongly up-regulated in human post-mortem frontal cortex brain samples of AD patients. Subsequent cell culture experiments revealed that elevated DAB1 level results in the deregulation of the cellular proteome. We found the proliferation associated protein 2G4 as well as the guanine monophosphate synthetase (GMPS) significantly up-regulated in DAB1 over-expressing cells. Both proteins can be involved in cellular transcription processes supporting the hypothesis that DAB1 acts via modification of the AICD-dependent transcriptionally active complex. Of note, expression of the three components of the putative transcription complex (AICD, FE65, and TIP60 (AFT)) also revealed deregulation of the GMPS protein in an opposite fashion. Our results point to a putative relevance of AICD-dependent mechanisms in AD, caused by protein abundance changes of AICD interacting proteins, as shown for DAB1 in this work.

Investigation of cellular detonation structure formation via linear stability theory and 2D and 3D numerical simulations

NASA Astrophysics Data System (ADS)

Borisov, S. P.; Kudryavtsev, A. N.

2017-10-01

Linear and nonlinear stages of the instability of a plane detonation wave (DW) and the subsequent process of formation of cellular detonation structure are investigated. A simple model with one-step irreversible chemical reaction is used. The linear analysis is employed to predict the DW front structure at the early stages of its formation. An emerging eigenvalue problem is solved with a global method using a Chebyshev pseudospectral method and the LAPACK software library. A local iterative shooting procedure is used for eigenvalue refinement. Numerical simulations of a propagation of a DW in plane and rectangular channels are performed with a shock capturing WENO scheme of 5th order. A special method of a computational domain shift is implemented in order to maintain the DW in the domain. It is shown that the linear analysis gives certain predictions about the DW structure that are in agreement with the numerical simulations of early stages of DW propagation. However, at later stages, a merger of detonation cells occurs so that their number is approximately halved. Computations of DW propagation in a square channel reveal two different types of spatial structure of the DW front, "rectangular" and "diagonal" types. A spontaneous transition from the rectangular to diagonal type of structure is observed during propagation of the DW.

Modulation of neuronal proteome profile in response to Japanese encephalitis virus infection.

PubMed

Sengupta, Nabonita; Ghosh, Sourish; Vasaikar, Suhas V; Gomes, James; Basu, Anirban

2014-01-01

In this study we have reported the in vivo proteomic changes during Japanese Encephalitis Virus (JEV) infection in combination with in vitro studies which will help in the comprehensive characterization of the modifications in the host metabolism in response to JEV infection. We performed a 2-DE based quantitative proteomic study of JEV-infected mouse brain as well as mouse neuroblastoma (Neuro2a) cells to analyze the host response to this lethal virus. 56 host proteins were found to be differentially expressed post JEV infection (defined as exhibiting ≥ 1.5-fold change in protein abundance upon JEV infection). Bioinformatics analyses were used to generate JEV-regulated host response networks which reported that the identified proteins were found to be associated with various cellular processes ranging from intracellular protein transport, cellular metabolism and ER stress associated unfolded protein response. JEV was found to invade the host protein folding machinery to sustain its survival and replication inside the host thereby generating a vigorous unfolded protein response, subsequently triggering a number of pathways responsible for the JEV associated pathologies. The results were also validated using a human cell line to correlate them to the human response to JEV. The present investigation is the first report on JEV-host interactome in in vivo model and will be of potential interest for future antiviral research in this field.

Formate generated by cellular oxidation of formaldehyde accelerates the glycolytic flux in cultured astrocytes.

PubMed

Tulpule, Ketki; Dringen, Ralf

2012-04-01

Formaldehyde is a neurotoxic compound that can be endogenously generated in the brain. Because astrocytes play a key role in metabolism and detoxification processes in brain, we have investigated the capacity of these cells to metabolize formaldehyde using primary astrocyte-rich cultures as a model system. Application of formaldehyde to these cultures resulted in the appearance of formate in cells and in a time-, concentration- and temperature-dependent disappearance of formaldehyde from the medium that was accompanied by a matching extracellular accumulation of formate. This formaldehyde-oxidizing capacity of astrocyte cultures is likely to be catalyzed by alcohol dehydrogenase 3 and aldehyde dehydrogenase 2, because the cells of the cultures contain the mRNAs of these formaldehyde-oxidizing enzymes. In addition, exposure to formaldehyde increased both glucose consumption and lactate production by the cells. Both the strong increase in the cellular formate content and the increase in glycolytic flux were only observed after application of formaldehyde to the cells, but not after treatment with exogenous methanol or formate. The accelerated lactate production was not additive to that obtained for azide, a known inhibitor of complex IV of the respiratory chain, and persisted after removal of formaldehyde after a formaldehyde exposure for 1.5 h. These data demonstrate that cultured astrocytes efficiently oxidize formaldehyde to formate, which subsequently enhances glycolytic flux, most likely by inhibition of mitochondrial respiration. Copyright © 2012 Wiley Periodicals, Inc.

Cell Therapy and Tissue Engineering Approaches for Cartilage Repair and/or Regeneration

PubMed Central

Mardones, Rodrigo; Jofré, Claudio M.; Minguell, José J.

2015-01-01

Articular cartilage injuries caused by traumatic, mechanical and/or by progressive degeneration result in pain, swelling, subsequent loss of joint function and finally osteoarthritis. Due to the peculiar structure of the tissue (no blood supply), chondrocytes, the unique cellular phenotype in cartilage, receive their nutrition through diffusion from the synovial fluid and this limits their intrinsic capacity for healing. The first cellular avenue explored for cartilage repair involved the in situ transplantation of isolated chondrocytes. Latterly, an improved alternative for the above reparative strategy involved the infusion of mesenchymal stem cells (MSC), which in addition to a self-renewal capacity exhibit a differentiation potential to chondrocytes, as well as a capability to produce a vast array of growth factors, cytokines and extracellular matrix compounds involved in cartilage development. In addition to the above and foremost reparative options up till now in use, other therapeutic options have been developed, comprising the design of biomaterial substrates (scaffolds) capable of sustaining MSC attachment, proliferation and differentiation. The implantation of these engineered platforms, closely to the site of cartilage damage, may well facilitate the initiation of an ‘in situ’ cartilage reparation process. In this mini-review, we examined the timely and conceptual development of several cell-based methods, designed to repair/regenerate a damaged cartilage. In addition to the above described cartilage reparative options, other therapeutic alternatives still in progress are portrayed. PMID:26019754

Cell Therapy and Tissue Engineering Approaches for Cartilage Repair and/or Regeneration.

PubMed

Mardones, Rodrigo; Jofré, Claudio M; Minguell, José J

2015-05-01

Articular cartilage injuries caused by traumatic, mechanical and/or by progressive degeneration result in pain, swelling, subsequent loss of joint function and finally osteoarthritis. Due to the peculiar structure of the tissue (no blood supply), chondrocytes, the unique cellular phenotype in cartilage, receive their nutrition through diffusion from the synovial fluid and this limits their intrinsic capacity for healing. The first cellular avenue explored for cartilage repair involved the in situ transplantation of isolated chondrocytes. Latterly, an improved alternative for the above reparative strategy involved the infusion of mesenchymal stem cells (MSC), which in addition to a self-renewal capacity exhibit a differentiation potential to chondrocytes, as well as a capability to produce a vast array of growth factors, cytokines and extracellular matrix compounds involved in cartilage development. In addition to the above and foremost reparative options up till now in use, other therapeutic options have been developed, comprising the design of biomaterial substrates (scaffolds) capable of sustaining MSC attachment, proliferation and differentiation. The implantation of these engineered platforms, closely to the site of cartilage damage, may well facilitate the initiation of an 'in situ' cartilage reparation process. In this mini-review, we examined the timely and conceptual development of several cell-based methods, designed to repair/regenerate a damaged cartilage. In addition to the above described cartilage reparative options, other therapeutic alternatives still in progress are portrayed.

Cellular mechanism of resistance of human colorectal adenocarcinoma cells against apoptosis-induction by Russell's Viper venom L-amino acid oxidase (Rusvinoxidase).

PubMed

Mukherjee, Ashis K; Saviola, Anthony J; Mackessy, Stephen P

2018-04-24

The present study highlights the cellular mechanism of resistance in human adenocarcinoma (Colo-205) cells against apoptosis induction by Rusvinoxidase, an L-amino acid oxidase purified from Russell's Viper venom (RVV). The significantly lower cytotoxicity as well as apoptotic activity of Rusvinoxidase towards Colo-205 cells (compared to MCF-7 breast cancer cells) is correlated with lower depletion of cellular glutathione content and increased down-regulation of catalase activity of Colo-205 cells following Rusvinoxidase treatment. Exposure to Rusvinoxidase subsequently diminished reactive oxygen species (ROS) production and failed to impair mitochondrial membrane potential, resulting in apoptosis induction resistance in Colo-205 cells. Further, higher expression levels of caspase 8, compared to caspase 9, indicate that Rusvinoxidase preferentially triggers the extrinsic pathway of apoptosis in Colo-205 cells. A time-dependent lower ratio of the relative expression of Bax and Bcl-xL (pro- and anti-apoptotic proteins) in Colo-205 cells, compared to our previous study on MCF-7 cells, unambiguously supports a higher cellular resistance mechanism in Colo-205 cells against Rusvinoxidase-induced apoptosis. Copyright © 2018. Published by Elsevier B.V.

Increased Foxo3a Nuclear Translocation and Activity is an Early Neuronal Response to βγ-Secretase-Mediated Processing of the Amyloid-β Protein Precursor: Utility of an AβPP-GAL4 Reporter Assay.

PubMed

Law, Bernard M; Guest, Amy L; Pullen, Matthew W J; Perkinton, Michael S; Williams, Robert J

2018-01-01

Sequential cleavage of the amyloid-β protein precursor (AβPP) by BACE1 (β-secretase) followed by theγ-secretase complex, is strongly implicated in Alzheimer's disease (AD) but the initial cellular responses to these cleavage events are not fully defined. β-secretase-mediated AβPP processing yields an extracellular domain (sAβPPβ) and a C-terminal fragment of AβPP of 99 amino acids (C99). Subsequent cleavage by γ-secretase produces amyloid-β (Aβ) and an AβPP intracellular domain (AICD). A cellular screen based on the generation of AICD from an AβPP-Gal4 fusion protein was adapted by introducing familial AD (FAD) mutations into the AβPP sequence and linking the assay to Gal4-UAS driven luciferase and GFP expression, to identify responses immediately downstream of AβPP processing in neurons with a focus on the transcription factor Foxo3a which has been implicated in neurodegeneration. The K670N/M671L, E682K, E693G, and V717I FAD mutations and the A673T protective mutation, were introduced into the AβPP sequence by site directed mutagenesis. When expressed in mouse cortical neurons, AβPP-Gal4-UAS driven luciferase and GFP expression was substantially reduced by γ-secretase inhibitors, lowered by β-secretase inhibitors, and enhanced by α-secretase inhibitors suggesting that AICD is a product of the βγ-secretase pathway. AβPP-Gal4-UAS driven GFP expression was exploited to identify individual neurons undergoing amyloidogenic AβPP processing, revealing increased nuclear localization of Foxo3a and enhanced Foxo3a-mediated transcription downstream of AICD production. Foxo3a translocation was not driven by AICD directly but correlated with reduced Akt phosphorylation. Collectively this suggests that βγ-secretase-mediated AβPP processing couples to Foxo3a which could be an early neuronal signaling response in AD.

The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells.

PubMed

Parker, Andrew; Cross, Sally H; Jackson, Ian J; Hardisty-Hughes, Rachel; Morse, Susan; Nicholson, George; Coghill, Emma; Bowl, Michael R; Brown, Steve D M

2015-12-01

Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss. © 2015. Published by The Company of Biologists Ltd.

Molecular and Cellular Biology Animations: Development and Impact on Student Learning

ERIC Educational Resources Information Center

McClean, Phillip; Johnson, Christina; Rogers, Roxanne; Daniels, Lisa; Reber, John; Slator, Brian M.; Terpstra, Jeff; White, Alan

2005-01-01

Educators often struggle when teaching cellular and molecular processes because typically they have only two-dimensional tools to teach something that plays out in four dimensions. Learning research has demonstrated that visualizing processes in three dimensions aids learning, and animations are effective visualization tools for novice learners…

Preparation of a micropatterned rigid-soft composite substrate for probing cellular rigidity sensing.

PubMed

Wong, Stephanie; Guo, Wei-hui; Hoffecker, Ian; Wang, Yu-li

2014-01-01

Substrate rigidity has been recognized as an important property that affects cellular physiology and functions. While the phenomenon has been well recognized, understanding the underlying mechanism may be greatly facilitated by creating a microenvironment with designed rigidity patterns. This chapter describes in detail an optimized method for preparing substrates with micropatterned rigidity, taking advantage of the ability to dehydrate polyacrylamide gels for micropatterning with photolithography, and subsequently rehydrate the gel to regain the original elastic state. While a wide range of micropatterns may be prepared, typical composite substrates consist of micron-sized islands of rigid photoresist grafted on the surface of polyacrylamide hydrogels of defined rigidity. These islands are displaced by cellular traction forces, for a distance determined by the size of the island, the rigidity of the underlying hydrogel, and the magnitude of traction forces. Domains of rigidity may be created using this composite material to allow systematic investigations of rigidity sensing and durotaxis. Copyright © 2014 Elsevier Inc. All rights reserved.

Targeting inflammation in pancreatic cancer: Clinical translation

PubMed Central

Steele, Colin William; Kaur Gill, Nina Angharad; Jamieson, Nigel Balfour; Carter, Christopher Ross

2016-01-01

Preclinical modelling studies are beginning to aid development of therapies targeted against key regulators of pancreatic cancer progression. Pancreatic cancer is an aggressive, stromally-rich tumor, from which few people survive. Within the tumor microenvironment cellular and extracellular components exist, shielding tumor cells from immune cell clearance, and chemotherapy, enhancing progression of the disease. The cellular component of this microenvironment consists mainly of stellate cells and inflammatory cells. New findings suggest that manipulation of the cellular component of the tumor microenvironment is possible to promote immune cell killing of tumor cells. Here we explore possible immunogenic therapeutic strategies. Additionally extracellular stromal elements play a key role in protecting tumor cells from chemotherapies targeted at the pancreas. We describe the experimental findings and the pitfalls associated with translation of stromally targeted therapies to clinical trial. Finally, we discuss the key inflammatory signal transducers activated subsequent to driver mutations in oncogenic Kras in pancreatic cancer. We present the preclinical findings that have led to successful early trials of STAT3 inhibitors in pancreatic adenocarcinoma. PMID:27096033

Untangling the origin of viruses and their impact on cellular evolution.

PubMed

Nasir, Arshan; Sun, Feng-Jie; Kim, Kyung Mo; Caetano-Anollés, Gustavo

2015-04-01

The origin and evolution of viruses remain mysterious. Here, we focus on the distribution of viral replicons in host organisms, their morphological features, and the evolution of highly conserved protein and nucleic acid structures. The apparent inability of RNA viral replicons to infect contemporary akaryotic species suggests an early origin of RNA viruses and their subsequent loss in akaryotes. A census of virion morphotypes reveals that advanced forms were unique to viruses infecting a specific supergroup, while simpler forms were observed in viruses infecting organisms in all forms of cellular life. Results hint toward an ancient origin of viruses from an ancestral virus harboring either filamentous or spherical virions. Finally, phylogenetic trees built from protein domain and tRNA structures in thousands of genomes suggest that viruses evolved via reductive evolution from ancient cells. The analysis presents a complete account of the evolutionary history of cells and viruses and identifies viruses as crucial agents influencing cellular evolution. © 2015 New York Academy of Sciences.

Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

NASA Astrophysics Data System (ADS)

Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya; Ookawara, Shigeo; Tanaka, Hozumi; Kobayashi, Eiji; Murakami, Takashi

2009-09-01

Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

Differential interference effects of negative emotional states on subsequent semantic and perceptual processing

PubMed Central

Gorlick, Marissa A.; Mather, Mara

2012-01-01

Past studies have revealed that encountering negative events interferes with cognitive processing of subsequent stimuli. The present study investigated whether negative events affect semantic and perceptual processing differently. Presentation of negative pictures produced slower reaction times than neutral or positive pictures in tasks that require semantic processing, such as natural/man-made judgments about drawings of objects, commonness judgments about objects, and categorical judgments about pairs of words. In contrast, negative picture presentation did not slow down judgments in subsequent perceptual processing (e.g., color judgments about words, and size judgments about objects). The subjective arousal level of negative pictures did not modulate the interference effects on semantic/perceptual processing. These findings indicate that encountering negative emotional events interferes with semantic processing of subsequent stimuli more strongly than perceptual processing, and that not all types of subsequent cognitive processing are impaired by negative events. PMID:22142207

Differential interference effects of negative emotional states on subsequent semantic and perceptual processing.

PubMed

Sakaki, Michiko; Gorlick, Marissa A; Mather, Mara

2011-12-01

Past studies have revealed that encountering negative events interferes with cognitive processing of subsequent stimuli. The present study investigates whether negative events affect semantic and perceptual processing differently. Presentation of negative pictures produced slower reaction times than neutral or positive pictures in tasks that require semantic processing, such as natural or man-made judgments about drawings of objects, commonness judgments about objects, and categorical judgments about pairs of words. In contrast, negative picture presentation did not slow down judgments in subsequent perceptual processing (e.g., color judgments about words, size judgments about objects). The subjective arousal level of negative pictures did not modulate the interference effects on semantic or perceptual processing. These findings indicate that encountering negative emotional events interferes with semantic processing of subsequent stimuli more strongly than perceptual processing, and that not all types of subsequent cognitive processing are impaired by negative events. (c) 2011 APA, all rights reserved.

Seeking a unified framework for cerebellar function and dysfunction: from circuit operations to cognition

PubMed Central

D'Angelo, Egidio; Casali, Stefano

2013-01-01

Following the fundamental recognition of its involvement in sensory-motor coordination and learning, the cerebellum is now also believed to take part in the processing of cognition and emotion. This hypothesis is recurrent in numerous papers reporting anatomical and functional observations, and it requires an explanation. We argue that a similar circuit structure in all cerebellar areas may carry out various operations using a common computational scheme. On the basis of a broad review of anatomical data, it is conceivable that the different roles of the cerebellum lie in the specific connectivity of the cerebellar modules, with motor, cognitive, and emotional functions (at least partially) segregated into different cerebro-cerebellar loops. We here develop a conceptual and operational framework based on multiple interconnected levels (a meta-levels hypothesis): from cellular/molecular to network mechanisms leading to generation of computational primitives, thence to high-level cognitive/emotional processing, and finally to the sphere of mental function and dysfunction. The main concept explored is that of intimate interplay between timing and learning (reminiscent of the “timing and learning machine” capabilities long attributed to the cerebellum), which reverberates from cellular to circuit mechanisms. Subsequently, integration within large-scale brain loops could generate the disparate cognitive/emotional and mental functions in which the cerebellum has been implicated. We propose, therefore, that the cerebellum operates as a general-purpose co-processor, whose effects depend on the specific brain centers to which individual modules are connected. Abnormal functioning in these loops could eventually contribute to the pathogenesis of major brain pathologies including not just ataxia but also dyslexia, autism, schizophrenia, and depression. PMID:23335884

Bioinformatics analysis reveals biophysical and evolutionary insights into the 3-nitrotyrosine post-translational modification in the human proteome

PubMed Central

Ng, John Y.; Boelen, Lies; Wong, Jason W. H.

2013-01-01

Protein 3-nitrotyrosine is a post-translational modification that commonly arises from the nitration of tyrosine residues. This modification has been detected under a wide range of pathological conditions and has been shown to alter protein function. Whether 3-nitrotyrosine is important in normal cellular processes or is likely to affect specific biological pathways remains unclear. Using GPS-YNO2, a recently described 3-nitrotyrosine prediction algorithm, a set of predictions for nitrated residues in the human proteome was generated. In total, 9.27 per cent of the proteome was predicted to be nitratable (27 922/301 091). By matching the predictions against a set of curated and experimentally validated 3-nitrotyrosine sites in human proteins, it was found that GPS-YNO2 is able to predict 73.1 per cent (404/553) of these sites. Furthermore, of these sites, 42 have been shown to be nitrated endogenously, with 85.7 per cent (36/42) of these predicted to be nitrated. This demonstrates the feasibility of using the predicted dataset for a whole proteome analysis. A comprehensive bioinformatics analysis was subsequently performed on predicted and all experimentally validated nitrated tyrosine. This found mild but specific biophysical constraints that affect the susceptibility of tyrosine to nitration, and these may play a role in increasing the likelihood of 3-nitrotyrosine to affect processes, including phosphorylation and DNA binding. Furthermore, examining the evolutionary conservation of predicted 3-nitrotyrosine showed that, relative to non-nitrated tyrosine residues, 3-nitrotyrosine residues are generally less conserved. This suggests that, at least in the majority of cases, 3-nitrotyrosine is likely to have a deleterious effect on protein function and less likely to be important in normal cellular function. PMID:23389939

Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses.

PubMed

Ozbun, Michelle A; Patterson, Nicole A

2014-08-01

Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection. Copyright © 2014 John Wiley & Sons, Inc.

Mitochondrial NUDIX hydrolases: A metabolic link between NAD catabolism, GTP and mitochondrial dynamics.

PubMed

Long, Aaron; Klimova, Nina; Kristian, Tibor

2017-10-01

NAD + catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD + catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD + pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics. Published by Elsevier Ltd.

Transposon mutagenesis identifies genes and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma

PubMed Central

Kodama, Takahiro; Newberg, Justin Y.; Kodama, Michiko; Rangel, Roberto; Yoshihara, Kosuke; Tien, Jean C.; Parsons, Pamela H.; Wu, Hao; Finegold, Milton J.; Copeland, Neal G.; Jenkins, Nancy A.

2016-01-01

Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets. PMID:27247392

Processing of the major autolysin of E. faecalis, AtlA, by the zinc-metalloprotease, GelE, impacts AtlA septal localization and cell separation.

PubMed

Stinemetz, Emily K; Gao, Peng; Pinkston, Kenneth L; Montealegre, Maria Camila; Murray, Barbara E; Harvey, Barrett R

2017-01-01

AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE), has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were developed as research tools. Flow cytometry and Western blot analysis suggests that in the presence of GelE, surface-bound AtlA exists primarily as a N-terminally truncated form whereas in the absence of GelE, the N-terminal domain of AtlA is retained. We identified the primary GelE cleavage site occurring near the transition between the T/E rich Domain I and catalytic region, Domain II via N-terminal sequencing. Truncation of AtlA had no effect on the peptidoglycan hydrolysis activity of AtlA. However, we observed that N-terminal cleavage was required for efficient AtlA-mediated cell division while unprocessed AtlA was unable to resolve dividing cells into individual units. Furthermore, we observed that the processed AtlA has the propensity to localize to the cell septum on wild-type cells whereas unprocessed AtlA in the ΔgelE strain were dispersed over the cell surface. Combined, these results suggest that AtlA septum localization and subsequent cell separation can be modulated by a single GelE-mediated N-terminal cleavage event, providing new insights into the post-translation modification of AtlA and the mechanisms governing chaining and cell separation.

Processing of the major autolysin of E. faecalis, AtlA, by the zinc-metalloprotease, GelE, impacts AtlA septal localization and cell separation

PubMed Central

Pinkston, Kenneth L.; Montealegre, Maria Camila; Murray, Barbara E.

2017-01-01

AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE), has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were developed as research tools. Flow cytometry and Western blot analysis suggests that in the presence of GelE, surface-bound AtlA exists primarily as a N-terminally truncated form whereas in the absence of GelE, the N-terminal domain of AtlA is retained. We identified the primary GelE cleavage site occurring near the transition between the T/E rich Domain I and catalytic region, Domain II via N-terminal sequencing. Truncation of AtlA had no effect on the peptidoglycan hydrolysis activity of AtlA. However, we observed that N-terminal cleavage was required for efficient AtlA-mediated cell division while unprocessed AtlA was unable to resolve dividing cells into individual units. Furthermore, we observed that the processed AtlA has the propensity to localize to the cell septum on wild-type cells whereas unprocessed AtlA in the ΔgelE strain were dispersed over the cell surface. Combined, these results suggest that AtlA septum localization and subsequent cell separation can be modulated by a single GelE-mediated N-terminal cleavage event, providing new insights into the post-translation modification of AtlA and the mechanisms governing chaining and cell separation. PMID:29049345

Inflammatory response and extracorporeal circulation.

PubMed

Kraft, Florian; Schmidt, Christoph; Van Aken, Hugo; Zarbock, Alexander

2015-06-01

Patients undergoing cardiac surgery with extracorporeal circulation (EC) frequently develop a systemic inflammatory response syndrome. Surgical trauma, ischaemia-reperfusion injury, endotoxaemia and blood contact to nonendothelial circuit compounds promote the activation of coagulation pathways, complement factors and a cellular immune response. This review discusses the multiple pathways leading to endothelial cell activation, neutrophil recruitment and production of reactive oxygen species and nitric oxide. All these factors may induce cellular damage and subsequent organ injury. Multiple organ dysfunction after cardiac surgery with EC is associated with an increased morbidity and mortality. In addition to the pathogenesis of organ dysfunction after EC, this review deals with different therapeutic interventions aiming to alleviate the inflammatory response and consequently multiple organ dysfunction after cardiac surgery. Copyright © 2015 Elsevier Ltd. All rights reserved.

Development of LC/MS/MS, high-throughput enzymatic and cellular assays for the characterization of compounds that inhibit kynurenine monooxygenase (KMO).

PubMed

Winkler, Dirk; Beconi, Maria; Toledo-Sherman, Leticia M; Prime, Michael; Ebneth, Andreas; Dominguez, Celia; Muñoz-Sanjuan, Ignacio

2013-09-01

Kynurenine monooxygenase (KMO) catalyzes the conversion of kynurenine to 3-hydroxykynurenine. Modulation of KMO activity has been implicated in several neurodegenerative diseases, including Huntington disease. Our goal is to develop potent and selective small-molecule KMO inhibitors with suitable pharmacokinetic characteristics for in vivo proof-of-concept studies and subsequent clinical development. We developed a comprehensive panel of biochemical and cell-based assays that use liquid chromatography/tandem mass spectrometry to quantify unlabeled kynurenine and 3-hydroxykynurenine. We describe assays to measure KMO inhibition in cell and tissue extracts, as well as cellular assays including heterologous cell lines and primary rat microglia and human peripheral blood mononuclear cells.

The effect of zinc on cellular immunity in chronic uremia.

PubMed

Antoniou, L D; Shalhoub, R J; Schechter, G P

1981-09-01

Delayed hypersensitivity to mumps was examined in 25 apparently well-nourished men receiving regular hemodialysis, each of whom had a history of mumps. A positive reaction was observed in eight of nine patients already under therapy with zinc added to the dialysis bath. In contrast, 11 of 16 untreated patients were anergic. Four of the anergic patients were subsequently treated with zinc resulting in restoration of sensitivity in three patients. There were no significant differences in lymphocyte, monocyte, or T-cell counts between the two groups of patients. Consequently, zinc probably acts by improving the function of one or more of these cell types. Protracted zinc deficiency may be a major cause of impaired cellular immunity in chronic renal failure.

Landslides, forest fires, and earthquakes: examples of self-organized critical behavior

NASA Astrophysics Data System (ADS)

Turcotte, Donald L.; Malamud, Bruce D.

2004-09-01

Per Bak conceived self-organized criticality as an explanation for the behavior of the sandpile model. Subsequently, many cellular automata models were found to exhibit similar behavior. Two examples are the forest-fire and slider-block models. Each of these models can be associated with a serious natural hazard: the sandpile model with landslides, the forest-fire model with actual forest fires, and the slider-block model with earthquakes. We examine the noncumulative frequency-area statistics for each natural hazard, and show that each has a robust power-law (fractal) distribution. We propose an inverse-cascade model as a general explanation for the power-law frequency-area statistics of the three cellular-automata models and their ‘associated’ natural hazards.

3D Printing Variable Stiffness Foams Using Viscous Thread Instability

PubMed Central

Lipton, Jeffrey I.; Lipson, Hod

2016-01-01

Additive manufacturing of cellular structures has numerous applications ranging from fabrication of biological scaffolds and medical implants, to mechanical weight reduction and control over mechanical properties. Various additive manufacturing processes have been used to produce open regular cellular structures limited only by the resolution of the printer. These efforts have focused on printing explicitly designed cells or explicitly planning offsets between strands. Here we describe a technique for producing cellular structures implicitly by inducing viscous thread instability when extruding material. This process allows us to produce complex cellular structures at a scale that is finer than the native resolution of the printer. We demonstrate tunable effective elastic modulus and density that span two orders of magnitude. Fine grained cellular structures allow for fabrication of foams for use in a wide range of fields ranging from bioengineering, to robotics to food printing. PMID:27503148

Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes

PubMed Central

2004-01-01

14-3-3 proteins exert an extraordinarily widespread influence on cellular processes in all eukaryotes. They operate by binding to specific phosphorylated sites on diverse target proteins, thereby forcing conformational changes or influencing interactions between their targets and other molecules. In these ways, 14-3-3s ‘finish the job’ when phosphorylation alone lacks the power to drive changes in the activities of intracellular proteins. By interacting dynamically with phosphorylated proteins, 14-3-3s often trigger events that promote cell survival – in situations from preventing metabolic imbalances caused by sudden darkness in leaves to mammalian cell-survival responses to growth factors. Recent work linking specific 14-3-3 isoforms to genetic disorders and cancers, and the cellular effects of 14-3-3 agonists and antagonists, indicate that the cellular complement of 14-3-3 proteins may integrate the specificity and strength of signalling through to different cellular responses. PMID:15167810

Lipids, lysosomes, and autophagy

PubMed Central

2016-01-01

Lipids are essential components of a cell providing energy substrates for cellular processes, signaling intermediates, and building blocks for biological membranes. Lipids are constantly recycled and redistributed within a cell. Lysosomes play an important role in this recycling process that involves the recruitment of lipids to lysosomes via autophagy or endocytosis for their degradation by lysosomal hydrolases. The catabolites produced are redistributed to various cellular compartments to support basic cellular function. Several studies demonstrated a bidirectional relationship between lipids and lysosomes that regulate autophagy. While lysosomal degradation pathways regulate cellular lipid metabolism, lipids also regulate lysosome function and autophagy. In this review, we focus on this bidirectional relationship in the context of dietary lipids and provide an overview of recent evidence of how lipid-overload lipotoxicity, as observed in obesity and metabolic syndrome, impairs lysosomal function and autophagy that may eventually lead to cellular dysfunction or cell death. PMID:27330054

SAOS-2 osteosarcoma cells bind fibroblasts via ICAM-1 and this is increased by tumour necrosis factor-α.

PubMed

David, Manu S; Kelly, Elizabeth; Cheung, Ivan; Xaymardan, Munira; Moore, Malcolm A S; Zoellner, Hans

2014-01-01

We recently reported exchange of membrane and cytoplasmic markers between SAOS-2 osteosarcoma cells and human gingival fibroblasts (h-GF) without comparable exchange of nuclear markers, while similar h-GF exchange was seen for melanoma and ovarian carcinoma cells. This process of "cellular sipping" changes phenotype such that cells sharing markers of both SAOS-2 and h-GF have morphology intermediate to that of either cell population cultured alone, evidencing increased tumour cell diversity without genetic change. TNF-α increases cellular sipping between h-GF and SAOS-2, and we here study binding of SAOS-2 to TNF-α treated h-GF to determine if increased cellular sipping can be accounted for by cytokine stimulated SAOS-2 binding. More SAOS-2 bound h-GF pe-seeded wells than culture plastic alone (p<0.001), and this was increased by h-GF pre-treatment with TNF-α (p<0.001). TNF-α stimulated binding was dose dependent and maximal at 1.16 nM (p<0.05) with no activity below 0.006 nM. SAOS-2 binding to h-GF was independent of serum, while the lipopolysaccharide antagonist Polymyxin B did not affect results, and TNF-α activity was lost on boiling. h-GF binding of SAOS-2 started to increase after 30min TNF-α stimulation and was maximal by 1.5 hr pre-treatment (p<0.001). h-GF retained maximal binding up to 6 hrs after TNF-α stimulation, but this was lost by 18 hrs (p<0.001). FACS analysis demonstrated increased ICAM-1 consistent with the time course of SAOS-2 binding, while antibody against ICAM-1 inhibited SAOS-2 adhesion (p<0.04). Pre-treating SAOS-2 with TNF-α reduced h-GF binding to background levels (p<0.003), and this opposite effect to h-GF cytokine stimulation suggests that the history of cytokine exposure of malignant cells migrating across different microenvironments can influence subsequent interactions with fibroblasts. Since cytokine stimulated binding was comparable in magnitude to earlier reported TNF-α stimulated cellular sipping, we conclude that TNF-α stimulated cellular sipping likely reflects increased SAOS-2 binding as opposed to enhanced exchange mechanisms.

Understanding Cellular Respiration: An Analysis of Conceptual Change in College Biology.

ERIC Educational Resources Information Center

Songer, Catherine J.; Mintzes, Joel J.

1994-01-01

Explores and documents the frequencies of conceptual difficulties confronted by college students (n=200) seeking to understand the basic processes of cellular respiration. Findings suggest that novices harbor a wide range of conceptual difficulties that constrain their understanding of cellular respiration and many of these conceptual problems…

Nanobodies and recombinant binders in cell biology.

PubMed

Helma, Jonas; Cardoso, M Cristina; Muyldermans, Serge; Leonhardt, Heinrich

2015-06-08

Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes. © 2015 Helma et al.

L1000CDS2: LINCS L1000 characteristic direction signatures search engine.

PubMed

Duan, Qiaonan; Reid, St Patrick; Clark, Neil R; Wang, Zichen; Fernandez, Nicolas F; Rouillard, Andrew D; Readhead, Ben; Tritsch, Sarah R; Hodos, Rachel; Hafner, Marc; Niepel, Mario; Sorger, Peter K; Dudley, Joel T; Bavari, Sina; Panchal, Rekha G; Ma'ayan, Avi

2016-01-01

The library of integrated network-based cellular signatures (LINCS) L1000 data set currently comprises of over a million gene expression profiles of chemically perturbed human cell lines. Through unique several intrinsic and extrinsic benchmarking schemes, we demonstrate that processing the L1000 data with the characteristic direction (CD) method significantly improves signal to noise compared with the MODZ method currently used to compute L1000 signatures. The CD processed L1000 signatures are served through a state-of-the-art web-based search engine application called L1000CDS 2 . The L1000CDS 2 search engine provides prioritization of thousands of small-molecule signatures, and their pairwise combinations, predicted to either mimic or reverse an input gene expression signature using two methods. The L1000CDS 2 search engine also predicts drug targets for all the small molecules profiled by the L1000 assay that we processed. Targets are predicted by computing the cosine similarity between the L1000 small-molecule signatures and a large collection of signatures extracted from the gene expression omnibus (GEO) for single-gene perturbations in mammalian cells. We applied L1000CDS 2 to prioritize small molecules that are predicted to reverse expression in 670 disease signatures also extracted from GEO, and prioritized small molecules that can mimic expression of 22 endogenous ligand signatures profiled by the L1000 assay. As a case study, to further demonstrate the utility of L1000CDS 2 , we collected expression signatures from human cells infected with Ebola virus at 30, 60 and 120 min. Querying these signatures with L1000CDS 2 we identified kenpaullone, a GSK3B/CDK2 inhibitor that we show, in subsequent experiments, has a dose-dependent efficacy in inhibiting Ebola infection in vitro without causing cellular toxicity in human cell lines. In summary, the L1000CDS 2 tool can be applied in many biological and biomedical settings, while improving the extraction of knowledge from the LINCS L1000 resource.

Real space flight travel is associated with ultrastructural changes, cytoskeletal disruption and premature senescence of HUVEC.

PubMed

Kapitonova, M Y; Muid, S; Froemming, G R A; Yusoff, W N W; Othman, S; Ali, A M; Nawawi, H M

2012-12-01

Microgravity, hypergravity, vibration, ionizing radiation and temperature fluctuations are major factors of outer space flight affecting human organs and tissues. There are several reports on the effect of space flight on different human cell types of mesenchymal origin while information regarding changes to vascular endothelial cells is scarce. Ultrastructural and cytophysiological features of macrovascular endothelial cells in outer space flight and their persistence during subsequent culturing were demonstrated in the present investigation. At the end of the space flight, endothelial cells displayed profound changes indicating cytoskeletal lesions and increased cell membrane permeability. Readapted cells of subsequent passages exhibited persisting cytoskeletal changes, decreased metabolism and cell growth indicating cellular senescence.

Pretreatment of high solid microbial sludges

DOEpatents

Rivard, C.J.; Nagle, N.J.

1998-07-28

A process and apparatus are disclosed for pretreating microbial sludges in order to enhance secondary anaerobic digestion. The pretreatment process involves disrupting the cellular integrity of municipal sewage sludge through a combination of thermal, explosive decompression and shear forces. The sludge is pressurized and pumped to a pretreatment reactor where it is mixed with steam to heat and soften the sludge. The pressure of the sludge is suddenly reduced and explosive decompression forces are imparted which partially disrupt the cellular integrity of the sludge. Shear forces are then applied to the sludge to further disrupt the cellular integrity of the sludge. Disrupting cellular integrity releases both soluble and insoluble organic constituents and thereby renders municipal sewage sludge more amenable to secondary anaerobic digestion. 1 fig.

Cellular Particle Dynamics simulation of biomechanical relaxation processes of multi-cellular systems

NASA Astrophysics Data System (ADS)

McCune, Matthew; Kosztin, Ioan

2013-03-01

Cellular Particle Dynamics (CPD) is a theoretical-computational-experimental framework for describing and predicting the time evolution of biomechanical relaxation processes of multi-cellular systems, such as fusion, sorting and compression. In CPD, cells are modeled as an ensemble of cellular particles (CPs) that interact via short range contact interactions, characterized by an attractive (adhesive interaction) and a repulsive (excluded volume interaction) component. The time evolution of the spatial conformation of the multicellular system is determined by following the trajectories of all CPs through numerical integration of their equations of motion. Here we present CPD simulation results for the fusion of both spherical and cylindrical multi-cellular aggregates. First, we calibrate the relevant CPD model parameters for a given cell type by comparing the CPD simulation results for the fusion of two spherical aggregates to the corresponding experimental results. Next, CPD simulations are used to predict the time evolution of the fusion of cylindrical aggregates. The latter is relevant for the formation of tubular multi-cellular structures (i.e., primitive blood vessels) created by the novel bioprinting technology. Work supported by NSF [PHY-0957914]. Computer time provided by the University of Missouri Bioinformatics Consortium.

In vivo cell biology in zebrafish - providing insights into vertebrate development and disease.

PubMed

Vacaru, Ana M; Unlu, Gokhan; Spitzner, Marie; Mione, Marina; Knapik, Ela W; Sadler, Kirsten C

2014-02-01

Over the past decades, studies using zebrafish have significantly advanced our understanding of the cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos that allow comprehensive imaging of embryogenesis combined with powerful genetic approaches. However, forward genetic screens in zebrafish have generated unanticipated findings that are mirrored by human genetic studies: disruption of genes implicated in basic cellular processes, such as protein secretion or cytoskeletal dynamics, causes discrete developmental or disease phenotypes. This is surprising because many processes that were assumed to be fundamental to the function and survival of all cell types appear instead to be regulated by cell-specific mechanisms. Such discoveries are facilitated by experiments in whole animals, where zebrafish provides an ideal model for visualization and manipulation of organelles and cellular processes in a live vertebrate. Here, we review well-characterized mutants and newly developed tools that underscore this notion. We focus on the secretory pathway and microtubule-based trafficking as illustrative examples of how studying cell biology in vivo using zebrafish has broadened our understanding of the role fundamental cellular processes play in embryogenesis and disease.

Neuro-Immune Mechanisms in Response to Venezuelan equine encephalitis Virus Infection

DTIC Science & Technology

2000-05-01

horses . They were subsequently shown to be previously unrecognized viral agents of severe equine encephalitis (Smith et al., 1997). One member of...iii ABSTRACT NEURO-IMMUNE MECHANISMS IN RESPONSE TO VENEZUELAN EQUINE ENCEPHALITIS VIRUS INFECTION Major Bruce A. Schoneboom directed by Franziska B...Grieder, DVM, Ph.D., Assistant Professor of Microbiology and Immunology, Molecular and Cellular Biology, and Neuroscience Venezuelan equine

Analysis of biological effects in human endothelial cells after stimulated microgravity

NASA Astrophysics Data System (ADS)

Min, Zhang; Sun, Yeqing; Xu, Dan

Space environment is characterized by strong radiation, ultra-high vacuum, weak magnetic field and microgravity. Among them, microgravity (10-4-10-6g) in space is different from gravity (1g) on earth, possibly causing visual disorders, muscle alterations, bone loss and dysfunction of cardiovascular systems. To study about microgravity environment, the most advanced rotary cell culture system (RCCS-1) was used to do stimulated microgravity (SMG) experiments in the ground. Up to now, most of studies focus on the biological effects under stimulated microgravity, but it is less known about the cellular response after stimulated microgravity. In the present study, we explored the subsequent effects of stimulated microgravity on human endothelial cells (HUVEC-C) after these cells were cultured on RCCS-1 for 48 hours. We co-cultured HUVEC-C cells with Hillex-microcarriers in 60-mm culture dishes for 24h, followed by transferring them to RCCS-1 so that cells remain to be the state of SMG. In parallel, HUVEC-C cells were co-cultured with microcarriers in the ground condition. We found that stimulated microgravity induced cytoskeleton remodeling, cell cycle G2/M arrest and cellular senescence, consistent with previous reports. To study the subsequent effects of stimulated microgravity, we make cells detach from microcarriers and observed various effects including cell growth, cell adhesion, cytoskeleton, cell cycle, apoptosis and senescence. The results showed that those cells undergoing stimulated microgravity appeared obvious growth inhibition, a transition from the decrease in cell adhesion ability and cytoskeleton remodeling within 24h to induction of apoptosis and senescence-like phenotype in the later time with slight changes in cell cycle. Analysis of protein expression in western blot demonstrated that apoptosis-related protein PTEN was up-regulated on the time-dependent pattern after stimulated microgravity, indicating that PTEN-PI3K-Akt pathway might play an important role in apoptosis. Our study suggests that stimulated microgravity has the subsequent biological effects of HUVEC-C, providing new insight of understanding the global effect of microgravity on cellular response in human endothelial cells.

High content screening in neurodegenerative diseases.

PubMed

Jain, Shushant; van Kesteren, Ronald E; Heutink, Peter

2012-01-06

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention. High-throughput screening (HTS) is an ideal methodology to address these issues. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS). HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. HCS has many advantages over HTS, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism. Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets. Copyright © 2012 Creative Commons Attribution License

Change Is Good: Variations in Common Biological Mechanisms in the Epsilonproteobacterial Genera Campylobacter and Helicobacter

PubMed Central

Gilbreath, Jeremy J.; Cody, William L.; Merrell, D. Scott; Hendrixson, David R.

2011-01-01

Summary: Microbial evolution and subsequent species diversification enable bacterial organisms to perform common biological processes by a variety of means. The epsilonproteobacteria are a diverse class of prokaryotes that thrive in diverse habitats. Many of these environmental niches are labeled as extreme, whereas other niches include various sites within human, animal, and insect hosts. Some epsilonproteobacteria, such as Campylobacter jejuni and Helicobacter pylori, are common pathogens of humans that inhabit specific regions of the gastrointestinal tract. As such, the biological processes of pathogenic Campylobacter and Helicobacter spp. are often modeled after those of common enteric pathogens such as Salmonella spp. and Escherichia coli. While many exquisite biological mechanisms involving biochemical processes, genetic regulatory pathways, and pathogenesis of disease have been elucidated from studies of Salmonella spp. and E. coli, these paradigms often do not apply to the same processes in the epsilonproteobacteria. Instead, these bacteria often display extensive variation in common biological mechanisms relative to those of other prototypical bacteria. In this review, five biological processes of commonly studied model bacterial species are compared to those of the epsilonproteobacteria C. jejuni and H. pylori. Distinct differences in the processes of flagellar biosynthesis, DNA uptake and recombination, iron homeostasis, interaction with epithelial cells, and protein glycosylation are highlighted. Collectively, these studies support a broader view of the vast repertoire of biological mechanisms employed by bacteria and suggest that future studies of the epsilonproteobacteria will continue to provide novel and interesting information regarding prokaryotic cellular biology. PMID:21372321

Change is good: variations in common biological mechanisms in the epsilonproteobacterial genera Campylobacter and Helicobacter.

PubMed

Gilbreath, Jeremy J; Cody, William L; Merrell, D Scott; Hendrixson, David R

2011-03-01

Microbial evolution and subsequent species diversification enable bacterial organisms to perform common biological processes by a variety of means. The epsilonproteobacteria are a diverse class of prokaryotes that thrive in diverse habitats. Many of these environmental niches are labeled as extreme, whereas other niches include various sites within human, animal, and insect hosts. Some epsilonproteobacteria, such as Campylobacter jejuni and Helicobacter pylori, are common pathogens of humans that inhabit specific regions of the gastrointestinal tract. As such, the biological processes of pathogenic Campylobacter and Helicobacter spp. are often modeled after those of common enteric pathogens such as Salmonella spp. and Escherichia coli. While many exquisite biological mechanisms involving biochemical processes, genetic regulatory pathways, and pathogenesis of disease have been elucidated from studies of Salmonella spp. and E. coli, these paradigms often do not apply to the same processes in the epsilonproteobacteria. Instead, these bacteria often display extensive variation in common biological mechanisms relative to those of other prototypical bacteria. In this review, five biological processes of commonly studied model bacterial species are compared to those of the epsilonproteobacteria C. jejuni and H. pylori. Distinct differences in the processes of flagellar biosynthesis, DNA uptake and recombination, iron homeostasis, interaction with epithelial cells, and protein glycosylation are highlighted. Collectively, these studies support a broader view of the vast repertoire of biological mechanisms employed by bacteria and suggest that future studies of the epsilonproteobacteria will continue to provide novel and interesting information regarding prokaryotic cellular biology.

RNA methylation in nuclear pre-mRNA processing.

PubMed

Covelo-Molares, Helena; Bartosovic, Marek; Vanacova, Stepanka

2018-06-19

Eukaryotic RNA can carry more than 100 different types of chemical modifications. Early studies have been focused on modifications of highly abundant RNA, such as ribosomal RNA and transfer RNA, but recent technical advances have made it possible to also study messenger RNA (mRNA). Subsequently, mRNA modifications, namely methylation, have emerged as key players in eukaryotic gene expression regulation. The most abundant and widely studied internal mRNA modification is N 6 -methyladenosine (m 6 A), but the list of mRNA chemical modifications continues to grow as fast as interest in this field. Over the past decade, transcriptome-wide studies combined with advanced biochemistry and the discovery of methylation writers, readers, and erasers revealed roles for mRNA methylation in the regulation of nearly every aspect of the mRNA life cycle and in diverse cellular, developmental, and disease processes. Although large parts of mRNA function are linked to its cytoplasmic stability and regulation of its translation, a number of studies have begun to provide evidence for methylation-regulated nuclear processes. In this review, we summarize the recent advances in RNA methylation research and highlight how these new findings have contributed to our understanding of methylation-dependent RNA processing in the nucleus. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Processing > Splicing Regulation/Alternative Splicing RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications. © 2018 The Authors. WIREs RNA published by Wiley Periodicals, Inc.

Role of Passive Diffusion, Transporters, and Membrane Trafficking-Mediated Processes in Cellular Drug Transport.

PubMed

Cocucci, E; Kim, J Y; Bai, Y; Pabla, N

2017-01-01

Intracellular drug accumulation is thought to be dictated by two major processes, passive diffusion through the lipid membrane or membrane transporters. The relative role played by these distinct processes remains actively debated. Moreover, the role of membrane-trafficking in drug transport remains underappreciated and unexplored. Here we discuss the distinct processes involved in cellular drug distribution and propose that better experimental models are required to elucidate the differential contributions of various processes in intracellular drug accumulation. © 2016 American Society for Clinical Pharmacology and Therapeutics.

Proteome-wide quantitative multiplexed profiling of protein expression: carbon-source dependency in Saccharomyces cerevisiae

PubMed Central

Paulo, Joao A.; O’Connell, Jeremy D.; Gaun, Aleksandr; Gygi, Steven P.

2015-01-01

The global proteomic alterations in the budding yeast Saccharomyces cerevisiae due to differences in carbon sources can be comprehensively examined using mass spectrometry–based multiplexing strategies. In this study, we investigate changes in the S. cerevisiae proteome resulting from cultures grown in minimal media using galactose, glucose, or raffinose as the carbon source. We used a tandem mass tag 9-plex strategy to determine alterations in relative protein abundance due to a particular carbon source, in triplicate, thereby permitting subsequent statistical analyses. We quantified more than 4700 proteins across all nine samples; 1003 proteins demonstrated statistically significant differences in abundance in at least one condition. The majority of altered proteins were classified as functioning in metabolic processes and as having cellular origins of plasma membrane and mitochondria. In contrast, proteins remaining relatively unchanged in abundance included those having nucleic acid–related processes, such as transcription and RNA processing. In addition, the comprehensiveness of the data set enabled the analysis of subsets of functionally related proteins, such as phosphatases, kinases, and transcription factors. As a resource, these data can be mined further in efforts to understand better the roles of carbon source fermentation in yeast metabolic pathways and the alterations observed therein, potentially for industrial applications, such as biofuel feedstock production. PMID:26399295

Membrane Assembly during the Infection Cycle of the Giant Mimivirus

PubMed Central

Mutsafi, Yael; Shimoni, Eyal; Shimon, Amir; Minsky, Abraham

2013-01-01

Although extensively studied, the structure, cellular origin and assembly mechanism of internal membranes during viral infection remain unclear. By combining diverse imaging techniques, including the novel Scanning-Transmission Electron Microscopy tomography, we elucidate the structural stages of membrane biogenesis during the assembly of the giant DNA virus Mimivirus. We show that this elaborate multistage process occurs at a well-defined zone localized at the periphery of large viral factories that are generated in the host cytoplasm. Membrane biogenesis is initiated by fusion of multiple vesicles, ∼70 nm in diameter, that apparently derive from the host ER network and enable continuous supply of lipid components to the membrane-assembly zone. The resulting multivesicular bodies subsequently rupture to form large open single-layered membrane sheets from which viral membranes are generated. Membrane generation is accompanied by the assembly of icosahedral viral capsids in a process involving the hypothetical major capsid protein L425 that acts as a scaffolding protein. The assembly model proposed here reveals how multiple Mimivirus progeny can be continuously and efficiently generated and underscores the similarity between the infection cycles of Mimivirus and Vaccinia virus. Moreover, the membrane biogenesis process indicated by our findings provides new insights into the pathways that might mediate assembly of internal viral membranes in general. PMID:23737745

Role of GATA transcription factor ELT-2 and p38 MAPK PMK-1 in recovery from acute P. aeruginosa infection in C. elegans

PubMed Central

Head, Brian P.; Olaitan, Abiola O.; Aballay, Alejandro

2017-01-01

ABSTRACT Infectious diseases caused by bacterial pathogens reduce the fitness of their associated host but are generally limited in duration. In order for the diseased host to regain any lost fitness upon recovery, a variety of molecular, cellular, and physiological processes must be employed. To better understand mechanisms underlying the recovery process, we have modeled an acute Pseudomonas aeruginosa infection in C. elegans using brief exposures to this pathogen and subsequent antibiotic treatment. To identify host genes altered during recovery from P. aeruginosa infection, we performed whole genome expression profiling. The analysis of this dataset indicated that the activity of the host immune system is down-regulated upon recovery and revealed shared and pathogen-specific host responses during recovery. We determined that the GATA transcription factor ELT-2 and the p38 MAP kinase PMK-1 are necessary for animals to successfully recover from an acute P. aeruginosa infection. In addition, we found that ELT-2 plays a more prominent and earlier role than PMK-1 during recovery. Our data sheds further light on the molecular mechanisms and transcriptional programs involved in recovery from an acute bacterial infection, which provides a better understanding of the entire infectious disease process. PMID:27600703

The bromodomain protein BRD4 regulates splicing during heat shock

PubMed Central

Hussong, Michelle; Kaehler, Christian; Kerick, Martin; Grimm, Christina; Franz, Alexandra; Timmermann, Bernd; Welzel, Franziska; Isensee, Jörg; Hucho, Tim; Krobitsch, Sylvia; Schweiger, Michal R.

2017-01-01

The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 (HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anti-cancer therapies. PMID:27536004

Systems biology of cellular membranes: a convergence with biophysics.

PubMed

Chabanon, Morgan; Stachowiak, Jeanne C; Rangamani, Padmini

2017-09-01

Systems biology and systems medicine have played an important role in the last two decades in shaping our understanding of biological processes. While systems biology is synonymous with network maps and '-omics' approaches, it is not often associated with mechanical processes. Here, we make the case for considering the mechanical and geometrical aspects of biological membranes as a key step in pushing the frontiers of systems biology of cellular membranes forward. We begin by introducing the basic components of cellular membranes, and highlight their dynamical aspects. We then survey the functions of the plasma membrane and the endomembrane system in signaling, and discuss the role and origin of membrane curvature in these diverse cellular processes. We further give an overview of the experimental and modeling approaches to study membrane phenomena. We close with a perspective on the converging futures of systems biology and membrane biophysics, invoking the need to include physical variables such as location and geometry in the study of cellular membranes. WIREs Syst Biol Med 2017, 9:e1386. doi: 10.1002/wsbm.1386 For further resources related to this article, please visit the WIREs website. © 2017 Wiley Periodicals, Inc.

Proteomic analysis of the response of α-ketoglutarate-producer Yarrowia lipolytica WSH-Z06 to environmental pH stimuli.

PubMed

Guo, Hongwei; Wan, Hui; Chen, Hongwen; Fang, Fang; Liu, Song; Zhou, Jingwen

2016-10-01

During bioproduction of short-chain carboxylates, a shift in pH is a common strategy for enhancing the biosynthesis of target products. Based on two-dimensional gel electrophoresis, comparative proteomics analysis of general and mitochondrial protein samples was used to investigate the cellular responses to environmental pH stimuli in the α-ketoglutarate overproducer Yarrowia lipolytica WSH-Z06. The lower environmental pH stimuli tensioned intracellular acidification and increased the level of reactive oxygen species (ROS). A total of 54 differentially expressed protein spots were detected, and 11 main cellular processes were identified to be involved in the cellular response to environmental pH stimuli. Slight decrease in cytoplasmic pH enhanced the cellular acidogenicity by elevating expression level of key enzymes in tricarboxylic acid cycle (TCA cycle). Enhanced energy biosynthesis, ROS elimination, and membrane potential homeostasis processes were also employed as cellular defense strategies to compete with environmental pH stimuli. Owing to its antioxidant role of α-ketoglutarate, metabolic flux shifted to α-ketoglutarate under lower pH by Y. lipolytica in response to acidic pH stimuli. The identified differentially expressed proteins provide clues for understanding the mechanisms of the cellular responses and for enhancing short-chain carboxylate production through metabolic engineering or process optimization strategies in combination with manipulation of environmental conditions.

Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

PubMed Central

Dougherty, W G; Semler, B L

1993-01-01

Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors. Images PMID:8302216

Polyelectrolyte multilayer-assisted fabrication of non-periodic silicon nanocolumn substrates for cellular interface applications

NASA Astrophysics Data System (ADS)

Lee, Seyeong; Kim, Dongyoon; Kim, Seong-Min; Kim, Jeong-Ah; Kim, Taesoo; Kim, Dong-Yu; Yoon, Myung-Han

2015-08-01

Recent advances in nanostructure-based biotechnology have resulted in a growing demand for vertical nanostructure substrates with elaborate control over the nanoscale geometry and a high-throughput preparation. In this work, we report the fabrication of non-periodic vertical silicon nanocolumn substrates via polyelectrolyte multilayer-enabled randomized nanosphere lithography. Owing to layer-by-layer deposited polyelectrolyte adhesives, uniformly-separated polystyrene nanospheres were securely attached on large silicon substrates and utilized as masks for the subsequent metal-assisted silicon etching in solution. Consequently, non-periodic vertical silicon nanocolumn arrays were successfully fabricated on a wafer scale, while each nanocolumn geometric factor, such as the diameter, height, density, and spatial patterning, could be fully controlled in an independent manner. Finally, we demonstrate that our vertical silicon nanocolumn substrates support viable cell culture with minimal cell penetration and unhindered cell motility due to the blunt nanocolumn morphology. These results suggest that vertical silicon nanocolumn substrates may serve as a useful cellular interface platform for performing a statistically meaningful number of cellular experiments in the fields of biomolecular delivery, stem cell research, etc.Recent advances in nanostructure-based biotechnology have resulted in a growing demand for vertical nanostructure substrates with elaborate control over the nanoscale geometry and a high-throughput preparation. In this work, we report the fabrication of non-periodic vertical silicon nanocolumn substrates via polyelectrolyte multilayer-enabled randomized nanosphere lithography. Owing to layer-by-layer deposited polyelectrolyte adhesives, uniformly-separated polystyrene nanospheres were securely attached on large silicon substrates and utilized as masks for the subsequent metal-assisted silicon etching in solution. Consequently, non-periodic vertical silicon nanocolumn arrays were successfully fabricated on a wafer scale, while each nanocolumn geometric factor, such as the diameter, height, density, and spatial patterning, could be fully controlled in an independent manner. Finally, we demonstrate that our vertical silicon nanocolumn substrates support viable cell culture with minimal cell penetration and unhindered cell motility due to the blunt nanocolumn morphology. These results suggest that vertical silicon nanocolumn substrates may serve as a useful cellular interface platform for performing a statistically meaningful number of cellular experiments in the fields of biomolecular delivery, stem cell research, etc. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr02384j

Amino acids and autophagy: cross-talk and co-operation to control cellular homeostasis.

PubMed

Carroll, Bernadette; Korolchuk, Viktor I; Sarkar, Sovan

2015-10-01

Maintenance of amino acid homeostasis is important for healthy cellular function, metabolism and growth. Intracellular amino acid concentrations are dynamic; the high demand for protein synthesis must be met with constant dietary intake, followed by cellular influx, utilization and recycling of nutrients. Autophagy is a catabolic process via which superfluous or damaged proteins and organelles are delivered to the lysosome and degraded to release free amino acids into the cytoplasm. Furthermore, autophagy is specifically activated in response to amino acid starvation via two key signaling cascades: the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and the general control nonderepressible 2 (GCN2) pathways. These pathways are key regulators of the integration between anabolic (amino acid depleting) and catabolic (such as autophagy which is amino acid replenishing) processes to ensure intracellular amino acid homeostasis. Here, we discuss the key roles that amino acids, along with energy (ATP, glucose) and oxygen, are playing in cellular growth and proliferation. We further explore how sophisticated methods are employed by cells to sense intracellular amino acid concentrations, how amino acids can act as a switch to dictate the temporal and spatial activation of anabolic and catabolic processes and how autophagy contributes to the replenishment of free amino acids, all to ensure cell survival. Relevance of these molecular processes to cellular and organismal physiology and pathology is also discussed.

Global analysis of bacterial transcription factors to predict cellular target processes.

PubMed

Doerks, Tobias; Andrade, Miguel A; Lathe, Warren; von Mering, Christian; Bork, Peer

2004-03-01

Whole-genome sequences are now available for >100 bacterial species, giving unprecedented power to comparative genomics approaches. We have applied genome-context methods to predict target processes that are regulated by transcription factors (TFs). Of 128 orthologous groups of proteins annotated as TFs, to date, 36 are functionally uncharacterized; in our analysis we predict a probable cellular target process or biochemical pathway for half of these functionally uncharacterized TFs.

Independent Risk Factors for Urinary Tract Infection and for Subsequent Bacteremia or Acute Cellular Rejection: A Single Center Report of 1166 Kidney Allograft Recipients

PubMed Central

Lee, John R.; Bang, Heejung; Dadhania, Darshana; Hartono, Choli; Aull, Meredith J.; Satlin, Michael; August, Phyllis; Suthanthiran, Manikkam; Muthukumar, Thangamani

2013-01-01

Background Urinary tract infection (UTI) is a frequent, serious complication in kidney allograft recipients. Methods We reviewed the records of 1166 kidney allograft recipients who received their allografts at our institution between January 2005 to December 2010 and determined the incidence of UTI during the first three months after transplantation (early UTI). We utilized Cox proportional hazard models to determine the risk factors for early UTI and whether early UTI was an independent risk factor for subsequent bacteremia or acute cellular rejection (ACR). Results UTI, defined as ≥105 bacterial colony forming units per milliliter of urine, developed in 247 (21%) of the 1166 recipients. Independent risk factors for the first episode of UTI were: female gender (hazard ratio [HR]: 2.9, 95% Confidence Intervals (CI): 2.2–3.7, P<0.001), prolonged use of Foley catheter (HR: 3.9, 95% CI: 2.8–5.4, P<0.001), ureteral stent (HR 1.4, 95% CI: 1.1–1.8, P=0.01), age (HR: 1.1, 95% CI: 1.0–1.2, P=0.03), and delayed graft function (HR:1.4, 95% CI: 1.0–1.9, P=0.06). Trimethoprim/sulfamethoxazole prophylaxis was associated with a reduced risk of UTI (HR: 0.6, 95% CI: 0.3–0.9, P=0.02). UTI was an independent risk factor for subsequent bacteremia (HR: 2.4, 95% CI: 1.2–4.8, P=0.01). Untreated, but not treated, UTI was associated with an increased risk of ACR (HR: 2.8, 95% CI: 1.3–6.2, P=0.01). Conclusions Female gender, prolonged use of Foley catheter, ureteral stent, age, and delayed graft function are independent risk factors for early UTI. UTI is independently associated with the development of bacteremia, and untreated UTI is associated with subsequent ACR. PMID:23917724

Independent risk factors for urinary tract infection and for subsequent bacteremia or acute cellular rejection: a single-center report of 1166 kidney allograft recipients.

PubMed

Lee, John R; Bang, Heejung; Dadhania, Darshana; Hartono, Choli; Aull, Meredith J; Satlin, Michael; August, Phyllis; Suthanthiran, Manikkam; Muthukumar, Thangamani

2013-10-27

Urinary tract infection (UTI) is a frequent, serious complication in kidney allograft recipients. We reviewed the records of 1166 kidney allograft recipients who received their allografts at our institution between January 2005 and December 2010 and determined the incidence of UTI during the first 3 months after transplantation (early UTI). We used Cox proportional hazards models to determine the risk factors for early UTI and whether early UTI was an independent risk factor for subsequent bacteremia or acute cellular rejection (ACR). UTI, defined as 10 or more bacterial colony-forming units/mL urine, developed in 247 (21%) of the 1166 recipients. Independent risk factors for the first episode of UTI were female gender (hazard ratio [HR], 2.9; 95% confidence intervals [CI], 2.2-3.7; P<0.001), prolonged use of Foley catheter (HR, 3.9; 95% CI, 2.8-5.4; P <0.001), ureteral stent (HR, 1.4; 95% CI, 1.1-1.8; P=0.01), age (HR, 1.1; 95% CI, 1.0-1.2; P=0.03), and delayed graft function (HR, 1.4; 95% CI, 1.0-1.9; P=0.06). Trimethoprim/sulfamethoxazole prophylaxis was associated with a reduced risk of UTI (HR, 0.6; 95% CI, 0.3-0.9; P=0.02). UTI was an independent risk factor for subsequent bacteremia (HR, 2.4; 95% CI, 1.2-4.8; P=0.01). Untreated UTI, but not treated UTI, was associated with an increased risk of ACR (HR, 2.8; 95% CI, 1.3-6.2; P=0.01). Female gender, prolonged use of Foley catheter, ureteral stent, age, and delayed graft function are independent risk factors for early UTI. UTI is independently associated with the development of bacteremia, and untreated UTI is associated with subsequent ACR.

Mitochondrial-associated metabolic disorders: foundations, pathologies and recent progress

PubMed Central

2013-01-01

Research in the last decade has revolutionized the way in which we view mitochondria. Mitochondria are no longer viewed solely as cellular powerhouses; rather, mitochondria are now understood to be vibrant, mobile structures, constantly undergoing fusion and fission, and engaging in intimate interactions with other cellular compartments and structures. Findings have implicated mitochondria in a wide variety of cellular processes and molecular interactions, such as calcium buffering, lipid flux, and intracellular signaling. As such, it does not come as a surprise that an increasing number of human pathologies have been associated with functional defects in mitochondria. The difficulty in understanding and treating human pathologies caused by mitochondrial dysfunction arises from the complex relationships between mitochondria and other cellular processes, as well as the genetic background of such diseases. This review attempts to provide a summary of the background knowledge and recent developments in mitochondrial processes relating to mitochondrial-associated metabolic diseases arising from defects or deficiencies in mitochondrial function, as well as insights into current and future avenues for investigation. PMID:24499129

An Internal Signal Sequence Directs Intramembrane Proteolysis of a Cellular Immunoglobulin Domain Protein*S⃞

PubMed Central

Robakis, Thalia; Bak, Beata; Lin, Shu-huei; Bernard, Daniel J.; Scheiffele, Peter

2008-01-01

Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signal peptidase (SP) is an enzyme with a well defined role in cleaving N-terminal signal sequences but no demonstrated function in the proteolysis of cellular precursor proteins. We provide evidence that SP mediates intraprotein cleavage of IgSF1, a large cellular Ig domain protein that is processed into two separate Ig domain proteins. In addition, our results suggest the involvement of signal peptide peptidase (SPP), an intramembrane protease, which acts on substrates that have been previously cleaved by SP. We show that IgSF1 is processed through sequential proteolysis by SP and SPP. Cleavage is directed by an internal signal sequence and generates two separate Ig domain proteins from a polytopic precursor. Our findings suggest that SP and SPP function are not restricted to N-terminal signal sequence cleavage but also contribute to the processing of cellular transmembrane proteins. PMID:18981173

Using Primary Literature in an Undergraduate Assignment: Demonstrating Connections among Cellular Processes

ERIC Educational Resources Information Center

Yeong, Foong May

2015-01-01

Learning basic cell biology in an essential module can be daunting to second-year undergraduates, given the depth of information that is provided in major molecular and cell biology textbooks. Moreover, lectures on cellular pathways are organised into sections, such that at the end of lectures, students might not see how various processes are…

Modifying Yeast Tolerance to Inhibitory Conditions of Ethanol Production Processes

PubMed Central

Caspeta, Luis; Castillo, Tania; Nielsen, Jens

2015-01-01

Saccharomyces cerevisiae strains having a broad range of substrate utilization, rapid substrate consumption, and conversion to ethanol, as well as good tolerance to inhibitory conditions are ideal for cost-competitive ethanol production from lignocellulose. A major drawback to directly design S. cerevisiae tolerance to inhibitory conditions of lignocellulosic ethanol production processes is the lack of knowledge about basic aspects of its cellular signaling network in response to stress. Here, we highlight the inhibitory conditions found in ethanol production processes, the targeted cellular functions, the key contributions of integrated -omics analysis to reveal cellular stress responses according to these inhibitors, and current status on design-based engineering of tolerant and efficient S. cerevisiae strains for ethanol production from lignocellulose. PMID:26618154

Cyclophilin A as a potential genetic adjuvant to improve HIV-1 Gag DNA vaccine immunogenicity by eliciting broad and long-term Gag-specific cellular immunity in mice.

PubMed

Hou, Jue; Zhang, Qicheng; Liu, Zheng; Wang, Shuhui; Li, Dan; Liu, Chang; Liu, Ying; Shao, Yiming

2016-01-01

Previous research has shown that host Cyclophilin A (CyPA) can promote dendritic cell maturation and the subsequent innate immune response when incorporated into an HIV-1 Gag protein to circumvent the resistance of dendritic cells to HIV-1 infection. This led us to hypothesize that CyPA may improve HIV-1 Gag-specific vaccine immunogenicity via binding with Gag antigen. The adjuvant effect of CyPA was evaluated using a DNA vaccine with single or dual expression cassettes. Mouse studies indicated that CyPA specifically and markedly promoted HIV-1 Gag-specific cellular immunity but not an HIV-1 Env-specific cellular response. The Gag/CyPA dual expression cassettes stimulated a greater Gag-specific cellular immune response, than Gag immunization alone. Furthermore, CyPA induced a broad Gag-specific T cell response and strong cellular immunity that lasted up to 5 months. In addition, CyPA skewed to cellular rather than humoral immunity. To investigate the mechanisms of the adjuvant effect, site-directed mutagenesis in CyPA, including active site residues H54Q and F60A resulted in mutants that were co-expressed with Gag in dual cassettes. The immune response to this vaccine was analyzed in vivo. Interestingly, the wild type CyPA markedly increased Gag cellular immunity, but the H54Q and F60A mutants drastically reduced CyPA adjuvant activation. Therefore, we suggest that the adjuvant effect of CyPA was based on Gag-CyPA-specific interactions. Herein, we report that Cyclophilin A can augment HIV-1 Gag-specific cellular immunity as a genetic adjuvant in multiplex DNA immunization strategies, and that activity of this adjuvant is specific, broad, long-term, and based on Gag-CyPA interaction.

Signals for the lysosome: a control center for cellular clearance and energy metabolism

PubMed Central

Settembre, Carmine; Fraldi, Alessandro; Medina, Diego L.

2015-01-01

Preface For a long time lysosomes were considered merely to be cellular “incinerators” involved in the degradation and recycling of cellular waste. However, there is now compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signaling and energy metabolism. Furthermore, the essential role of lysosomes in the autophagic pathway puts these organelles at the crossroads of several cellular processes, with significant implications for health and disease. The identification of a master gene, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy, has revealed how the lysosome adapts to environmental cues, such as starvation, and suggests novel therapeutic strategies for modulating lysosomal function in human disease. PMID:23609508

Piezo Proteins: Regulators of Mechanosensation and Other Cellular Processes*

PubMed Central

Bagriantsev, Sviatoslav N.; Gracheva, Elena O.; Gallagher, Patrick G.

2014-01-01

Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature. PMID:25305018

Administrative and research policies required to bring cellular therapies from the research laboratory to the patient's bedside.

PubMed

Yim, Robyn

2005-10-01

The research process is a balance between the inherent risks of new discoveries and the risks of research participant safety. Conflicts of interest, inherent to the research process, as well as those introduced by emerging cellular therapies, have the potential to compromise safety. The relationship of trust between the researcher and the clinical trial participant facilitates objective decision making, in the best interest of both parties. In the setup of each clinical trial, investigators incorporate ethical, political, legal, financial, and regulatory considerations as protocols are established. Responsibility to abide by these decisions ensures a systematic process and safeguards participants in this process. The integrity of the research process is strengthened by identifying potential conflicting issues with the guiding principles established in the protocols, which may threaten the objectivity of involved parties and jeopardize safety of the participants. The rapid pace and changing paradigms of new discoveries in cellular therapies exaggerate existing conflicts and introduce new ones. Ethical issues raised by emerging cellular therapies include the division of opinions regarding the use of embryonic and fetal tissue to develop stem cell lines for research, the individual versus professional conscience of a researcher, overselling of outcomes as a result of the researcher's desire to be the first to discover a cellular therapy, and therapeutic misconception resulting from a participant's desire for a miracle cure. The basic ethical issue of whether stem cells should be utilized as a cellular therapy raises heated debates because some believe that it is not acceptable to use fetal material as a source of research material for future cures and others feel equally as strong that inaction is unethical because it results in needless suffering and death owing to the absence of this research. Political issues include the divergent position statements of presidential administrations on cellular therapy, variations in individual state laws, and states becoming involved in research funding, such as California's Proposition 71. Legal concerns include expanding private litigation with diversity of lawsuits, expanding lists of defendants, and the use of class-action lawsuits in research cases. Ownership issues also arise in terms of intellectual property, patents, and ownership of stem cells collected from minors, as in umbilical cord blood donations. Situations that challenge the regulatory processes established to ensure participant safety include differences in reporting requirements for private- and public-funded research and the lack of adequate funding and resources to implement and support the institutional review board (IRB) process. Financial considerations influence the development of clinical protocols, because funding is often limited. Financial incentives, personal investment in companies funding research activities, and fundraising pressures may present potential conflicts. In addition, the increasing role of emerging biotechnology start-up companies and pharmaceutical companies in clinical research introduces additional financial considerations. Administrative policies are needed to address these possible conflicts and ensure research participant safety as cellular therapies progress from the research laboratories to the patient's bedside. Administrative policies to ensure minimum standards of quality for emerging products before human clinical trials, policies to enforce consistent reporting requirements for private and public cellular research, policies to minimize financial conflicts of interest, policies to strengthen implementation of the existing IRB process and to structure into the process a consistent, systematic review of these identified conflicts, and policies to limit private litigation will help to preserve the objectivity of the review process and ultimately increase participant safety.

Encoding-related brain activity dissociates between the recollective processes underlying successful recall and recognition: a subsequent-memory study.

PubMed

Sadeh, Talya; Maril, Anat; Goshen-Gottstein, Yonatan

2012-07-01

The subsequent-memory (SM) paradigm uncovers brain mechanisms that are associated with mnemonic activity during encoding by measuring participants' neural activity during encoding and classifying the encoding trials according to performance in the subsequent retrieval phase. The majority of these studies have converged on the notion that the mechanism supporting recognition is mediated by familiarity and recollection. The process of recollection is often assumed to be a recall-like process, implying that the active search for the memory trace is similar, if not identical, for recall and recognition. Here we challenge this assumption and hypothesize - based on previous findings obtained in our lab - that the recollective processes underlying recall and recognition might show dissociative patterns of encoding-related brain activity. To this end, our design controlled for familiarity, thereby focusing on contextual, recollective processes. We found evidence for dissociative neurocognitive encoding mechanisms supporting subsequent-recall and subsequent-recognition. Specifically, the contrast of subsequent-recognition versus subsequent-recall revealed activation in the Parahippocampal cortex (PHc) and the posterior hippocampus--regions associated with contextual processing. Implications of our findings and their relation to current cognitive models of recollection are discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

Green tea catechin intervention of reactive oxygen species-mediated ERK pathway activation and chronically induced breast cell carcinogenesis

PubMed Central

Rathore, Kusum; Choudhary, Shambhunath; Odoi, Agricola; Wang, Hwa-Chain R.

2012-01-01

Long-term exposure to low doses of environmental carcinogens contributes to sporadic human breast cancers. Epidemiologic and experimental studies indicate that green tea catechins (GTCs) may intervene with breast cancer development. We have been developing a chronically induced breast cell carcinogenesis model wherein we repeatedly expose non-cancerous, human breast epithelial MCF10A cells to bioachievable picomolar concentrations of environmental carcinogens, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P), to progressively induce cellular acquisition of cancer-associated properties, as measurable end points. The model is then used as a target to identify non-cytotoxic preventive agents effective in suppression of cellular carcinogenesis. Here, we demonstrate, for the first time, a two-step strategy that initially used end points that were transiently induced by short-term exposure to NNK and B[a]P as targets to detect GTCs capable of blocking the acquisition of cancer-associated properties and subsequently used end points constantly induced by long-term exposure to carcinogens as targets to verify GTCs capable of suppressing carcinogenesis. We detected that short-term exposure to NNK and B[a]P resulted in elevation of reactive oxygen species (ROS), leading to Raf-independent extracellular signal-regulated kinase (ERK) pathway activation and subsequent induction of cell proliferation and DNA damage. These GTCs, at non-cytotoxic levels, were able to suppress chronically induced cellular carcinogenesis by blocking carcinogen-induced ROS elevation, ERK activation, cell proliferation and DNA damage in each exposure cycle. Our model may help accelerate the identification of preventive agents to intervene in carcinogenesis induced by long-term exposure to environmental carcinogens, thereby safely and effectively reducing the health risk of sporadic breast cancer. PMID:22045026

Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

PubMed

Hough, Chris; Radu, Maria; Doré, Jules J E

2012-01-01

The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-β/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-β-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

Bartonella entry mechanisms into mammalian host cells.

PubMed

Eicher, Simone C; Dehio, Christoph

2012-08-01

The Gram-negative genus Bartonella comprises arthropod-borne pathogens that typically infect mammals in a host-specific manner. Bartonella bacilliformis and Bartonella quintana are human-specific pathogens, while several zoonotic bartonellae specific for diverse animal hosts infect humans as an incidental host. Clinical manifestations of Bartonella infections range from mild symptoms to life-threatening disease. Following transmission by blood-sucking arthropods or traumatic contact with infected animals, bartonellae display sequential tropisms towards endothelial and possibly other nucleated cells and erythrocytes, the latter in a host-specific manner. Attachment to the extracellular matrix (ECM) and to nucleated cells is mediated by surface-exposed bacterial adhesins, in particular trimeric autotransporter adhesins (TAAs). The subsequent engulfment of the pathogen into a vacuolar structure follows a unique series of events whereby the pathogen avoids the endolysosomal compartments. For Bartonella henselae and assumingly most other species, the infection process is aided at different steps by Bartonella effector proteins (Beps). They are injected into host cells through the type IV secretion system (T4SS) VirB/D4 and subvert host cellular functions to favour pathogen uptake. Bacterial binding to erythrocytes is mediated by Trw, another T4SS, in a strictly host-specific manner, followed by pathogen-forced uptake involving the IalB invasin and subsequent replication and persistence within a membrane-bound intra-erythrocytic compartment. © 2012 Blackwell Publishing Ltd.

An Evaluation of the Efficacy of a Laboratory Exercise on Cellular Respiration

ERIC Educational Resources Information Center

Scholer, Anne-Marie; Hatton, Mary

2008-01-01

This study is an analysis of the effectiveness of a faculty-designed laboratory experience about a difficult topic, cellular respiration. The activity involves a hands-on model of the cellular-respiration process, making use of wooden ball-and-stick chemistry models and small toy trucks on a table top model of the mitochondrion. Students…

Electrospraying of microfluidic encapsulated cells for the fabrication of cell-laden electrospun hybrid tissue constructs.

PubMed

Weidenbacher, L; Abrishamkar, A; Rottmar, M; Guex, A G; Maniura-Weber, K; deMello, A J; Ferguson, S J; Rossi, R M; Fortunato, G

2017-12-01

The fabrication of functional 3D tissues is a major goal in tissue engineering. While electrospinning is a promising technique to manufacture a structure mimicking the extracellular matrix, cell infiltration into electrospun scaffolds remains challenging. The robust and in situ delivery of cells into such biomimetic scaffolds would potentially enable the design of tissue engineered constructs with spatial control over cellular distribution but often solvents employed in the spinning process are problematic due to their high cytotoxicity. Herein, microfluidic cell encapsulation is used to establish a temporary protection vehicle for the in situ delivery of cells for the development of a fibrous, cell-laden hybrid biograft. Therefore a layer-by-layer process is used by alternating fiber electrospinning and cell spraying procedures. Both encapsulation and subsequent electrospraying of capsules has no negative effect on the viability and myogenic differentiation of murine myoblast cells. Propidium iodide positive stained cells were analyzed to quantify the amount of dead cells and the presence of myosin heavy chain positive cells after the processes was shown. Furthermore, encapsulation successfully protects cells from cytotoxic solvents (such as dimethylformamide) during in situ delivery of the cells into electrospun poly(vinylidene fluoride-co-hexafluoropropylene) scaffolds. The resulting cell-populated biografts demonstrate the clear potential of this approach in the creation of viable tissue engineering constructs. Infiltration of cells and their controlled spatial distribution within fibrous electrospun membranes is a challenging task but allows for the development of functional highly organized 3D hybrid tissues. Combining polymer electrospinning and cell electrospraying in a layer-by-layer approach is expected to overcome current limitations of reduced cell infiltration after traditional static seeding. However, organic solvents, used during the electrospinning process, impede often major issues due to their high cytotoxicity. Utilizing microfluidic encapsulation as a mean to embed cells within a protective polymer casing enables the controlled deposition of viable cells without interfering with the cellular phenotype. The presented techniques allow for novel cell manipulation approaches being significant for enhanced 3D tissue engineering based on its versatility in terms of material and cell selection. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Hypothesis: The Psychedelic Ayahuasca Heals Traumatic Memories via a Sigma 1 Receptor-Mediated Epigenetic-Mnemonic Process.

PubMed

Inserra, Antonio

2018-01-01

Ayahuasca ingestion modulates brain activity, neurotransmission, gene expression and epigenetic regulation. N,N -Dimethyltryptamine (DMT, one of the alkaloids in Ayahuasca) activates sigma 1 receptor (SIGMAR1) and others. SIGMAR1 is a multi-faceted stress-responsive receptor which promotes cell survival, neuroprotection, neuroplasticity, and neuroimmunomodulation. Simultaneously, monoamine oxidase inhibitors (MAOIs) also present in Ayahuasca prevent the degradation of DMT. One peculiarity of SIGMAR1 activation and MAOI activity is the reversal of mnemonic deficits in pre-clinical models. Since traumatic memories in post-traumatic stress disorder (PTSD) are often characterised by "repression" and PTSD patients ingesting Ayahuasca report the retrieval of such memories, it cannot be excluded that DMT-mediated SIGMAR1 activation and the concomitant MAOIs effects during Ayahuasca ingestion might mediate such "anti-amnesic" process. Here I hypothesise that Ayahuasca, via hyperactivation of trauma and emotional memory-related centres, and via its concomitant SIGMAR1- and MAOIs- induced anti-amnesic effects, facilitates the retrieval of traumatic memories, in turn making them labile (destabilised). As Ayahuasca alkaloids enhance synaptic plasticity, increase neurogenesis and boost dopaminergic neurotransmission, and those processes are involved in memory reconsolidation and fear extinction, the fear response triggered by the memory can be reprogramed and/or extinguished. Subsequently, the memory is stored with this updated significance. To date, it is unclear if new memories replace, co-exist with or bypass old ones. Although the mechanisms involved in memory are still debated, they seem to require the involvement of cellular and molecular events, such as reorganisation of homo and heteroreceptor complexes at the synapse, synaptic plasticity, and epigenetic re-modulation of gene expression. Since SIGMAR1 mobilises synaptic receptor, boosts synaptic plasticity and modulates epigenetic processes, such effects might be involved in the reported healing of traumatic memories in PTSD patients. If this theory proves to be true, Ayahuasca could come to represent the only standing pharmacological treatment which targets traumatic memories in PTSD. Lastly, since SIGMAR1 activation triggers both epigenetic and immunomodulatory programmes, the mechanism here presented could help understanding and treating other conditions in which the cellular memory is dysregulated, such as cancer, diabetes, autoimmune and neurodegenerative pathologies and substance addiction.

Aggregation of gold nanoparticles followed by methotrexate release enables Raman imaging of drug delivery into cancer cells

NASA Astrophysics Data System (ADS)

Durgadas, C. V.; Sharma, C. P.; Paul, W.; Rekha, M. R.; Sreenivasan, K.

2012-09-01

This study refers an aqueous synthesis of methotrexate (MTX)-conjugated gold nanoparticles (GNPs), their interaction with HepG2 cells, and the use of Raman imaging to observe cellular internalization and drug delivery. GNPs of average size 3.5-5 nm were stabilized using the amine terminated bifunctional biocompatible copolymer and amended by conjugating MTX, an anticancer drug. The nanoparticles were released MTX at a faster rate in acidic pH and subsequently found to form aggregates. The Raman signals of cellular components were found to be enhanced by the aggregated particles enabling the mapping to visualize site-specific drug delivery. The methodology seems to have potential in optimizing the characteristics of nanodrug carriers for emptying the cargo precisely at specified sites.

'Til Eph do us part': intercellular signaling via Eph receptors and ephrin ligands guides cerebral cortical development from birth through maturation.

PubMed

North, Hilary A; Clifford, Meredith A; Donoghue, Maria J

2013-08-01

Eph receptors, the largest family of surface-bound receptor tyrosine kinases and their ligands, the ephrins, mediate a wide variety of cellular interactions in most organ systems throughout both development and maturity. In the forming cerebral cortex, Eph family members are broadly and dynamically expressed in particular sets of cortical cells at discrete times. Here, we review the known functions of Eph-mediated intercellular signaling in the generation of progenitors, the migration of maturing cells, the differentiation of neurons, the formation of functional connections, and the choice between life and death during corticogenesis. In synthesizing these results, we posit a signaling paradigm in which cortical cells maintain a life history of Eph-mediated intercellular interactions that guides subsequent cellular decision-making.

Fast and accurate automated cell boundary determination for fluorescence microscopy

NASA Astrophysics Data System (ADS)

Arce, Stephen Hugo; Wu, Pei-Hsun; Tseng, Yiider

2013-07-01

Detailed measurement of cell phenotype information from digital fluorescence images has the potential to greatly advance biomedicine in various disciplines such as patient diagnostics or drug screening. Yet, the complexity of cell conformations presents a major barrier preventing effective determination of cell boundaries, and introduces measurement error that propagates throughout subsequent assessment of cellular parameters and statistical analysis. State-of-the-art image segmentation techniques that require user-interaction, prolonged computation time and specialized training cannot adequately provide the support for high content platforms, which often sacrifice resolution to foster the speedy collection of massive amounts of cellular data. This work introduces a strategy that allows us to rapidly obtain accurate cell boundaries from digital fluorescent images in an automated format. Hence, this new method has broad applicability to promote biotechnology.

Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model

PubMed Central

Berlinguer, Fiammetta; Madeddu, Manuela; Pasciu, Valeria; Succu, Sara; Spezzigu, Antonio; Satta, Valentina; Mereu, Paolo; Leoni, Giovanni G; Naitana, Salvatore

2009-01-01

Currently, the assessment of sperm function in a raw or processed semen sample is not able to reliably predict sperm ability to withstand freezing and thawing procedures and in vivo fertility and/or assisted reproductive biotechnologies (ART) outcome. The aim of the present study was to investigate which parameters among a battery of analyses could predict subsequent spermatozoa in vitro fertilization ability and hence blastocyst output in a goat model. Ejaculates were obtained by artificial vagina from 3 adult goats (Capra hircus) aged 2 years (A, B and C). In order to assess the predictive value of viability, computer assisted sperm analyzer (CASA) motility parameters and ATP intracellular concentration before and after thawing and of DNA integrity after thawing on subsequent embryo output after an in vitro fertility test, a logistic regression analysis was used. Individual differences in semen parameters were evident for semen viability after thawing and DNA integrity. Results of IVF test showed that spermatozoa collected from A and B lead to higher cleavage rates (0 < 0.01) and blastocysts output (p < 0.05) compared with C. Logistic regression analysis model explained a deviance of 72% (p < 0.0001), directly related with the mean percentage of rapid spermatozoa in fresh semen (p < 0.01), semen viability after thawing (p < 0.01), and with two of the three comet parameters considered, i.e tail DNA percentage and comet length (p < 0.0001). DNA integrity alone had a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). The model proposed here represents one of the many possible ways to explain differences found in embryo output following IVF with different semen donors and may represent a useful tool to select the most suitable donors for semen cryopreservation. PMID:19900288

Immunogenicity and safety of xenogeneic vascular endothelial growth factor receptor-2 DNA vaccination in mice and dogs

PubMed Central

Denies, Sofie; Cicchelero, Laetitia; Polis, Ingeborgh; Sanders, Niek N.

2016-01-01

Vascular endothelial growth factor receptor-2 (VEGFR-2) is an attractive target in oncology due to its crucial role in angiogenesis. In this study a DNA vaccine coding for human VEGFR-2 was evaluated in healthy mice and dogs, administered by intradermal injection and electroporation. In mice, three doses and vaccination schedules were evaluated. Cellular immune responses were measured by intracellular IFN-gamma staining and a cytotoxicity assay and antibodies by ELISA. Safety was assessed by measuring regulatory T cells and myeloid derived suppressor cells and a wound healing assay. The vaccine was subsequently evaluated in dogs, which were vaccinated three times with 100μg. Cellular immune responses were measured by intracellular IFN-gamma staining and antibodies by a flow cytometric assay. In mice, maximal cellular responses were observed after two vaccinations with 5μg. Humoral responses continued to increase with higher dose and number of vaccinations. No abnormalities in the measured safety parameters were observed. The vaccine was also capable of eliciting a cellular and humoral immune response in dogs. No adverse effects were observed, but tolerability of the electroporation was poor. This study will facilitate the evaluation of the vaccine in tumor bearing animals, ranging from rodent models to dogs with spontaneous tumors. PMID:26871296

[Cell signaling pathways interaction in cellular proliferation: Potential target for therapeutic interventionism].

PubMed

Valdespino-Gómez, Víctor Manuel; Valdespino-Castillo, Patricia Margarita; Valdespino-Castillo, Víctor Edmundo

2015-01-01

Nowadays, cellular physiology is best understood by analysing their interacting molecular components. Proteins are the major components of the cells. Different proteins are organised in the form of functional clusters, pathways or networks. These molecules are ordered in clusters of receptor molecules of extracellular signals, transducers, sensors and biological response effectors. The identification of these intracellular signaling pathways in different cellular types has required a long journey of experimental work. More than 300 intracellular signaling pathways have been identified in human cells. They participate in cell homeostasis processes for structural and functional maintenance. Some of them participate simultaneously or in a nearly-consecutive progression to generate a cellular phenotypic change. In this review, an analysis is performed on the main intracellular signaling pathways that take part in the cellular proliferation process, and the potential use of some components of these pathways as target for therapeutic interventionism are also underlined. Copyright © 2015 Academia Mexicana de Cirugía A.C. Published by Masson Doyma México S.A. All rights reserved.

Terminal addition in a cellular world.

PubMed

Torday, J S; Miller, William B

2018-07-01

Recent advances in our understanding of evolutionary development permit a reframed appraisal of Terminal Addition as a continuous historical process of cellular-environmental complementarity. Within this frame of reference, evolutionary terminal additions can be identified as environmental induction of episodic adjustments to cell-cell signaling patterns that yield the cellular-molecular pathways that lead to differing developmental forms. Phenotypes derive, thereby, through cellular mutualistic/competitive niche constructions in reciprocating responsiveness to environmental stresses and epigenetic impacts. In such terms, Terminal Addition flows according to a logic of cellular needs confronting environmental challenges over space-time. A reconciliation of evolutionary development and Terminal Addition can be achieved through a combined focus on cell-cell signaling, molecular phylogenies and a broader understanding of epigenetic phenomena among eukaryotic organisms. When understood in this manner, Terminal Addition has an important role in evolutionary development, and chronic disease might be considered as a form of 'reverse evolution' of the self-same processes. Copyright © 2017. Published by Elsevier Ltd.

Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane.

PubMed

Stone, Matthew B; Shelby, Sarah A; Veatch, Sarah L

2017-06-14

Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.

Nano/microvehicles for efficient delivery and (bio)sensing at the cellular level

PubMed Central

Esteban-Fernández de Ávila, B.; Yáñez-Sedeño, P.

2017-01-01

A perspective review of recent strategies involving the use of nano/microvehicles to address the key challenges associated with delivery and (bio)sensing at the cellular level is presented. The main types and characteristics of the different nano/microvehicles used for these cellular applications are discussed, including fabrication pathways, propulsion (catalytic, magnetic, acoustic or biological) and navigation strategies, and relevant parameters affecting their propulsion performance and sensing and delivery capabilities. Thereafter, selected applications are critically discussed. An emphasis is made on enhancing the extra- and intra-cellular biosensing capabilities, fast cell internalization, rapid inter- or intra-cellular movement, efficient payload delivery and targeted on-demand controlled release in order to greatly improve the monitoring and modulation of cellular processes. A critical discussion of selected breakthrough applications illustrates how these smart multifunctional nano/microdevices operate as nano/microcarriers and sensors at the intra- and extra-cellular levels. These advances allow both the real-time biosensing of relevant targets and processes even at a single cell level, and the delivery of different cargoes (drugs, functional proteins, oligonucleotides and cells) for therapeutics, gene silencing/transfection and assisted fertilization, while overcoming challenges faced by current affinity biosensors and delivery vehicles. Key challenges for the future and the envisioned opportunities and future perspectives of this remarkably exciting field are discussed. PMID:29147499

Cellular events in adhesion formation due to thermal trauma.

PubMed

Kaplun, A; Aronson, M; Halperin, B; Griffel, B

1984-01-01

Consequent to thermal traumatization of the intestinal wall of the mouse, histopathological events ensue which lead to peritoneal adhesion formation. In the first 48 h, the main pathological findings are of a necrotic and inflammatory nature, but subsequently fibroplasia is the main feature, as evidenced by the appearance of spindle-shaped cells followed by fibroblasts. Factors essential for and contributing to the formation of adhesions are described.

Partial Gravity Biological Tether Experiment on the Deep Space Gateway

NASA Astrophysics Data System (ADS)

Wallace, S.; Graham, L.

2018-02-01

A tether-based partial gravity bacterial biological experiment represents a viable biological experiment to investigate the fundamental internal cellular processes between altered levels of gravity and cellular adaption.

A Highly Accurate Face Recognition System Using Filtering Correlation

NASA Astrophysics Data System (ADS)

Watanabe, Eriko; Ishikawa, Sayuri; Kodate, Kashiko

2007-09-01

The authors previously constructed a highly accurate fast face recognition optical correlator (FARCO) [E. Watanabe and K. Kodate: Opt. Rev. 12 (2005) 460], and subsequently developed an improved, super high-speed FARCO (S-FARCO), which is able to process several hundred thousand frames per second. The principal advantage of our new system is its wide applicability to any correlation scheme. Three different configurations were proposed, each depending on correlation speed. This paper describes and evaluates a software correlation filter. The face recognition function proved highly accurate, seeing that a low-resolution facial image size (64 × 64 pixels) has been successfully implemented. An operation speed of less than 10 ms was achieved using a personal computer with a central processing unit (CPU) of 3 GHz and 2 GB memory. When we applied the software correlation filter to a high-security cellular phone face recognition system, experiments on 30 female students over a period of three months yielded low error rates: 0% false acceptance rate and 2% false rejection rate. Therefore, the filtering correlation works effectively when applied to low resolution images such as web-based images or faces captured by a monitoring camera.

Therapeutic peptides: new arsenal against drug resistant pathogens.

PubMed

Mok, Wendy W K; Li, Yingfu

2014-01-01

Our incessant tug-of-war with multidrug resistant pathogenic bacteria has prompted researchers to explore novel methods of designing therapeutics in order to defend ourselves against infectious diseases. Combined advances in whole genome analysis, bioinformatics algorithms, and biochemical techniques have led to the discovery and subsequent characterization of an abundant array of functional small peptides in microorganisms and multicellular organisms. Typically classified as having 10 to 100 amino acids, many of these peptides have been found to have dual activities, executing important defensive and regulatory functions in their hosts. In higher organisms, such as mammals, plants, and fungi, host defense peptides have been shown to have immunomodulatory and antimicrobial properties. In microbes, certain growth-inhibiting peptides have been linked to the regulation of diverse cellular processes. Examples of these processes include quorum sensing, stress response, cell differentiation, biofilm formation, pathogenesis, and multidrug tolerance. In this review, we will present a comprehensive overview of the discovery, characteristics, and functions of host- and bacteria-derived peptides with antimicrobial activities. The advantages and possible shortcomings of using these peptides as antimicrobial agents and targets will also be discussed. We will further examine current efforts in engineering synthetic peptides to be used as therapeutics and/or drug delivery vehicles.

The population ecology of male gametophytes: the link between pollination and seed production.

PubMed

Harder, Lawrence D; Aizen, Marcelo A; Richards, Shane A

2016-05-01

The fate of male gametophytes after pollen reaches stigmas links pollination to ovule fertilisation, governing subsequent siring success and seed production. Although male gametophyte performance primarily involves cellular processes, an ecological analogy may expose insights into the nature and implications of male gametophyte success. We elaborate this analogy theoretically and present empirical examples that illustrate associated insights. Specifically, we consider pollen loads on stigmas as localised populations subject to density-independent mortality and density-dependent processes as they traverse complex stylar environments. Different combinations of the timing of pollen-tube access to limiting stylar resources (simultaneous or sequential), the tube distribution among resources (repulsed or random) and the timing of density-independent mortality relative to competition (before or after) create signature relations of mean pollen-tube success and its variation among pistils to pollen receipt. Using novel nonlinear regression analyses (two-moment regression), we illustrate contrasting relations for two species, demonstrating that variety in these relations is a feature of reproductive diversity among angiosperms, rather than merely a theoretical curiosity. Thus, the details of male gametophyte ecology should shape sporophyte reproductive success and hence the dynamics and structure of angiosperm populations. © 2016 John Wiley & Sons Ltd/CNRS.

Embryonic maturation of epidermal Merkel cells is controlled by a redundant transcription factor network.

PubMed

Perdigoto, Carolina N; Bardot, Evan S; Valdes, Victor J; Santoriello, Francis J; Ezhkova, Elena

2014-12-01

Merkel cell-neurite complexes are located in touch-sensitive areas of the mammalian skin and are involved in recognition of the texture and shape of objects. Merkel cells are essential for these tactile discriminations, as they generate action potentials in response to touch stimuli and induce the firing of innervating afferent nerves. It has been shown that Merkel cells originate from epidermal stem cells, but the cellular and molecular mechanisms of their development are largely unknown. In this study, we analyzed Merkel cell differentiation during development and found that it is a temporally regulated maturation process characterized by a sequential activation of Merkel cell-specific genes. We uncovered key transcription factors controlling this process and showed that the transcription factor Atoh1 is required for initial Merkel cell specification. The subsequent maturation steps of Merkel cell differentiation are controlled by cooperative function of the transcription factors Sox2 and Isl1, which physically interact and work to sustain Atoh1 expression. These findings reveal the presence of a robust transcriptional network required to produce functional Merkel cells that are required for tactile discrimination. © 2014. Published by The Company of Biologists Ltd.

The role of water molecules in computational drug design.

PubMed

de Beer, Stephanie B A; Vermeulen, Nico P E; Oostenbrink, Chris

2010-01-01

Although water molecules are small and only consist of two different atom types, they play various roles in cellular systems. This review discusses their influence on the binding process between biomacromolecular targets and small molecule ligands and how this influence can be modeled in computational drug design approaches. Both the structure and the thermodynamics of active site waters will be discussed as these influence the binding process significantly. Structurally conserved waters cannot always be determined experimentally and if observed, it is not clear if they will be replaced upon ligand binding, even if sufficient space is available. Methods to predict the presence of water in protein-ligand complexes will be reviewed. Subsequently, we will discuss methods to include water in computational drug research. Either as an additional factor in automated docking experiments, or explicitly in detailed molecular dynamics simulations, the effect of water on the quality of the simulations is significant, but not easily predicted. The most detailed calculations involve estimates of the free energy contribution of water molecules to protein-ligand complexes. These calculations are computationally demanding, but give insight in the versatility and importance of water in ligand binding.

Investigation of the stallion sperm proteome by mass spectrometry.

PubMed

Swegen, Aleona; Curry, Benjamin J; Gibb, Zamira; Lambourne, Sarah R; Smith, Nathan D; Aitken, R John

2015-03-01

Stallion spermatozoa continue to present scientific and clinical challenges with regard to the biological mechanisms responsible for their survival and function. In particular, deeper understanding of sperm energy metabolism, defence against oxidative damage and cell-cell interactions should improve fertility assessment and the application of advanced reproductive technologies in the equine species. In this study, we used highly sensitive LC-MS/MS technology and sequence database analysis to identify and characterise the proteome of Percoll-isolated ejaculated equine spermatozoa, with the aim of furthering our understanding of this cell's complex biological machinery. We were able to identify 9883 peptides comprising 1030 proteins, which were subsequently attributed to 975 gene products. Gene ontology analysis for molecular and cellular processes revealed new information about the metabolism, antioxidant defences and receptors of stallion spermatozoa. Mitochondrial proteins and those involved in catabolic processes constituted dominant categories. Several enzymes specific to β-oxidation of fatty acids were identified, and further experiments were carried out to ascertain their functional significance. Inhibition of carnitine palmitoyl transferase 1, a rate-limiting enzyme of β-oxidation, reduced motility parameters, indicating that β-oxidation contributes to maintenance of motility in stallion spermatozoa. © 2015 Society for Reproduction and Fertility.

Wireless Phone Threat Assessment and New Wireless Technology Concerns for Aircraft Navigation Radios

NASA Technical Reports Server (NTRS)

Ely, Jay J.; Nguyen, Truong X.; Koppen, Sandra V.; Beggs, John H.; Salud, Maria Theresa P.

2003-01-01

To address the concern for cellular phone electromagnetic interference to aircraft radios, a radiated emission measurement process was developed for two dominant digital standards of wireless handsets. Spurious radiated emissions were efficiently characterized from devices tested in either a semi-anechoic or reverberation chamber, in terms of effective radiated power. Eight representative handsets (four from each digital standard) were commanded to operate while varying their radio transmitter parameters (power, modulation, etc.). This report provides a detailed description of the measurement process and resulting data, which may subsequently be used by others as a basis of consistent evaluation of other portable transmitters using a variety of wireless transmission protocols. Aircraft interference path loss and navigation radio interference threshold data from numerous reference documents, standards, and NASA partnerships were compiled. Using these data, a preliminary risk assessment is provided for wireless phone interference to aircraft Localizer, Glideslope, Very High Frequency Omni directional Range, and Global Positioning Satellite radio receivers on typical transport airplanes. The report identifies where existing data for device emissions, interference path loss, and navigation radio interference thresholds need to be extended for an accurate risk assessment for wireless transmitters in aircraft.

The expression of PTEN in the development of mouse cochlear lateral wall.

PubMed

Dong, Y; Sui, L; Yamaguchi, F; Kamitori, K; Hirata, Y; Hossain, A; Noguchi, C; Katagi, A; Nishio, M; Suzuki, A; Lou, X; Tokuda, M

2014-01-31

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that regulates various cell processes including proliferation, growth, synaptogenesis, neural and glioma stem/progenitor cell renewal. In addition, PTEN can regulate sensory cell proliferation and differentiation of hair bundles in the mammalian cochlea. In this study we use immunofluorescence, Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) to reveal the expression of PTEN in the developing cochlear lateral wall, which is crucial for regulating K(+) homeostasis. Relatively high levels of PTEN are initially expressed in the marginal cells (MCs) of the lateral wall at embryonic day (E) 17.5 when they start to differentiate. Similarly high levels are subsequently expressed in differentiating root cells (RCs) at postnatal day (P) 3 and then in spiral ligament fibrocytes (SLFs) at P 10. In the mature cochlea, PTEN expression is low or undetectable in MCs and SLFs but it remains high in RCs and their processes. The expression pattern for PTEN in the developing lateral wall suggests that it plays a critical role in the differentiation of the cellular pathways that regulate K(+) homeostasis in the cochlea. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Cellular self-assembly and biomaterials-based organoid models of development and diseases.

PubMed

Shah, Shivem B; Singh, Ankur

2017-04-15

Organogenesis and morphogenesis have informed our understanding of physiology, pathophysiology, and avenues to create new curative and regenerative therapies. Thus far, this understanding has been hindered by the lack of a physiologically relevant yet accessible model that affords biological control. Recently, three-dimensional ex vivo cellular cultures created through cellular self-assembly under natural extracellular matrix cues or through biomaterial-based directed assembly have been shown to physically resemble and recapture some functionality of target organs. These "organoids" have garnered momentum for their applications in modeling human development and disease, drug screening, and future therapy design or even organ replacement. This review first discusses the self-organizing organoids as materials with emergent properties and their advantages and limitations. We subsequently describe biomaterials-based strategies used to afford more control of the organoid's microenvironment and ensuing cellular composition and organization. In this review, we also offer our perspective on how multifunctional biomaterials with precise spatial and temporal control could ultimately bridge the gap between in vitro organoid platforms and their in vivo counterparts. Several notable reviews have highlighted PSC-derived organoids and 3D aggregates, including embryoid bodies, from a development and cellular assembly perspective. The focus of this review is to highlight the materials-based approaches that cells, including PSCs and others, adopt for self-assembly and the controlled development of complex tissues, such as that of the brain, gut, and immune system. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Cellular uptake and retention measurements of alkylphosphocholines in the SK-BR-3 breast cancer and Molt-4 leukemia cell line using capillary gas chromatography.

PubMed

Brochez, V; Van Heuverswyn, D; Diniz, J A; De Potter, C R; Van den Eeckhout, E G

1999-05-01

The determination of cellular content of octadecylphosphocholine (D-19391) and hexadecylphosphocholine (HePC, D-18506), two anticancer agents of the alkylphosphocholine group, using capillary gas chromatography is described. The compounds' cytotoxicity was first determined by the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium] assay, being indicative for the concentration used in the uptake and retention measurements. D-19391 was added to the SK-BR-3 breast cancer cell line and HePC to the Molt-4 leukemia cell line in concentrations of, respectively, 18.6 and 15.0 microM, during a 36-h incubation period at 37 degrees C, 5% CO2. HePC uptake in the leukemia cells was followed by a 24-h reversibility test in drug-free medium. Subsequently, sample clean-up was performed on a weak cation-exchange column. For the quantitative analysis, HePC was used as internal standard for the D-19391 measurements and vice versa. Derivatization of the samples with trimethylsilylbromide was followed by capillary gas chromatographic analysis. From these data we conclude that our uptake results are quite similar with those of a previous study of HePC cellular uptake in the more resistant Caco-2T colon cancer cell line. Without having investigated the mechanism that underlies the cellular uptake results obtained, our study points to no direct correlation between the compounds' cellular uptake and their cytotoxic effects.

Determination of the mechanical properties of solid and cellular polymeric dosage forms by diametral compression.

PubMed

Blaesi, Aron H; Saka, Nannaji

2016-07-25

At present, the immediate-release solid dosage forms, such as the oral tablets and capsules, are granular solids. They release drug rapidly and have adequate mechanical properties, but their manufacture is fraught with difficulties inherent in processing particulate matter. Such difficulties, however, could be overcome by liquid-based processing. Therefore, we have recently introduced polymeric cellular (i.e., highly porous) dosage forms prepared from a melt process. Experiments have shown that upon immersion in a dissolution medium, the cellular dosage forms with polyethylene glycol (PEG) as excipient and with predominantly open-cell topology disintegrate by exfoliation, thus enabling rapid drug release. If the volume fraction of voids of the open-cell structures is too large, however, their mechanical strength is adversely affected. At present, the common method for determining the tensile strength of brittle, solid dosage forms (such as select granular forms) is the diametral compression test. In this study, the theory of diametral compression is first refined to demonstrate that the relevant mechanical properties of ductile and cellular solids (i.e., the elastic modulus and the yield strength) can also be extracted from this test. Diametral compression experiments are then conducted on PEG-based solid and cellular dosage forms. It is found that the elastic modulus and yield strength of the open-cell structures are about an order of magnitude smaller than those of the non-porous solids, but still are substantially greater than the stiffness and strength requirements for handling the dosage forms manually. This work thus demonstrates that melt-processed polymeric cellular dosage forms that release drug rapidly can be designed and manufactured to have adequate mechanical properties. Copyright © 2016. Published by Elsevier B.V.

Translating neurotrophic and cellular plasticity: from pathophysiology to improved therapeutics for bipolar disorder

PubMed Central

Soeiro-de-Souza, M. G.; Dias, V. V.; Figueira, M. L.; Forlenza, O. V.; Gattaz, W. F.; Zarate, C. A.; Machado-Vieira, R.

2014-01-01

Objective Bipolar disorder (BD) likely involves, at a molecular and cellular level, dysfunctions of critical neurotrophic, cellular plasticity and resilience pathways and neuroprotective processes. Therapeutic properties of mood stabilizers are presumed to result from a restoration of the function of these altered pathways and processes through a wide range of biochemical and molecular effects. We aimed to review the altered pathways and processes implicated in BD, such as neurotrophic factors, mitogen-activated protein kinases, Bcl-2, phosphoinositol signaling, intracellular calcium and glycogen synthase kinase-3. Methods We undertook a literature search of recent relevant journal articles, book chapter and reviews on neurodegeneration and neuroprotection in BD. Search words entered were ‘brain-derived neurotrophic factor,’ ‘Bcl-2,’ ‘mitogen-activated protein kinases,’ ‘neuroprotection,’ ‘calcium,’ ‘bipolar disorder,’ ‘mania,’ and ‘depression.’ Results The most consistent and replicated findings in the pathophysiology of BD may be classified as follows: i) calcium dysregulation, ii) mitochondrial/endoplasmic reticulum dysfunction, iii) glial and neuronal death/atrophy and iv) loss of neurotrophic/plasticity effects in brain areas critically involved in mood regulation. In addition, the evidence supports that treatment with mood stabilizers; in particular, lithium restores these pathophysiological changes. Conclusion Bipolar disorder is associated with impairments in neurotrophic, cellular plasticity and resilience pathways as well as in neuroprotective processes. The evidence supports that treatment with mood stabilizers, in particular lithium, restores these pathophysiological changes. Studies that attempt to prevent (intervene before the onset of the molecular and cellular changes), treat (minimize severity of these deficits over time), and rectify (reverse molecular and cellular deficits) are promising therapeutic strategies for developing improved treatments for bipolar disorder. PMID:22676371

Translating neurotrophic and cellular plasticity: from pathophysiology to improved therapeutics for bipolar disorder.

PubMed

Soeiro-de-Souza, M G; Dias, V V; Figueira, M L; Forlenza, O V; Gattaz, W F; Zarate, C A; Machado-Vieira, R

2012-11-01

Bipolar disorder (BD) likely involves, at a molecular and cellular level, dysfunctions of critical neurotrophic, cellular plasticity and resilience pathways and neuroprotective processes. Therapeutic properties of mood stabilizers are presumed to result from a restoration of the function of these altered pathways and processes through a wide range of biochemical and molecular effects. We aimed to review the altered pathways and processes implicated in BD, such as neurotrophic factors, mitogen-activated protein kinases, Bcl-2, phosphoinositol signaling, intracellular calcium and glycogen synthase kinase-3. We undertook a literature search of recent relevant journal articles, book chapter and reviews on neurodegeneration and neuroprotection in BD. Search words entered were 'brain-derived neurotrophic factor,''Bcl-2,''mitogen-activated protein kinases,''neuroprotection,''calcium,''bipolar disorder,''mania,' and 'depression.' The most consistent and replicated findings in the pathophysiology of BD may be classified as follows: i) calcium dysregulation, ii) mitochondrial/endoplasmic reticulum dysfunction, iii) glial and neuronal death/atrophy and iv) loss of neurotrophic/plasticity effects in brain areas critically involved in mood regulation. In addition, the evidence supports that treatment with mood stabilizers; in particular, lithium restores these pathophysiological changes. Bipolar disorder is associated with impairments in neurotrophic, cellular plasticity and resilience pathways as well as in neuroprotective processes. The evidence supports that treatment with mood stabilizers, in particular lithium, restores these pathophysiological changes. Studies that attempt to prevent (intervene before the onset of the molecular and cellular changes), treat (minimize severity of these deficits over time), and rectify (reverse molecular and cellular deficits) are promising therapeutic strategies for developing improved treatments for bipolar disorder. © 2012 John Wiley & Sons A/S.

In vivo cell biology in zebrafish – providing insights into vertebrate development and disease

PubMed Central

Vacaru, Ana M.; Unlu, Gokhan; Spitzner, Marie; Mione, Marina; Knapik, Ela W.; Sadler, Kirsten C.

2014-01-01

ABSTRACT Over the past decades, studies using zebrafish have significantly advanced our understanding of the cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos that allow comprehensive imaging of embryogenesis combined with powerful genetic approaches. However, forward genetic screens in zebrafish have generated unanticipated findings that are mirrored by human genetic studies: disruption of genes implicated in basic cellular processes, such as protein secretion or cytoskeletal dynamics, causes discrete developmental or disease phenotypes. This is surprising because many processes that were assumed to be fundamental to the function and survival of all cell types appear instead to be regulated by cell-specific mechanisms. Such discoveries are facilitated by experiments in whole animals, where zebrafish provides an ideal model for visualization and manipulation of organelles and cellular processes in a live vertebrate. Here, we review well-characterized mutants and newly developed tools that underscore this notion. We focus on the secretory pathway and microtubule-based trafficking as illustrative examples of how studying cell biology in vivo using zebrafish has broadened our understanding of the role fundamental cellular processes play in embryogenesis and disease. PMID:24481493

Cell phenotypic variation in normal and damaged tendons

PubMed Central

Clegg, Peter D; Strassburg, Sandra; Smith, Roger K

2007-01-01

Injuries to tendons are common in both human athletes as well as in animals, such as the horse, which are used for competitive purposes. Furthermore, such injuries are also increasing in prevalence in the ageing, sedentary population. Tendon diseases often respond poorly to treatment and require lengthy periods of rehabilitation. The tendon has a unique extracellular matrix, which has developed to withstand the mechanical demands of such tensile-load bearing structures. Following injury, any repair process is inadequate and results in tissue that is distinct from original tendon tissue. There is growing evidence for the key role of the tendon cell (tenocyte) in both the normal physiological homeostasis and regulation of the tendon matrix and the pathological derangements that occur in disease. In particular, the tenocyte is considered to have a major role in effecting the subclinical matrix degeneration that is thought to occur prior to clinical disease, as well as in the severe degradative events that occur in the tendon at the onset of clinical disease. Furthermore, the tenocyte is likely to have a central role in the production of the biologically inadequate fibrocartilaginous repair tissue that develops subsequent to tendinopathy. Understanding the biology of the tenocyte is central to the development of appropriate interventions and drug therapies that will either prevent the onset of disease, or lead to more rapid and appropriate repair of injured tendon. Central to this is a full understanding of the proteolytic response in the tendon in disease by such enzymes as metalloproteinases, as well as the control of the inappropriate fibrocartilaginous differentiation. Finally, it is important that we understand the role of both intrinsic and extrinsic cellular elements in the repair process in the tendon subsequent to injury. PMID:17696903

A meta-analysis to evaluate the cellular processes regulated by the interactome of endogenous and over-expressed estrogen receptor alpha.

PubMed

Simões, Joana; Amado, Francisco M; Vitorino, Rui; Helguero, Luisa A

2015-01-01

The nature of the proteins complexes that regulate ERα subcellular localization and activity is still an open question in breast cancer biology. Identification of such complexes will help understand development of endocrine resistance in ER+ breast cancer. Mass spectrometry (MS) has allowed comprehensive analysis of the ERα interactome. We have compared six published works analyzing the ERα interactome of MCF-7 and HeLa cells in order to identify a shared or different pathway-related fingerprint. Overall, 806 ERα interacting proteins were identified. The cellular processes were differentially represented according to the ERα purification methodology, indicating that the methodologies used are complementary. While in MCF-7 cells, the interactome of endogenous and over-expressed ERα essentially represents the same biological processes and cellular components, the proteins identified were not over-lapping; thus, suggesting that the biological response may differ as the regulatory/participating proteins in these complexes are different. Interestingly, biological processes uniquely associated to ERα over-expressed in HeLa cell line included L-serine biosynthetic process, cellular amino acid biosynthetic process and cell redox homeostasis. In summary, all the approaches analyzed in this meta-analysis are valid and complementary; in particular, for those cases where the processes occur at low frequency with normal ERα levels, and can be identified when the receptor is over-expressed. However special effort should be put into validating these findings in cells expressing physiological ERα levels.

Synthetic Biology: Tools to Design, Build, and Optimize Cellular Processes

PubMed Central

Young, Eric; Alper, Hal

2010-01-01

The general central dogma frames the emergent properties of life, which make biology both necessary and difficult to engineer. In a process engineering paradigm, each biological process stream and process unit is heavily influenced by regulatory interactions and interactions with the surrounding environment. Synthetic biology is developing the tools and methods that will increase control over these interactions, eventually resulting in an integrative synthetic biology that will allow ground-up cellular optimization. In this review, we attempt to contextualize the areas of synthetic biology into three tiers: (1) the process units and associated streams of the central dogma, (2) the intrinsic regulatory mechanisms, and (3) the extrinsic physical and chemical environment. Efforts at each of these three tiers attempt to control cellular systems and take advantage of emerging tools and approaches. Ultimately, it will be possible to integrate these approaches and realize the vision of integrative synthetic biology when cells are completely rewired for biotechnological goals. This review will highlight progress towards this goal as well as areas requiring further research. PMID:20150964

Functional analysis of alpha5beta1 integrin and lipid rafts in invasion of epithelial cells by Porphyromonas gingivalis using fluorescent beads coated with bacterial membrane vesicles.

PubMed

Tsuda, Kayoko; Furuta, Nobumichi; Inaba, Hiroaki; Kawai, Shinji; Hanada, Kentaro; Yoshimori, Tamotsu; Amano, Atsuo

2008-01-01

Porphyromonas gingivalis, a periodontal pathogen, was previously suggested to exploit alpha5beta1 integrin and lipid rafts to invade host cells. However, it is unknown if the functional roles of these host components are distinct from one another during bacterial invasion. In the present study, we analyzed the mechanisms underlying P. gingivalis invasion, using fluorescent beads coated with bacterial membrane vesicles (MV beads). Cholesterol depletion reagents including methyl-beta-cyclodextrin (MbetaCD) drastically inhibited the entry of MV beads into epithelial cells, while they were less effective on bead adhesion to the cells. Bead entry was also abolished in CHO cells deficient in sphingolipids, components of lipid rafts, whereas adhesion was negligibly influenced. Following MbetaCD treatment, downstream events leading to actin polymerization were abolished; however, alpha5beta1 integrin was recruited to beads attached to the cell surface. Dominant-negative Rho GTPase Rac1 abolished cellular engulfment of the beads, whereas dominant-negative Cdc42 did not. Following cellular interaction with the beads, Rac1 was found to be translocated to the lipid rafts fraction, which was inhibited by MbetaCD. These results suggest that alpha5beta1 integrin, independent of lipid rafts, promotes P. gingivalis adhesion to epithelial cells, while the subsequent uptake process requires lipid raft components for actin organization, with Rho GTPase Rac1.

Activation of Membrane Fusion by Murine Leukemia Viruses Is Controlled in cis or in trans by Interactions between the Receptor-Binding Domain and a Conserved Disulfide Loop of the Carboxy Terminus of the Surface Glycoprotein

PubMed Central

Lavillette, Dimitri; Boson, Bertrand; Russell, Stephen J.; Cosset, François-Loïc

2001-01-01

Cell entry of retroviruses is initiated by the recognition of cellular receptors and the subsequent membrane fusion between viral and cellular membranes. These two steps are mediated by the surface (SU) and transmembrane (TM) subunits of the retroviral envelope glycoprotein (Env), respectively. Determinants regulating membrane fusion have been described throughout SU and TM, but the processes coupling receptor recognition to fusion are still elusive. Here we establish that a critical interaction is formed between the receptor-binding domain (RBD) and the major disulfide loop of the carboxy-terminal domain (C domain) of the murine leukemia virus SU. Receptor binding causes an alteration of this interaction and, in turn, promotes further events of Env fusion activation. We characterize mutations which, by lowering this interaction and reducing the compatibility between the RBD and C domains of Env glycoprotein chimeras, affect both Env fusogenicity and sensitivity to receptor interference. Additionally, we demonstrate that suboptimal interactions in such mutant Env proteins can be compensated in trans by soluble RBDs in a manner that depends on their compatibility with the C domain. Our results therefore indicate that RBD/C domain interactions may occur in cis, via the proper RBD of the viral Env itself, or in trans, via a distinct RBD expressed by virion-free Env glycoproteins expressed endogenously by the infected cells or provided by neighboring Env trimers. PMID:11264358

Effects of overnight refrigeration on the microscopic evaluation of sputum.

PubMed Central

Penn, R L; Silberman, R

1984-01-01

Microscopic evaluation of sputum permits selection of specimens suitable for culture, assessment of likely pathogens, and the best interpretation of culture results. We prospectively evaluated 50 sputum specimens which were promptly submitted to our clinical laboratory; smears and cultures were performed both immediately and after 20 h of refrigeration. Specimens were grouped according to the numbers of squamous epithelial cells and neutrophils per low-power field present on coded Gram-stained smears. The numbers of bacteria in five oil immersion fields were used to characterize smears for predominant, mixed, or scanty forms. After refrigeration, only three specimens changed group from a definite loss of squamous epithelial cells, and only two changed group from a definite loss of neutrophils. Based on cellular composition, the majority of samples would have been processed identically both before and after refrigeration. In contrast, organism forms detected on smears and their relative quantities were dramatically altered after refrigeration. A predominant smear form was gained in 11 and lost in 8 refrigerated specimens. The frequent changes on smears observed overall resulted from both increases and decreases in numbers of bacteria and yeasts. The majority of sputum culture results were insignificantly affected by the refrigeration of specimens. We conclude that 20 h of refrigeration renders sputum useless for the microscopic evaluation of potential pathogens and the subsequent interpretation of culture results. However, overnight refrigeration does not affect the determination from smears of sputum suitability for culture based on cellular composition. PMID:6699145

Lipids and lipid binding proteins: a perfect match.

PubMed

Glatz, Jan F C

2015-02-01

Lipids serve a great variety of functions, ranging from structural components of biological membranes to signaling molecules affecting various cellular functions. Several of these functions are related to the unique physico-chemical properties shared by all lipid species, i.e., their hydrophobicity. The latter, however, is accompanied by a poor solubility in an aqueous environment and thus a severe limitation in the transport of lipids in aqueous compartments such as blood plasma and the cellular soluble cytoplasm. Specific proteins which can reversibly and non-covalently associate with lipids, designated as lipid binding proteins or lipid chaperones, greatly enhance the aqueous solubility of lipids and facilitate their transport between tissues and within tissue cells. Importantly, transport of lipids across biological membranes also is facilitated by specific (membrane-associated) lipid binding proteins. Together, these lipid binding proteins determine the bio-availability of their ligands, and thereby markedly influence the subsequent processing, utilization, or signaling effect of lipids. The bio-availability of specific lipid species thus is governed by the presence of specific lipid binding proteins, the affinity of these proteins for distinct lipid species, and the presence of competing ligands (including pharmaceutical compounds). Recent studies suggest that post-translational modifications of lipid binding proteins may have great impact on lipid-protein interactions. As a result, several levels of regulation exist that together determine the bio-availability of lipid species. This short review discusses the significance of lipid binding proteins and their potential application as targets for therapeutic intervention. Copyright © 2014 Elsevier Ltd. All rights reserved.

Chronic wheel running affects cocaine-induced c-Fos expression in brain reward areas in rats.

PubMed

Zlebnik, Natalie E; Hedges, Valerie L; Carroll, Marilyn E; Meisel, Robert L

2014-03-15

Emerging evidence from human and animal studies suggests that exercise is a highly effective treatment for drug addiction. However, most work has been done in behavioral models, and the effects of exercise on the neurobiological substrates of addiction have not been identified. Specifically, it is unknown whether prior exercise exposure alters neuronal activation of brain reward circuitry in response to drugs of abuse. To investigate this hypothesis, rats were given 21 days of daily access to voluntary wheel running in a locked or unlocked running wheel. Subsequently, they were challenged with a saline or cocaine (15 mg/kg, i.p.) injection and sacrificed for c-Fos immunohistochemistry. The c-Fos transcription factor is a measure of cellular activity and was used to quantify cocaine-induced activation of reward-processing areas of the brain: nucleus accumbens (NAc), caudate putamen (CPu), medial prefrontal cortex (mPFC), and orbitofrontal cortex (OFC). The mean fold change in cocaine-induced c-Fos cell counts relative to saline-induced c-Fos cell counts was significantly higher in exercising compared to control rats in the NAc core, dorsomedial and dorsolateral CPu, the prelimbic area, and the OFC, indicating differential cocaine-specific cellular activation of brain reward circuitry between exercising and control animals. These results suggest neurobiological mechanisms by which voluntary wheel running attenuates cocaine-motivated behaviors and provide support for exercise as a novel treatment for drug addiction. Copyright © 2013 Elsevier B.V. All rights reserved.

Comparison of specimen adequacy and smear quality in oral smears prepared by manual liquid-based cytology and conventional methods

PubMed Central

Shukla, Surabhi; Einstein, A; Shukla, Abhilasha; Mishra, Deepika

2015-01-01

Background: Liquid-based cytology (LBC), recommended in the mass screening of potentially malignant cervical and oral lesions, suffers from high cost owing to the use of expensive automated devices and materials. Considering the need for cost-effective LBC techniques, we evaluated the efficacy of an inexpensive manual LBC (MLBC) technique against conventional cytological technique in terms of specimen adequacy and smear quality of oral smears. Materials and Methods: Cytological samples were collected from 21 patients using a cytobrush device. After preparation of a conventional smear, the brush containing the remaining sample was immersed in the preservative vial. The preserved material was processed by an MLBC technique and subsequently, direct smears were made from the prepared cell button. Both conventional and MLBC smears were stained by routine Papanicolaou technique and evaluated by an independent observer for the thickness of the smear, cellular distribution, resolution/clarity of cells, cellular staining characteristics and the presence of unsatisfactory background/artifacts. Each parameter was graded as satisfactory; or satisfactory, but limited; or unsatisfactory. Chi-square test was used to compare the values obtained (significance set at P ≤ 0.05). Results: MLBC technique produced a significant number of satisfactory smears with regard to cell distribution, clarity/resolution, staining characteristics and background/artifacts compared to conventional methods. Conclusions: MLBC is a cost-effective cytological technique that may produce oral smears with excellent cytomorphology and longer storage life. PMID:26980958

Aggregation of luteinizing hormone receptors in granulosa cells: a possible mechanism of desensitization to the hormone.

PubMed Central

Amsterdam, A; Berkowitz, A; Nimrod, A; Kohen, F

1980-01-01

The temporal relationship between redistribution of receptors to lutropin (luteinizing hormone)/human chorionic gonadotropin in cultured rat ovarian granulosa cells and the cellular response to hormonal challenge were studied. Visualization of receptor-bound human chorionic gonadotropin by indirect immunofluorescence, with hormone-specific antibodies after fixation with 2% formaldehyde, revealed the existence of small clusters around the entire cell circumference 5--20 min after exposure to the hormone at 37 degrees C. Such small receptor aggregates were also evident if hormone incubation was at 4 degrees C or if cells were fixed with 2% formaldehyde before incubation. Larger clusters were evident after prolonged incubation with the hormone (2--4 hr) at 37 degrees C. The later change coincided with diminished cyclic AMP accumulation in respose to challenge with fresh hormone. When the fixation step was omitted and antibodies to human chorionic gonadotropin were applied after hormonal binding, acceleration of both receptor clustering and the desensitization process was observed. This maneuver also induced capping of the hormone receptors. In contrast, monovalent Fab' fragments of the antibodies were without effect. Internalization of the bound hormone in lysosomes, and subsequent degradation, was evident 8 hr after hormonal application and was not accelerated by the antibodies. It is suggested that clustering of the luteinizing hormone receptors may play a role in cellular responsiveness to the hormone. Massive aggregation of the receptors may desensitize the cell by interferring with coupling to adenylate cyclase. Images PMID:6251459

An epidemiological modeling and data integration framework.

PubMed

Pfeifer, B; Wurz, M; Hanser, F; Seger, M; Netzer, M; Osl, M; Modre-Osprian, R; Schreier, G; Baumgartner, C

2010-01-01

In this work, a cellular automaton software package for simulating different infectious diseases, storing the simulation results in a data warehouse system and analyzing the obtained results to generate prediction models as well as contingency plans, is proposed. The Brisbane H3N2 flu virus, which has been spreading during the winter season 2009, was used for simulation in the federal state of Tyrol, Austria. The simulation-modeling framework consists of an underlying cellular automaton. The cellular automaton model is parameterized by known disease parameters and geographical as well as demographical conditions are included for simulating the spreading. The data generated by simulation are stored in the back room of the data warehouse using the Talend Open Studio software package, and subsequent statistical and data mining tasks are performed using the tool, termed Knowledge Discovery in Database Designer (KD3). The obtained simulation results were used for generating prediction models for all nine federal states of Austria. The proposed framework provides a powerful and easy to handle interface for parameterizing and simulating different infectious diseases in order to generate prediction models and improve contingency plans for future events.

ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease.

PubMed

Nakano, Masaki; Imamura, Hiromi; Sasaoka, Norio; Yamamoto, Masamichi; Uemura, Norihito; Shudo, Toshiyuki; Fuchigami, Tomohiro; Takahashi, Ryosuke; Kakizuka, Akira

2017-08-01

Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances), which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors) are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist) against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor) were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Biomimetic approaches to modulate cellular adhesion in biomaterials: A review.

PubMed

Rahmany, Maria B; Van Dyke, Mark

2013-03-01

Natural extracellular matrix (ECM) proteins possess critical biological characteristics that provide a platform for cellular adhesion and activation of highly regulated signaling pathways. However, ECM-based biomaterials can have several limitations, including poor mechanical properties and risk of immunogenicity. Synthetic biomaterials alleviate the risks associated with natural biomaterials but often lack the robust biological activity necessary to direct cell function beyond initial adhesion. A thorough understanding of receptor-mediated cellular adhesion to the ECM and subsequent signaling activation has facilitated development of techniques that functionalize inert biomaterials to provide a biologically active surface. Here we review a range of approaches used to modify biomaterial surfaces for optimal receptor-mediated cell interactions, as well as provide insights into specific mechanisms of downstream signaling activation. In addition to a brief overview of integrin receptor-mediated cell function, so-called "biomimetic" techniques reviewed here include (i) surface modification of biomaterials with bioadhesive ECM macromolecules or specific binding motifs, (ii) nanoscale patterning of the materials and (iii) the use of "natural-like" biomaterials. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Molecular and cellular aspects of rhabdovirus entry.

PubMed

Albertini, Aurélie A V; Baquero, Eduard; Ferlin, Anna; Gaudin, Yves

2012-01-01

Rhabdoviruses enter the cell via the endocytic pathway and subsequently fuse with a cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion is triggered via a low-pH induced structural rearrangement. G is an atypical fusion protein as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The elucidation of the atomic structures of these two conformations for the vesicular stomatitis virus (VSV) G has revealed that it is different from the previously characterized class I and class II fusion proteins. In this review, the pre- and post-fusion VSV G structures are presented in detail demonstrating that G combines the features of the class I and class II fusion proteins. In addition to these similarities, these G structures also reveal some particularities that expand our understanding of the working of fusion machineries. Combined with data from recent studies that revealed the cellular aspects of the initial stages of rhabdovirus infection, all these data give an integrated view of the entry pathway of rhabdoviruses into their host cell.

Molecular and Cellular Aspects of Rhabdovirus Entry

PubMed Central

Albertini, Aurélie A. V.; Baquero, Eduard; Ferlin, Anna; Gaudin, Yves

2012-01-01

Rhabdoviruses enter the cell via the endocytic pathway and subsequently fuse with a cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion is triggered via a low-pH induced structural rearrangement. G is an atypical fusion protein as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The elucidation of the atomic structures of these two conformations for the vesicular stomatitis virus (VSV) G has revealed that it is different from the previously characterized class I and class II fusion proteins. In this review, the pre- and post-fusion VSV G structures are presented in detail demonstrating that G combines the features of the class I and class II fusion proteins. In addition to these similarities, these G structures also reveal some particularities that expand our understanding of the working of fusion machineries. Combined with data from recent studies that revealed the cellular aspects of the initial stages of rhabdovirus infection, all these data give an integrated view of the entry pathway of rhabdoviruses into their host cell. PMID:22355455

Elucidating dynamic metabolic physiology through network integration of quantitative time-course metabolomics

DOE PAGES

Bordbar, Aarash; Yurkovich, James T.; Paglia, Giuseppe; ...

2017-04-07

In this study, the increasing availability of metabolomics data necessitates novel methods for deeper data analysis and interpretation. We present a flux balance analysis method that allows for the computation of dynamic intracellular metabolic changes at the cellular scale through integration of time-course absolute quantitative metabolomics. This approach, termed “unsteady-state flux balance analysis” (uFBA), is applied to four cellular systems: three dynamic and one steady-state as a negative control. uFBA and FBA predictions are contrasted, and uFBA is found to be more accurate in predicting dynamic metabolic flux states for red blood cells, platelets, and Saccharomyces cerevisiae. Notably, only uFBAmore » predicts that stored red blood cells metabolize TCA intermediates to regenerate important cofactors, such as ATP, NADH, and NADPH. These pathway usage predictions were subsequently validated through 13C isotopic labeling and metabolic flux analysis in stored red blood cells. Utilizing time-course metabolomics data, uFBA provides an accurate method to predict metabolic physiology at the cellular scale for dynamic systems.« less

The segment polarity network is a robust developmental module

NASA Astrophysics Data System (ADS)

von Dassow, George; Meir, Eli; Munro, Edwin M.; Odell, Garrett M.

2000-07-01

All insects possess homologous segments, but segment specification differs radically among insect orders. In Drosophila, maternal morphogens control the patterned activation of gap genes, which encode transcriptional regulators that shape the patterned expression of pair-rule genes. This patterning cascade takes place before cellularization. Pair-rule gene products subsequently `imprint' segment polarity genes with reiterated patterns, thus defining the primordial segments. This mechanism must be greatly modified in insect groups in which many segments emerge only after cellularization. In beetles and parasitic wasps, for instance, pair-rule homologues are expressed in patterns consistent with roles during segmentation, but these patterns emerge within cellular fields. In contrast, although in locusts pair-rule homologues may not control segmentation, some segment polarity genes and their interactions are conserved. Perhaps segmentation is modular, with each module autonomously expressing a characteristic intrinsic behaviour in response to transient stimuli. If so, evolution could rearrange inputs to modules without changing their intrinsic behaviours. Here we suggest, using computer simulations, that the Drosophila segment polarity genes constitute such a module, and that this module is resistant to variations in the kinetic constants that govern its behaviour.

Psychedelics Recruit Multiple Cellular Types and Produce Complex Transcriptional Responses Within the Brain.

PubMed

Martin, David A; Nichols, Charles D

2016-09-01

There has recently been a resurgence of interest in psychedelics, substances that profoundly alter perception and cognition and have recently demonstrated therapeutic efficacy to treat anxiety, depression, and addiction in the clinic. The receptor mechanisms that drive their molecular and behavioral effects involve activation of cortical serotonin 5-HT 2A receptors, but the responses of specific cellular populations remain unknown. Here, we provide evidence that a small subset of 5-HT 2A -expressing excitatory neurons is directly activated by psychedelics and subsequently recruits other select cell types including subpopulations of inhibitory somatostatin and parvalbumin GABAergic interneurons, as well as astrocytes, to produce distinct and regional responses. To gather data regarding the response of specific neuronal populations, we developed methodology for fluorescence-activated cell sorting (FACS) to segregate and enrich specific cellular subtypes in the brain. These methods allow for robust neuronal sorting based on cytoplasmic epitopes followed by downstream nucleic acid analysis, expanding the utility of FACS in neuroscience research. Copyright © 2016 Forschungsgesellschaft für Arbeitsphysiologie und Arbeitschutz e.V. Published by Elsevier B.V. All rights reserved.

Elucidating dynamic metabolic physiology through network integration of quantitative time-course metabolomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bordbar, Aarash; Yurkovich, James T.; Paglia, Giuseppe

In this study, the increasing availability of metabolomics data necessitates novel methods for deeper data analysis and interpretation. We present a flux balance analysis method that allows for the computation of dynamic intracellular metabolic changes at the cellular scale through integration of time-course absolute quantitative metabolomics. This approach, termed “unsteady-state flux balance analysis” (uFBA), is applied to four cellular systems: three dynamic and one steady-state as a negative control. uFBA and FBA predictions are contrasted, and uFBA is found to be more accurate in predicting dynamic metabolic flux states for red blood cells, platelets, and Saccharomyces cerevisiae. Notably, only uFBAmore » predicts that stored red blood cells metabolize TCA intermediates to regenerate important cofactors, such as ATP, NADH, and NADPH. These pathway usage predictions were subsequently validated through 13C isotopic labeling and metabolic flux analysis in stored red blood cells. Utilizing time-course metabolomics data, uFBA provides an accurate method to predict metabolic physiology at the cellular scale for dynamic systems.« less

Continuous transport of a small fraction of plasma membrane cholesterol to endoplasmic reticulum regulates total cellular cholesterol

PubMed Central

Infante, Rodney Elwood; Radhakrishnan, Arun

2017-01-01

Cells employ regulated transport mechanisms to ensure that their plasma membranes (PMs) are optimally supplied with cholesterol derived from uptake of low-density lipoproteins (LDL) and synthesis. To date, all inhibitors of cholesterol transport block steps in lysosomes, limiting our understanding of post-lysosomal transport steps. Here, we establish the cholesterol-binding domain 4 of anthrolysin O (ALOD4) as a reversible inhibitor of cholesterol transport from PM to endoplasmic reticulum (ER). Using ALOD4, we: (1) deplete ER cholesterol without altering PM or overall cellular cholesterol levels; (2) demonstrate that LDL-derived cholesterol travels from lysosomes first to PM to meet cholesterol needs, and subsequently from PM to regulatory domains of ER to suppress activation of SREBPs, halting cholesterol uptake and synthesis; and (3) determine that continuous PM-to-ER cholesterol transport allows ER to constantly monitor PM cholesterol levels, and respond rapidly to small declines in cellular cholesterol by activating SREBPs, increasing cholesterol uptake and synthesis. DOI: http://dx.doi.org/10.7554/eLife.25466.001 PMID:28414269

Current State-of-the-Art 3D Tissue Models and Their Compatibility with Live Cell Imaging.

PubMed

Bardsley, Katie; Deegan, Anthony J; El Haj, Alicia; Yang, Ying

2017-01-01

Mammalian cells grow within a complex three-dimensional (3D) microenvironment where multiple cells are organized and surrounded by extracellular matrix (ECM). The quantity and types of ECM components, alongside cell-to-cell and cell-to-matrix interactions dictate cellular differentiation, proliferation and function in vivo. To mimic natural cellular activities, various 3D tissue culture models have been established to replace conventional two dimensional (2D) culture environments. Allowing for both characterization and visualization of cellular activities within possibly bulky 3D tissue models presents considerable challenges due to the increased thickness and subsequent light scattering features of such 3D models. In this chapter, state-of-the-art methodologies used to establish 3D tissue models are discussed, first with a focus on both scaffold-free and scaffold-based 3D tissue model formation. Following on, multiple 3D live cell imaging systems, mainly optical imaging modalities, are introduced. Their advantages and disadvantages are discussed, with the aim of stimulating more research in this highly demanding research area.

Enhanced cellular uptake of size-separated lipophilic silicon nanoparticles

NASA Astrophysics Data System (ADS)

Kusi-Appiah, Aubrey E.; Mastronardi, Melanie L.; Qian, Chenxi; Chen, Kenneth K.; Ghazanfari, Lida; Prommapan, Plengchart; Kübel, Christian; Ozin, Geoffrey A.; Lenhert, Steven

2017-03-01

Specific size, shape and surface chemistry influence the biological activity of nanoparticles. In the case of lipophilic nanoparticles, which are widely used in consumer products, there is evidence that particle size and formulation influences skin permeability and that lipophilic particles smaller than 6 nm can embed in lipid bilayers. Since most nanoparticle synthetic procedures result in mixtures of different particles, post-synthetic purification promises to provide insights into nanostructure-function relationships. Here we used size-selective precipitation to separate lipophilic allyl-benzyl-capped silicon nanoparticles into monodisperse fractions within the range of 1 nm to 5 nm. We measured liposomal encapsulation and cellular uptake of the monodisperse particles and found them to have generally low cytotoxicities in Hela cells. However, specific fractions showed reproducibly higher cytotoxicity than other fractions as well as the unseparated ensemble. Measurements indicate that the cytotoxicity mechanism involves oxidative stress and the differential cytotoxicity is due to enhanced cellular uptake by specific fractions. The results indicate that specific particles, with enhanced suitability for incorporation into lipophilic regions of liposomes and subsequent in vitro delivery to cells, are enriched in certain fractions.

Role of Complement on Broken Surfaces After Trauma.

PubMed

Huber-Lang, Markus; Ignatius, Anita; Brenner, Rolf E

2015-01-01

Activation of both the complement and coagulation cascade after trauma and subsequent local and systemic inflammatory response represent a major scientific and clinical problem. After severe tissue injury and bone fracture, exposure of innate immunity to damaged cells and molecular debris is considered a main trigger of the posttraumatic danger response. However, the effects of cellular fragments (e.g., histones) on complement activation remain enigmatic. Furthermore, direct effects of "broken" bone and cartilage surfaces on the fluid phase response of complement and its interaction with key cells of connective tissues are still unknown. Here, we summarize data suggesting direct and indirect complement activation by extracellular and cellular danger associated molecular patterns. In addition, key complement components and the corresponding receptors (such as C3aR, C5aR) have been detected on "exposed surfaces" of the damaged regions. On a cellular level, multiple effects of complement activation products on osteoblasts, osteoclasts, chondrocytes and mesenchymal stem cells have been found.In conclusion, the complement system may be activated by trauma-altered surfaces and is crucially involved in connective tissue healing and posttraumatic systemic inflammatory response.

Death-domain associated protein-6 (DAXX) mediated apoptosis in hantavirus infection is counter-balanced by activation of interferon-stimulated nuclear transcription factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khaiboullina, Svetlana F., E-mail: sv.khaiboullina@gmail.com; Morzunov, Sergey P.; Boichuk, Sergei V.

2013-09-01

Hantaviruses are negative strand RNA species that replicate predominantly in the cytoplasm. They also activate numerous cellular responses, but their involvement in nuclear processes is yet to be established. Using human umbilical vein endothelial cells (HUVECs), this study investigates the molecular finger-print of nuclear transcription factors during hantavirus infection. The viral-replication-dependent activation of pro-myelocytic leukemia protein (PML) was followed by subsequent localization in nuclear bodies (NBs). PML was also found in close proximity to activated Sp100 nuclear antigen and interferon-stimulated gene 20 kDa protein (ISG-20), but co-localization with death-domain associated protein-6 (DAXX) was not observed. These data demonstrate that hantavirusmore » triggers PML activation and localization in NBs in the absence of DAXX-PLM-NB co-localization. The results suggest that viral infection interferes with DAXX-mediated apoptosis, and expression of interferon-activated Sp100 and ISG-20 proteins may indicate intracellular intrinsic antiviral attempts.« less

A Summary of the Biological Basis of Frailty.

PubMed

Fielding, Roger A

2015-01-01

Frailty has been defined as a geriatric syndrome that is characterized by a reduction in the physiological reserve required for an individual to respond to endogenous and exogenous stressors. Using a discrete definition of frailty that includes sedentariness, involuntary weight loss, fatigue, poor muscle strength, and slow gait speed, 'frailty' has been associated with increased disability, postsurgical complications, and increased mortality. Despite the strong associations between frailty and subsequent poor outcomes, limited attention to this common geriatric condition has been paid in clinical settings. A more fundamental basic understanding of the biological factors that contribute to the frailty phenotype has begun to emerge. Multiple underlying biological factors such as dysregulation of inflammatory processes, genomic instability, oxidative stress, mitochondrial dysfunction, and cellular senescence appear to contribute to the clinical presentation of frailty. This chapter summarizes the papers presented on the biological basis of frailty from the 83rd Nestlé Nutrition Institute Workshop on 'Frailty, Pathophysiology, Phenotype and Patient Care' held in Barcelona, Spain, in March 2014. © 2015 Nestec Ltd., Vevey/S. Karger AG, Basel.

Ras history

PubMed Central

2010-01-01

Although the roots of Ras sprouted from the rich history of retrovirus research, it was the discovery of mutationally activated RAS genes in human cancer in 1982 that stimulated an intensive research effort to understand Ras protein structure, biochemistry and biology. While the ultimate goal has been developing anti-Ras drugs for cancer treatment, discoveries from Ras have laid the foundation for three broad areas of science. First, they focused studies on the origins of cancer to the molecular level, with the subsequent discovery of genes mutated in cancer that now number in the thousands. Second, elucidation of the biochemical mechanisms by which Ras facilitates signal transduction established many of our fundamental concepts of how a normal cell orchestrates responses to extracellular cues. Third, Ras proteins are also founding members of a large superfamily of small GTPases that regulate all key cellular processes and established the versatile role of small GTP-binding proteins in biology. We highlight some of the key findings of the last 28 years. PMID:21686117

The polarity protein Baz forms a platform for the centrosome orientation during asymmetric stem cell division in the Drosophila male germline.

PubMed

Inaba, Mayu; Venkei, Zsolt G; Yamashita, Yukiko M

2015-03-20

Many stem cells divide asymmetrically in order to balance self-renewal with differentiation. The essence of asymmetric cell division (ACD) is the polarization of cells and subsequent division, leading to unequal compartmentalization of cellular/extracellular components that confer distinct cell fates to daughter cells. Because precocious cell division before establishing cell polarity would lead to failure in ACD, these two processes must be tightly coupled; however, the underlying mechanism is poorly understood. In Drosophila male germline stem cells, ACD is prepared by stereotypical centrosome positioning. The centrosome orientation checkpoint (COC) further serves to ensure ACD by preventing mitosis upon centrosome misorientation. In this study, we show that Bazooka (Baz) provides a platform for the correct centrosome orientation and that Baz-centrosome association is the key event that is monitored by the COC. Our work provides a foundation for understanding how the correct cell polarity may be recognized by the cell to ensure productive ACD.

Biological pathways involved in the development of inflammatory bowel disease.

PubMed

Zemljic, Mateja; Pejkovic, Bozena; Krajnc, Ivan; Lipovsek, Saska

2014-10-01

Apoptosis, autophagy and necrosis are three distinct functional types of the mammalian cell death network. All of them are characterized by a number of cell's morphological changes. The inappropriate induction of cell death is involved in the pathogenesis of a number of diseases.Pathogenesis of inflammatory bowel diseases (ulcerative colitis, Crohn's disease) includes an abnormal immunological response to disturbed intestinal microflora. One of the most important reason in pathogenesis of chronic inflammatory disease and subsequent multiple organ pathology is a barrier function of the gut, regulating cellular viability. Recent findings have begun to explain the mechanisms by which intestinal epithelial cells are able to survive in such an environment and how loss of normal regulatory processes may lead to inflammatory bowel disease (IBD).This review focuses on the regulation of biological pathways in development and homeostasis in IBD. Better understanding of the physiological functions of biological pathways and their influence on inflammation, immunity, and barrier function will simplify our expertice of homeostasis in the gastrointestinal tract and in upgrading diagnosis and treatment.

Lessons from natural and artificial polyploids in higher plants.

PubMed

Hegarty, M; Coate, J; Sherman-Broyles, S; Abbott, R; Hiscock, S; Doyle, J

2013-01-01

Polyploidy in higher plants is a major source of genetic novelty upon which selection may act to drive evolution, as evidenced by the widespread success of polyploid species in the wild. However, research into the effects of polyploidy can be confounded by the entanglement of several processes: genome duplication, hybridisation (allopolyploidy is frequent in plants) and subsequent evolution. The discovery of the chemical agent colchicine, which can be used to produce artificial polyploids on demand, has enabled scientists to unravel these threads and understand the complex genomic changes involved in each. We present here an overview of lessons learnt from studies of natural and artificial polyploids, and from comparisons between the 2, covering basic cellular and metabolic consequences through to alterations in epigenetic gene regulation, together with 2 in-depth case studies in Senecio and Glycine. See also the sister article focusing on animals by Arai and Fujimoto in this themed issue. Copyright © 2013 S. Karger AG, Basel.

Understanding How Astronauts Adapt to Space and to Earth: Anatomical Studies of Central Vestibular Adaptation

NASA Technical Reports Server (NTRS)

Holstein, Gay; Vasques, Marilyn; Aquilina, Rudy (Technical Monitor)

2002-01-01

Significant changes take place in the nervous systems of astronauts during and following exposure to microgravity. These changes, particularly in the part of the brain that controls balance, the vestibular system, can cause sensations of rotation, dizziness, and vertigo, as well as space adaptation syndrome. Adaptation to the microgravity environment usually occurs within one week, and a subsequent re-adaptation period of several days is often required upon return to Earth. In order to realize long-term spaceflight, effective countermeasures for these symptoms must be developed. The structural changes that take place in one of the vestibular regions of the brain (the cerebellar cortex) during the process of adaptation to Earth's gravity remain unclear and are the subject of an experiment being conducted on STS-107 by Dr. Gay Holstein of the Mount Sinai School of Medicine in New York. Using the rat as a model, Dr. Holstein and her team will seek to identify the cellular changes underlying the vestibular changes experienced by astronauts.

Structural insights into 5‧ flap DNA unwinding and incision by the human FAN1 dimer

NASA Astrophysics Data System (ADS)

Zhao, Qi; Xue, Xiaoyu; Longerich, Simonne; Sung, Patrick; Xiong, Yong

2014-12-01

Human FANCD2-associated nuclease 1 (FAN1) is a DNA structure-specific nuclease involved in the processing of DNA interstrand crosslinks (ICLs). FAN1 maintains genomic stability and prevents tissue decline in multiple organs, yet it confers ICL-induced anti-cancer drug resistance in several cancer subtypes. Here we report three crystal structures of human FAN1 in complex with a 5‧ flap DNA substrate, showing that two FAN1 molecules form a head-to-tail dimer to locate the lesion, orient the DNA and unwind a 5‧ flap for subsequent incision. Biochemical experiments further validate our model for FAN1 action, as structure-informed mutations that disrupt protein dimerization, substrate orientation or flap unwinding impair the structure-specific nuclease activity. Our work elucidates essential aspects of FAN1-DNA lesion recognition and a unique mechanism of incision. These structural insights shed light on the cellular mechanisms underlying organ degeneration protection and cancer drug resistance mediated by FAN1.

Determining protein complex connectivity using a probabilistic deletion network derived from quantitative proteomics.

PubMed

Sardiu, Mihaela E; Gilmore, Joshua M; Carrozza, Michael J; Li, Bing; Workman, Jerry L; Florens, Laurence; Washburn, Michael P

2009-10-06

Protein complexes are key molecular machines executing a variety of essential cellular processes. Despite the availability of genome-wide protein-protein interaction studies, determining the connectivity between proteins within a complex remains a major challenge. Here we demonstrate a method that is able to predict the relationship of proteins within a stable protein complex. We employed a combination of computational approaches and a systematic collection of quantitative proteomics data from wild-type and deletion strain purifications to build a quantitative deletion-interaction network map and subsequently convert the resulting data into an interdependency-interaction model of a complex. We applied this approach to a data set generated from components of the Saccharomyces cerevisiae Rpd3 histone deacetylase complexes, which consists of two distinct small and large complexes that are held together by a module consisting of Rpd3, Sin3 and Ume1. The resulting representation reveals new protein-protein interactions and new submodule relationships, providing novel information for mapping the functional organization of a complex.

Mechanisms and regulation of DNA replication initiation in eukaryotes

PubMed Central

Parker, Matthew W.; Botchan, Michael R.; Berger, James M.

2017-01-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a given cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the Origin Recognition Complex (ORC), and subsequent activation of the helicase by incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms. PMID:28094588

Mechanisms and regulation of DNA replication initiation in eukaryotes.

PubMed

Parker, Matthew W; Botchan, Michael R; Berger, James M

2017-04-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.

Biocompatibility of platinum-metallized silicone rubber: in vivo and in vitro evaluation.

PubMed

Vince, V; Thil, M A; Veraart, C; Colin, I M; Delbeke, J

2004-01-01

Silicone rubber is commonly used for biomedical applications, including implanted cuff electrodes for both recording and stimulation of peripheral nerves. This study was undertaken to evaluate the consequences of a new platinum metallization method on the biocompatibility of silicone rubber cuff electrodes. This method was introduced in order to allow the manufacture of spiral nerve cuff electrodes with a large number of contacts. The metallization process, implying silicone coating with poly(methyl methacrylate) (PMMA), its activation by an excimer laser and subsequent electroless metal deposition, led to a new surface microtexture. The neutral red cytotoxicity assay procedure was first applied in vitro on BALB/c 3T3 fibroblasts in order to analyze the cellular response elicited by the studied material. An in vivo assay was then performed to investigate the tissue reaction after chronic subcutaneous implantation of the metallized material. Results demonstrate that silicone rubber biocompatibility is not altered by the new platinum metallization method.

CtBP impedes JNK- and Upd/STAT-driven cell fate misspecifications in regenerating Drosophila imaginal discs

PubMed Central

Worley, Melanie I; Alexander, Larissa A

2018-01-01

Regeneration following tissue damage often necessitates a mechanism for cellular re-programming, so that surviving cells can give rise to all cell types originally found in the damaged tissue. This process, if unchecked, can also generate cell types that are inappropriate for a given location. We conducted a screen for genes that negatively regulate the frequency of notum-to-wing transformations following genetic ablation and regeneration of the wing pouch, from which we identified mutations in the transcriptional co-repressor C-terminal Binding Protein (CtBP). When CtBP function is reduced, ablation of the pouch can activate the JNK/AP-1 and JAK/STAT pathways in the notum to destabilize cell fates. Ectopic expression of Wingless and Dilp8 precede the formation of the ectopic pouch, which is subsequently generated by recruitment of both anterior and posterior cells near the compartment boundary. Thus, CtBP stabilizes cell fates following damage by opposing the destabilizing effects of the JNK/AP-1 and JAK/STAT pathways. PMID:29372681